US20080175869A1

US20080175869A1 - Mycobacterial sulfation pathway proteins and methods of use thereof

Info

Publication number: US20080175869A1
Application number: US11/874,536
Authority: US
Inventors: Carolyn R. Bertozzi; Spencer J. WILLIAMS; Joseph D. MOUGOUS
Original assignee: University of California
Current assignee: University of California
Priority date: 2001-04-20
Filing date: 2007-10-18
Publication date: 2008-07-24
Also published as: US7344703B2; US6974580B2; US20030180321A1; US20040249131A1; US20060188517A1; US6863895B2

Abstract

Novel mycobacterial sulfation pathway proteins and polypeptides related thereto, as well as nucleic acid compositions encoding the same, are provided. The subject polypeptide and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications. Also provided are methods of inhibiting growth and/or virulence of a pathogenic mycobacterium, and methods of treating disease conditions associated with a pathogenic mycobacterium, particularly by administering an inhibitor of a mycobacterial sulfation pathway protein. The present invention further provides genetically modified mycobacteria having a defect in a sulfation pathway enzyme gene; and immunogenic compositions that include such genetically modified mycobacteria.

Description

CROSS-REFERENCE

This application is a continuation-in-part application of U.S. patent application Ser. No. 10/126,279, filed Apr. 19, 2002, which claims the benefit of U.S. Provisional Patent Application No. 60/285,394, filed Apr. 20, 2001, and U.S. Provisional Patent Application No. 60/345,953, filed Oct. 26, 2001, which applications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under grant no. R01 GM59907-01 awarded by the National Institutes of Health. The U.S. government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention is in the field of mycobacterial proteins, and in particular, mycobacterial sulfation pathway proteins.

BACKGROUND OF THE INVENTION

Mycobacteria are a significant cause of morbidity and mortality, particularly among immunocompromised or elderly individuals and in countries with limited medical resources. Ninety-five percent of human infections are caused by seven species: Mycobacterium tuberculosis, M. avium (also known as the mycobacterium avium complex or M. avium-intracellulare), M. leprae, M. kansasii, M. fortuitum, M. chelonae, and M. absecessus. The most common mycobacterial infections in the United States are pulmonary infections by M. tuberculosis or M. avium. Such mycobacterial infections have been of increasing concern over the past decade, particularly in light of the increasing incidence of multi-drug resistant strains.
Mycobacterium tuberculosis (Mtub) is the causative agent of the disease tuberculosis in humans. Estimates indicate that one-third of the world's population, including 10 million in the U.S., are infected with M. tuberculosis, with 8 million new cases and 3 million deaths reported world wide each year. Although incidence of tuberculosis steadily decreased since the early 1900s, this trend changed in 1984 with increased immigration from endemic countries and increased infection among homeless individuals, drug and alcohol abusers, prisoners, and HIV-infected individuals. The increasing occurrence of drug-resistant strains requires continued research into new and more effective treatments.
M. avium infection poses the greatest health risk to immunocompromised individuals, and is one of the most common opportunistic infections in patients with AIDS (Horsburgh (1991) New Eng. J. Med. 324:1332-1338). In contrast with disease in other patients, M. avium infection can be very serious in immunocompromised individuals (e.g., AIDS patients, who have a low CD4+ T-cell count (Crowe, et al. (1991) J. AIDS 4:770-776)), and can result in disseminated infection in which virtually no organ is spared.
Treatment of mycobacterial infections is complicated and difficult. For example, treatment of M. tuberculosis and of M. avium infections requires a combination of relatively toxic agents, usually three different drugs, for at least six months. The toxicity and intolerability of these medications usually result in low compliance and inadequate treatment, which in turn increases the chance of therapeutic failure and enhances the selection for drug-resistant organisms. Treatment of mycobacterial infections is further complicated in pregnant women, patients with pre-existing liver or renal diseases, and immunocompromised patients, e.g., AIDS patients.
Sulfotransferases are enzymes that catalyze the transfer of a sulfate from a donor compound to an acceptor compound, usually placing the sulfate moiety at a specific location on the acceptor compound. In mycobacteria, the most notable sulfated compounds identified to date are the “sulfatides” of Mtub. Sulfatides are a closely related set of sulfated glycolipids. They are characterized by a common trehalose-2-sulfate core disaccharide. Sulfatide-1 (sulfolipid-1 or SL-1), the most abundant of the sulfatides, has been extensively studied both structurally and biologically. The molecule consists of a 2,3,6,6′-tetra-O-acyl-trehalose-2′-sulfate. Other members of the family differ in the number and type of the acyl substituents, but not in the core sulfated disaccharide. Reported biological properties of the purified SL-1 include its ability to inhibit macrophage phagosome/lysosome fusion, to enhance the secretion of TNF-α, to inhibit macrophage priming, and to activate human neutrophils.
Recently, a second set of sulfated structures have been identified and characterized in Mycobacteria. A sulfate group has been found in an ester linkage to a sugar residue of a mycobacterial glycopeptidolipid (GPL), in one case at the 2-position of a 3,4-di-O-methylrhamnose in the GPL of M. fortuitum, and in another case at the 4-position of a 6-deoxy-talose in a GPL of a drug-resistant strain of M. avium.
To date, numerous virulence factors and potential drug targets have been studied in Mtub and Mav. No single genetic or metabolic entity, however, has yet to be identified as solely or even mostly responsible for the organisms' ability to cause disease in humans. In particular, information regarding the enzymes responsible for synthesizing sulfated macromolecules in mycobacteria is needed. As such, there is continued interest in identifying additional genes and gene products in Mycobacterium species that can serve as diagnostic tools, and as targets for therapeutic intervention.
Literature Bloom and Murray (1999) Science 257:105-1064 Daffe and Draper (1998) Adv. Microb. Physiol. 39:149-152; Hemmerich and Rosen (2000) Glycobiol. 10:848-856; Goren et al. (1976) Proc. Natl. Acad. Sci. USA 73:2510-2514; Bronzna et al. (1991) Infect. Immun. 59:2542-2548; Pabst et al. (1988) J. Immunol. 140:634-640; Zhang et al. (1991) J. Immunol. 146:2730-2736; Lopez Marin et al. (1992) Biochem. 31:11106-11111; Khoo et al. (1999) J. Biol. Chem. 274:9778-9785; Tsukamara and Mizuno (1981) Microbiol. Immunol. 25:215; Cole et al. (1998) Nature 393:537-544; U.S. Pat. No. 6,046,002.

SUMMARY OF THE INVENTION

BRIEF DESCRIPTIONS OF THE DRAWING

FIG. 1 i-iii provides an alignment of the amino acid sequences of mycobacterial sulfotransferases.

FIG. 2 provides an alignment of the amino acid sequences of mycobacterial sulfotransferases. The sequences of Mycobacterium avium glycosyl sulfotransferases correspond to the sequences in FIG. 1 as follows: identified as AST1 (mav_—62); AST2 (mav_—4); AST3 (mav_—16); AST4 (mav_—144); AST5 (mav_—93); AST6 (mav_—131); AST7 (mav_—130); AST8 (mav_—304).

FIG. 3 provides the nucleotide sequence of Rv2267c (SEQ ID NO:20).

FIG. 4 provides the nucleotide sequence of Rv3529c (SEQ ID NO:14).

FIG. 5 provides the nucleotide sequence of Rv1373 (SEQ ID NO:24).

FIG. 6 provides the nucleotide sequence of AST1 (SEQ ID NO:16; mav_—62).

FIG. 7 provides the nucleotide sequence of AST2 (SEQ ID NO:7; mav_—4).

FIG. 8 provides the nucleotide sequence of AST3 (SEQ ID NO:3; mav_—16).

FIG. 9 provides the nucleotide sequence of AST4 (SEQ ID NO: 11; mav_—144).

FIG. 10 provides the nucleotide sequence of AST5 (SEQ ID NO:9; mav_—93).

FIG. 11 provides the nucleotide sequence of AST6 (SEQ ID NO:5; mav_—131).

FIG. 12 provides the nucleotide sequence of AST7 (SEQ ID NO:1; mav_—130).

FIG. 13 provides the nucleotide sequence of AST8 (SEQ ID NO:22; mav_—304).

FIG. 14 provides the amino acid sequence of an APS reductase from M. tuberculosis H37Rv (SEQ ID NO:27).

FIG. 15 provides the amino acid sequence of an APS reductase from M. smegmatis mc²155 (SEQ ID NO:28).

FIG. 16 provides the amino acid sequence of an APS reductase from M. avium (SEQ ID NO:29).

FIG. 17 provides an alignment of the amino acid sequences of APS reductases from M. tuberculosis, M. smegmatis, and M. avium.

FIG. 18 depicts complementation of E. coli JM81A by M. tuberculosis CysH.

FIG. 19 provides the amino acid sequence of an APS kinase from M. smegmatis mc²155 (SEQ ID NO:31).

FIG. 20 provides the amino acid sequence of an APS kinase from M. avium (SEQ ID NO:32).

FIG. 21 a depicts a sulfation assimilation pathway used by M. tuberculosis, M. smegmatis, and M. avium. FIG. 21 b depicts sulfate assimilation pathways in plants and bacteria.

FIG. 22 depicts a screen for inhibitors of APS reductase and APS kinase.

FIG. 23 depicts a growth curve for JM81A; JM81A complemented with CysC; JM81A complemented with CysH; in the presence and absence of DMSO.

FIG. 24 depicts Fourier transform ion cyclotron resonance mass spectroscopy (FT-ICR MS) analysis of M. tuberculosis extracts.

FIG. 25 depicts survival of mice infected with M. tuberculosis wild-type H37Rv or mutant M. tuberculosis H37RvΔCysH.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term “polynucleotide” includes single-, double-stranded and triple helical molecules. “Oligonucleotide” generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.
The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art. Nucleic acids may be naturally occurring, e.g. DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O— phosphorothioate, 3′-CH₂-5′-O-phosphonate and 3′—NH-5′-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.
Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2′-OH of the ribose sugar may be altered to form 2′-O— methyl or 2′-O-allyl sugars, which provides resistance to degradation without comprising affinity.
Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine for deoxycytidine. 5-propynyl-2′-deoxyuridine and 5-propynyl-2′-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
A “substantially isolated” or “isolated” polynucleotide is one that is substantially free of the sequences with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of the materials with which it is associated in nature. As used herein, an “isolated” polynucleotide also refers to recombinant polynucleotides, which, by virtue of origin or manipulation: (1) are not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) are linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
Hybridization reactions can be performed under conditions of different “stringency”. Conditions that increase stringency of a hybridization reaction of widely known and published in the art. See, for example, Sambrook et al. (1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1 ×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water. Examples of stringent conditions are hybridization and washing at 50° C. or higher and in 0.1×SSC (9 mM NaCl/0.9 mM sodium citrate).
Stringent hybridization conditions are, for example, 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5 mM sodium citrate) or lower. Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention.
“T_m” is the temperature in degrees Celsius at which 50% of a polynucleotide duplex made of complementary strands hydrogen bonded in anti-parallel direction by Watson-Crick base pairing dissociates into single strands under conditions of the experiment. T_mmay be predicted according to a standard formula, such as:
T _m=81.5+16.6 log [X ⁺]+0.41(% G/C)−0.61(% F)−600/L
where [X⁺] is the cation concentration (usually sodium ion, Na⁺) in mol/L; (% G/C) is the number of G and C residues as a percentage of total residues in the duplex; (% F) is the percent formamide in solution (wt/vol); and L is the number of nucleotides in each strand of the duplex.
Stringent conditions for both DNA/DNA and DNA/RNA hybridization are as described by Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, herein incorporated by reference. For example, see page 7.52 of Sambrook et al.
A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at http://ww.ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)
Of interest is the BestFit program using the local homology algorithm of Smith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters:


	Mismatch Penalty:	1.00;
	Gap Penalty:	1.00;
	Gap Size Penalty:	0.33; and
	Joining Penalty:	30.0.

One parameter for determining percent sequence identity is the “percentage of the alignment region length” where the strongest alignment is found. The percentage of the alignment region length is calculated by counting the number of residues of the individual sequence found in the region of strongest alignment. This number is divided by the total residue length of the target or query polynucleotide sequence to find a percentage.
An example is shown below:
where a=guanine; b=cytosine; c=thymine; and d=adenine.
The region of alignment begins at residue 9 and ends at residue 19. The total length of the target sequence is 20 residues. The percent of the alignment region length is 11 divided by 20 or 55%, for example.
Percent sequence identity is calculated by counting the number of residue matches between the target and query polynucleotide sequence and dividing total number of matches by the number of residues of the target or query sequence found in the region of strongest alignment. For the example above, the percent identity would be 10 matches divided by 11 residues, or approximately, 90.9%.
The percent of the alignment region length is typically at least about 55% of total length of the sequence, more typically at least about 58%, and even more typically at least about 60% of the total residue length of the sequence. Usually, percent length of the alignment region can be as great as about 62%, more usually as great as about 64% and even more usually as great as about 66%.
The term “host cell” includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a “recombinant host cell.”
The term “binds specifically,” in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide i.e., epitope of a subject polypeptide. Antibody binding to an epitope on a specific mycobacterial sulfation pathway polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest, e.g., binds more strongly to an epitope on a specific mycobacterial sulfation pathway polypeptide than to an epitope on a different mycobacterial sulfation pathway polypeptide so that by adjusting binding conditions the antibody binds almost exclusively to an epitope of the specific mycobacterial sulfation pathway polypeptide and not to any other epitope on the mycobacterial sulfation pathway polypeptide, and not to any other mycobacterial sulfation pathway polypeptide which does not comprise the epitope. In some embodiments, an antibody of the invention binds to a mycobacterial sulfation pathway polypeptide of one species, but not another, and thus can distinguish between sulfation pathway polypeptides from two mycobacterial species. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g. by use of appropriate controls. In general, antibodies of the invention which bind to a specific mycobacterial sulfation pathway polypeptide with a binding affinity of 10⁷mole/liter or more, preferably 10⁸mole/liter or more are said to bind specifically to the specific mycobacterial sulfation pathway polypeptide. In general, an antibody with a binding affinity of 10⁶mole/liter or less is not useful in that it will not bind an antigen at a detectable level using conventional methodology currently used.
A “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
“Treatment” or “treating” as used herein means any therapeutic intervention in a subject, usually a mammalian subject, generally a human subject, including: (i) prevention, that is, causing the clinical symptoms not to develop, e.g., preventing infection and/or preventing progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active (ongoing) infection so that pathogen load is decreased to the degree that it is no longer harmful, which decrease can include complete elimination of an infectious dose of the pathogen from the subject; and/or (iii) relief, that is, causing the regression of clinical symptoms, e.g., causing a relief of fever, inflammation, and/or other symptoms caused by an infection.
The term “effective amount” or “therapeutically effective amount” means a dosage sufficient to provide for treatment for the disease state being treated or to otherwise provide the desired effect (e.g., induction of an effective immune response). The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease (e.g., the species of the infecting pathogen), and the treatment being effected. In the case of an intracellular pathogen infection, an “effective amount” is that amount necessary to substantially improve the likelihood of treating the infection, in particular that amount which improves the likelihood of successfully preventing infection or eliminating infection when it has occurred.
By “subject” or “individual” or “patient” or “host” is meant any subject for whom or which therapy is desired. Human subjects are of particular interest. Other subjects may include non-human primates, cattle, sheep, goats, dogs, cats, birds (e.g., chickens or other poultry), guinea pigs, rabbits, rats, mice, horses, and so on. Of particular interest are subjects having or susceptible to intracellular pathogen infection, particularly mycobacterial infection, more particularly to infection by M. tuberculosis, M. avium, and the like.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the mycobacterium” includes reference to one or more mycobacteria and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Polypeptide Compositions

Novel mycobacterial sulfation pathway polypeptides, as well as polypeptide compositions related thereto, are provided. The subject sulfation pathway polypeptides are present in other than their natural environment, e.g., they are isolated. The term polypeptide composition as used herein refers to both the full length mycobacterial protein as well as portions or fragments thereof. Also included in this term are variations of the naturally occurring mycobacterial protein, where such variations are homologous or substantially similar to the naturally occurring protein, as described in greater detail below, as well as corresponding homologs from other mycobacterial species.
Mycobacterial sulfation pathway polypeptides are polypeptides that are components of a biosynthetic pathway whose end product is a sulfated glycopeptidolipid or a sulfated glycolipid found in a mycobacterium. Mycobacterial sulfation pathway polypeptides of the invention include, but are not limited to, sulfotransferases, ATP sulfurylases; adenylyl phosphosulfate (APS) reductases; 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductases; APS kinases; sulfatases; and sulfate transporters.
In the following description of the subject invention, the term M-ST is used to refer to mycobacterial sulfotransferases. A mycobacterial sulfotransferase of the invention comprises one or more of the following motifs: (1) a 5′-phosphosulfate binding loop; (2) a 3′-phosphate binding motif; and (3) a conserved RYEDL motif (SEQ ID NO:52). The 5′-phosphosulfate binding loop and the 3′-phosphate binding motif are necessary to bind the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). PAPS is a universal sulfotransferase substrate that serves as the sulfate donor.
In particular embodiments, a mycobacterial sulfation pathway protein, e.g., an M-ST polypeptide, of the invention has an amino acid sequence of any one of the proteins identified as mav_—130 (SEQ ID NO:2); mav_—16 (SEQ ID NO:4); mav_—131 (SEQ ID NO:6); mav_—4 (SEQ ID NO:8); mav_—93 (SEQ ID NO:10); mav_—144 (SEQ ID NO:12); mbov_—334 (SEQ ID NO:13); mtub_rv3529c (SEQ ID NO:15); mav_—62 (SEQ ID NO:17); mav_tb_—2056 (SEQ ID NO: 18); mbov_—479 (SEQ ID NO: 19); mtub_rv2267c (SEQ ID NO:21); and mav_—304 (SEQ ID NO:23) in FIG. 1; and rv1373 (SEQ ID NO:25) in FIG. 2. In some embodiments, an M-ST polypeptide of the invention has the sequence identified as “consensus” (SEQ ID NO:26) in FIG. 2.
Also provided are M-ST homologs. The subject M-ST homologs have a sequence that is substantially identical to Mav-130 (as shown in FIG. 1), having the amino acid sequence set forth in SEQ ID NO:02, where by “substantially identical” is meant that the protein has an amino acid sequence identity to the sequence set forth in SEQ ID NO:02 of at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95, at least about 98%, or at least about 99%.
The mycobacterial sulfation pathway proteins of the subject invention (e.g. M-ST, etc.) are present in a non-naturally occurring environment, e.g. are separated from their naturally occurring environment. In certain embodiments, the subject proteins are present in a composition that is enriched for subject protein as compared to its naturally occurring environment. For example, purified subject protein is provided, where by purified is meant that subject protein is present in a composition that is substantially free of non-subject proteins, where by substantially free is meant that less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of non-subject proteins. The proteins of the subject invention may also be present as an isolate, by which is meant that the protein is substantially free of other proteins and other naturally occurring biologic molecules, such as oligosaccharides, lipids commonly found in mycobacteria, polynucleotides and fragments thereof, and the like, where substantially free in this instance means that less than 70%, usually less than 60% and more usually less than 50%, less than about 40%, less than about 30%, or less than about 20%, of the composition containing the isolated protein is some other naturally occurring biological molecule. In certain embodiments, the proteins are present in substantially pure form, where by substantially pure form is meant at least 95%, usually at least 97% and more usually at least 99% pure.
In addition to the naturally occurring proteins, polypeptides which vary from the naturally occurring proteins are also provided. By “an M-ST” polypeptide is meant an amino acid sequence encoded by an open reading frame (ORF) of an M-ST polynucleotide, described in greater detail below, including the full length M-ST protein and fragments thereof, particularly biologically active fragments and/or fragments corresponding to functional domains, e.g. acceptor binding site (postulated to be the most 5′ consensus region, the donor binding site, e.g. RYEDL, and the like; and including fusions of the subject polypeptides to other proteins or parts thereof. Thus, in some embodiments, an M-ST polypeptide comprises at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, or at least about 300, contiguous amino acids of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 15, 17, 18, 19, 21, 23, and 25. In many embodiments, an M-ST polypeptide of the invention comprises the complete amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 15, 17, 18, 19, 21, 23, and 25.
Also provided are polypeptides that include an amino acid sequence of any one of SEQ ID NOs: 27, 28, and 29, depicted in FIGS. 14-17. Polypeptides of interest that include an amino acid sequence of any one of SEQ ID NOs: 27, 28, and 29 are those that exhibit APS reductase activity. Also provided are polypeptides that include an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 28, and 29, depicted in FIGS. 14-17. Also provided are polypeptides that include at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, or at least about 225 contiguous amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 27, 28, and 29, depicted in FIGS. 14-17.
Also provided are polypeptides that include an amino acid sequence of any one of SEQ ID NOs: 31 and 32, depicted in FIGS. 19, and 20, respectively. Polypeptides of interest that include an amino acid sequence of any one of SEQ ID NOs: 31 and 32 are those that exhibit APS kinase activity. Also provided are polypeptides that include an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 31 and 32, depicted in FIGS. 19, and 20, respectively. Also provided are polypeptides that include at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, or at least about 600 contiguous amino acids of the amino acid sequence set forth in any one of SEQ ID NOs: 31 and 32, depicted in FIGS. 19, and 20, respectively.
Also provided are mutants of a mycobacterial sulfation pathway polypeptide, e.g., an M-ST, an APS reductase, an APS kinase, etc. In some embodiments, mutants have altered physical characteristics, compared to a “wild-type” or naturally occurring mycobacterial sulfation pathway polypeptide. Physical characteristics of a mutant mycobacterial sulfation pathway polypeptide of the invention include one or more of the following: (1) increased solubility in aqueous solution; (2) correct folding during translation; (3) mutations that alter antigenicity; and (4) mutations that increase or decrease enzyme turnover. Mutants can be generated using well-known techniques for mutagenesis of a nucleic acid molecule. Random mutagenesis of a polynucleotide comprising a nucleotide sequence encoding a mycobacterial sulfation pathway polypeptide can be carried out, using techniques that are standard in the art, and the polypeptides encoded thereby evaluated for various physical properties described above. Mutants can also be selected for various physical properties.
For example, one can select for properly folded mutants in the following manner. Following random mutagenesis of a polynucleotide comprising a nucleotide sequence encoding a mycobacterial sulfation pathway polypeptide, the polynucleotide can be cloned into an expression vector comprising a nucleotide sequence encoding a detectable marker protein, e.g., a chromoprotein or fluoroprotein (fluorescent protein) (e.g., green fluorescent protein from Aequorea victoria; or any fluorescent protein from, e.g., an anthozoan species) such that a fusion protein is encoded. Fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a “humanized” version of a GFP, e.g., wherein codons of the naturally-occurring nucleotide sequence are changed to more closely match human codon bias; a GFP derived from Aequoria Victoria or a derivative thereof, e.g., a “humanized” derivative such as Enhanced GFP, which are available commercially, e.g., from Clontech, Inc.; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP (hrGFP) (Stratagene); any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like. Where the fusion partner is an enzyme that yields a detectable product, the product can be detected using an appropriate means, e.g., β-galactosidase can, depending on the substrate, yield colored product, which is detected spectrophotometrically, or a fluorescent product; luciferase can yield a luminescent product detectable with a luminometer; etc.
The fusion protein comprises the mycobacterial sulfation pathway protein fused in-frame to the detectable marker protein. After transfection into a suitable host cell, e.g., Mycobacterium smegmatis, E. coli, and the like) colonies are examined visually for the presence of the detectable marker protein. If the detectable marker protein is detectable, e.g., it fluoresces or is colored, then it is likely properly folded. The mycobacterial sulfation pathway polypeptide is therefore also likely to be properly folded.
The subject proteins and polypeptides may be obtained from naturally occurring sources or synthetically produced. Where obtained from naturally occurring sources, the source chosen will generally depend on the species from which the protein is to be derived. For example, Mtub sulfotransferase is generally derived from Mycobacterium tuberculosis. The subject proteins may also be derived from synthetic means, e.g. by expressing a recombinant gene encoding protein of interest in a suitable host, as described in greater detail below. Any convenient protein purification procedures may be employed, where suitable protein purification methodologies are described in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example, a lysate may prepared from the original source, e.g. a mycobacterium or the expression host, and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

Polynucleotide Compositions; Recombinant Vectors; Host Cells

Also provided are polynucleotide compositions encoding mycobacterial sulfation pathway proteins (e.g., M-ST and the like) or fragments thereof, where the nucleotide sequence of the polynucleotide differs from a wild-type or naturally occurring polynucleotide that comprises a nucleotide sequence encoding a mycobacterial sulfation pathway protein. The invention further provides recombinant vectors comprising a subject polynucleotide, as well as host cells comprising a subject polynucleotide and host cells comprising a subject recombinant vector.
By mycobacterial sulfation pathway polynucleotide composition is meant a composition comprising a sequence of polynucleotide having an open reading frame that encodes mycobacterial sulfation pathway polypeptide of the invention, and is capable, under appropriate conditions, of being transcribed and translated such that a mycobacterial sulfation pathway polypeptide is produced. Also encompassed in this term are polynucleotides that are homologous or substantially similar or identical to the polynucleotides encoding mycobacterial sulfation pathway polypeptides. Thus, the subject invention provides genes encoding mycobacterial sulfation pathway polypeptides and homologs thereof.
The nucleotide sequences set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 14, 16, 20, 22, and 24 encode polypeptides identified as SEQ ID NO:2, 4, 6, 8, 10, 12, 15, 17, 21, 23, and 25, respectively. In all embodiments, the nucleotide sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 14, 16, 20, 22, and 24 are specifically excluded. Polynucleotides of the invention comprise nucleotide sequences that differ in nucleotide sequence from the sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 14, 16, 20, 22, and 24 by at least about 5%.
In some embodiments a mycobacterial sulfation pathway polynucleotide of the invention shares from about 50% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80% from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, nucleotide sequence identity to the coding region of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 14, 16, 20, 22, and 24. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using default settings). The sequences provided herein are essential for recognizing related and homologous proteins in database searches.
In some embodiments, a mycobacterial sulfation pathway polynucleotide of the invention encodes a mycobacterial sulfation pathway polypeptide. In some of these embodiments, a mycobacterial sulfation pathway polynucleotide of the invention comprises a nucleotide sequence that encodes a polypeptide comprising at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, or at least about 300, contiguous amino acids of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 15, 17, 21, 23, and 25. In many embodiments, a mycobacterial sulfation pathway polynucleotide of the invention comprises a nucleotide sequence that encodes a polypeptide comprising the complete amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 15, 17, 21, 23, and 25.
In other embodiments, a mycobacterial sulfation pathway polynucleotide includes a nucleotide sequence that encodes a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with any one of SEQ ID NOs:27, 28, 29, 30, 31, and 32.
In other embodiments, a mycobacterial sulfation pathway polynucleotide includes a nucleotide sequence that encodes a polypeptide that includes at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, or at least about 225 contiguous amino acids of the amino acid sequence set forth in any one of SEQ ID NO:27, 28, and 29, depicted in FIGS. 14-17.
In other embodiments, a mycobacterial sulfation pathway polynucleotide includes a nucleotide sequence that encodes a polypeptide that includes at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, or at least about 600 contiguous amino acids of the amino acid sequence set forth in any one of SEQ ID NOs:31 and 32, depicted in FIGS. 19, and 20, respectively.
Mycobacterial sulfation pathway polynucleotides of the invention differ from wild-type mycobacterial polynucleotides. The subject polynucleotides are typically generated by random or directed mutagenesis of wild-type mycobacterial sulfation pathway polynucleotides. The source of wild-type mycobacterial sulfation pathway polynucleotide is any mycobacterial species, e.g., by M. tuberculosis, M. avium (or M. avium-intracellulare), M. leprae (particularly M. leprae infection leading to tuberculoid leprosy), M. kansasii, M. fortuitum, M. chelonae, and M. absecessus.
Nucleic acids encoding the polypeptides of the subject invention may be cDNA or genomic DNA or a fragment thereof. The term “mycobacterial sulfation pathway gene” refers to the open reading frame encoding specific mycobacterial sulfation pathway proteins and polypeptides, as well as adjacent 5′ and 3′ non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
The term “cDNA” as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are coding regions, as well as 3′ and 5′ non-coding regions. Normally mRNA species have a sequence of a continuous open reading frame encoding a mycobacterial sulfation pathway protein.
A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences that are normally present in a native chromosome. It may further include the 3′ and 5′ untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5′ or 3′ end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3′ or 5′, or internal regulatory sequences, contains sequences required for proper expression (e.g., expression during a specific phase of growth or exposure to a regulator of expression).
The mycobacterial sulfation pathway genes are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a mycobacterial sulfation pathway sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically “recombinant”, i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
In addition to the plurality of uses described in greater detail in following sections, the subject nucleic acid compositions find use in the preparation of all or a portion of the subject polypeptides, as described above. For expression, an expression cassette may be employed. The expression vector will provide a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to a mycobacterial sulfation pathway gene, or may be derived from exogenous sources.
Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Expression vectors may be used for the production of fusion proteins, where the exogenous fusion peptide provides additional functionality, i.e. increased protein synthesis, stability, reactivity with defined antisera, an enzyme marker, e.g. β-galactosidase, a fluoroprotein, a chromoprotein, etc.
Expression vectors for introducing exogenous coding sequences into mycobacteria are known in the art, any of which can be used herein. See, e.g., U.S. Pat. No. 5,968,733; U.S. Pat. No. 6,074,866; U.S. Pat. No. 6,015,696; Triccas et al. (1998) FEMS Microbiol. Lett. 167:151-156; and DasGupta et al. (1998) Biochem. Biophys. Res. Commun. 246:797-804. Examples of expression vectors include those that utilize Hsp60 promoters, the promoter normally associated with the coding region for the specific protein, the glutamine synthase promoter, or the inducible acetamidase promoter. Many of these promoters are used in the pMS series of vectors. These vectors often include the Hyg (hygromycin) resistance gene. Vectors can provide for inducible expression of a protein, by using an inducible promoter, e.g., the acetamidase promoter (inducible by adding acetamide to the culture medium), and the like.
Expression cassettes may be prepared comprising a transcription initiation region, the gene or fragment thereof, and a transcriptional termination region. Of particular interest is the use of sequences that allow for the expression of functional epitopes or domains, usually at least about 8 amino acids in length, more usually at least about 15 amino acids in length, to about 25 amino acids, and up to the complete open reading frame of the gene. After introduction of the DNA, the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.
Subject proteins and polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as Mycobacterium smegmatis, E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g., mammals, e.g. COS 7 cells, may be used as the expression host cells. Of particular interest in many embodiments is the use of non-pathogenic strains of mycobacteria, e.g., Mycobacterium smegmatis, Mycobacterium bovis-BCG (Bacille Calmette Guerin), and the like. Small peptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete mycobacterial sulfation pathway protein sequence may be used to identify and investigate parts of the protein important for function.

Antibodies Specific for a Mycobacterial Sulfation Pathway Polypeptide of the Invention

The invention provides antibodies that are specific for a subject mycobacterial sulfation pathway polypeptide. Suitable antibodies are obtained by immunizing a host animal with peptides comprising all or a portion of the subject protein. Suitable host animals include mouse, rat sheep, goat, hamster, rabbit, etc.
The immunogen may comprise the complete protein, or fragments and derivatives thereof. Preferred immunogens comprise all or a part of one of the subject proteins, where these residues contain the post-translation modifications, such as glycosylation, found on the native target protein. Immunogens comprising one or more epitopes are produced in a variety of ways known in the art, e.g. expression of cloned genes using conventional recombinant methods, isolation from mycobacteria, etc.
For preparation of polyclonal antibodies, the first step is immunization of the host animal with a subject protein, where the subject protein will preferably be in substantially pure form, comprising less than about 1% contaminant. The immunogen may comprise the complete subject protein, fragments or derivatives thereof. To increase the immune response of the host animal, the subject protein may be combined with an adjuvant, where suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like. The subject protein may also be conjugated to synthetic carrier proteins or synthetic antigens. A variety of hosts may be immunized to produce the polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and the like. The subject protein is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages. Following immunization, the blood from the host will be collected, followed by separation of the serum from the blood cells. The Ig present in the resultant antiserum may be further fractionated using known methods, such as ammonium salt fractionation, DEAE chromatography, and the like.
Monoclonal antibodies are produced by conventional techniques. Generally, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma cells. The plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells. Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity. Suitable animals for production of monoclonal antibodies to the mycobacterial protein include mouse, rat, hamster, etc. The antibody may be purified from the hybridoma cell supernatants or ascites fluid by conventional techniques, e.g. affinity chromatography using protein according to the subject invention bound to an insoluble support, protein A sepharose, etc.
The antibody may be produced as a single chain, instead of the normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine. The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
For in vivo use, particularly for injection into humans, it is desirable to decrease the antigenicity of the antibody. An immune response of a recipient against the blocking agent will potentially decrease the period of time that the therapy is effective. Methods of humanizing antibodies are known in the art. The humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example International Patent Applications WO 90/10077 and WO 90/04036). Alternatively, the antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190).
The use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.
In yet other embodiments, the antibodies may be fully human antibodies. For example, xenogeneic antibodies which are identical to human antibodies may be employed. By xenogeneic human antibodies is meant antibodies that are the same has human antibodies, i.e. they are fully human antibodies, with exception that they are produced using a non-human host which has been genetically engineered to express human antibodies. See e.g. WO 98/50433; WO 98,24893 and WO 99/53049, the disclosures of which are herein incorporated by reference.
Antibody fragments, such as Fv, F(ab′)₂and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab′)₂fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.
Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g. SV-40 early promoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Ig promoters, etc.

Genetically Altered Mycobacteria

The invention further provides genetically altered mycobacteria. A polynucleotide of the invention, or a wild-type mycobacterial sulfation pathway polynucleotide, or other polynucleotide, can be used to genetically alter a mycobacterium. In some embodiments, a genetically altered mycobacterium over-expresses a sulfation pathway enzyme. In some embodiments, the invention provides knock-out mutants, where an endogenous mycobacterial sulfation pathway gene is functionally disabled via homologous recombination. Such genetically altered mycobacteria are attenuated, i.e., their ability to invade and infect is reduced. A subject genetically modified mycobacterium is therefore useful in immunogenic compositions, e.g., as vaccines. A subject genetically modified mycobacterium is also useful in cell-based screening assays (described below), where a subject genetically modified mycobacterium that has a functionally disabled sulfation pathway gene is useful as a control.
Homologous recombination is carried out using well-established techniques. Exogenous DNA, which includes DNA homologous to genomic DNA of the recipient mycobacterium (homologous DNA), as well as DNA which is not homologous to genomic DNA of the recipient mycobacterium (nonhomologous DNA), is introduced into a mycobacterium. Exogenous DNA is integrated into genomic DNA. The DNA construct includes homologous DNA for targeting into a homologous genomic locus and DNA which acts to knock out (inactivate) or activate a resident (endogenous) mycobacterial gene. In the case of inactivation, the mycobacterial gene is “knocked out”, in the sense that it is rendered inactive by addition of DNA whose presence interferes with its ability to function, by removal or replacement of sequences necessary for it to be functional or by its complete removal from the mycobacterial genome. Methods of homologous recombination in mycobacteria are described in detail in Ganjam et al. (1991) Proc. Natl. Acad. Sci. USA 88:5433-5437; Aldovini et al. (1993) J. Bacteriol. 175:7282-7289, which are incorporated herein by reference.
Knock-out by homologous recombination are performed using established techniques. See, e.g., U.S. Pat. No. 6,136,324. General protocols for generating knockouts are provided in the Examples section. For example, an allelic replacement method can be performed, as described in the Examples, using well-known techniques. See, e.g., Parish and Stoker (2000) Microbiology 146(8):1969-75.
Any other method of genetically modifying a mycobacterium, such that is functionally disabled sulfation pathway enzyme gene is generated, can be used. Standard methods include random and site-specific mutagenesis. Random or site-specific mutagenesis is used to generate mutants in a transcriptional or translation control element, in a coding region, and the like, to generate a genetically modified mycobacterium that has a functionally disabled sulfation pathway enzyme gene.
In general, a subject genetically modified mycobacterium has a functionally disabled sulfation pathway gene. A subject genetically modified, attenuated mycobacterium typically has a genetic modification in one or more of a sulfotransferase gene, an ATP sulfurylase gene, an APS reductase gene, a PAPS reductase gene, an APS kinase gene, a sulfatase gene, and a sulfate transporter gene, such that the gene is functionally disabled. A “functionally disabled sulfation pathway gene” is a sulfation pathway gene that is genetically altered such that the level of protein encoded by the gene is at background levels (e.g., undetectable, or at or near the lower limit of detection), or is undetectable; such that the protein encoded by the gene is produced but is non-functional; such that the encoded protein produced and is functional but is produced at levels that are too low to be effective in carrying out the normal function of the protein in the bacterium; or such that the encoded protein produced is functional but is produced at levels that are lower than normal (e.g., lower than wild-type levels) such that bacterial virulence is attenuated.
A subject genetically modified mycobacterium that has a functionally disabled sulfation pathway enzyme gene exhibits reduced virulence as a result of the functional disablement of the gene. Virulence in a subject genetically modified mycobacterium is reduced by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (e.g., 95%, 99%, 100%), compared with a wild-type mycobacterium of the same species and not having the genetic modification, e.g., a wild-type mycobacterium that is virulent.
In some embodiments, the LD₅₀of a subject genetically modified mycobacterium is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, or at least about 250-fold, or more, higher than the LD₅₀of a wild-type mycobacterium of the same species and not having the genetic modification.
Virulence is determined using any known assay. The term “virulence” encompasses two features of a pathogenic organism: its infectivity (i.e., the ability to colonize a host) and the severity of the disease produced. Virulence can be expressed as the LD₅₀, i.e., the dose that will kill 50% of inoculated animals within a given time. Virulence can also be expressed as transmissibility, i.e., the ability of a bacterium to cause a demonstrable infection in a given animal host. Transmissibility is usually detected by culture methods. The dose required is the ID₅₀, the infection dose in 50% of animals. Virulence can also be expressed as communicability. Virulence can be tested using any known assay, including, but not limited to, mouse colony formation assay, in which the number of mycobacterial colonies in the lung of infected mice is counted at various time points after infection; and macrophage infectivity assays. Other laboratory animals such as rabbits and guinea pigs can also be used. Virulence can also be determined in a cell culture assay using macrophages. Bacteria are incubated with cultured macrophages and the number of bacteria that enter the macrophages determined by washing the macrophages, lysing them, culturing their contents on plates, and counting “colony forming units.”
In particular embodiments, a subject genetically altered mycobacterium has a functionally disabled APS reductase gene. In other particular embodiments, a subject genetically modified mycobacterium has a functionally disabled APS kinase gene. In still other particular embodiments, a subject genetically altered mycobacterium has a functionally disabled sulfotransferase gene. Examples of such mycobacteria are found in Examples 3, 6, and 9. In some embodiments, a genetically altered mycobacterium is a strain that is normally pathogenic, but exhibits reduced virulence by virtue of the genetic modification. In particular embodiments, a subject genetically altered mycobacterium is M. tuberculosis. The present invention also provides immunogenic compositions comprising genetically altered mycobacterium, which compositions are described in more detail below.

Methods

The invention further provides screening methods and therapeutic methods. Screening methods identify agents that reduce an activity of a mycobacterial sulfation pathway polypeptide. Therapeutic methods of the invention include methods of treating a mycobacterial infection in an individual, methods of reducing viability of a pathogenic mycobacterium, methods of reducing virulence of a pathogenic mycobacterium, and methods of increasing a protective immune response to a mycobacterium.

Screening Assays

The present invention further provides in vitro screening assays to identify agents that modulate an activity of a component of a mycobacterial sulfation pathway, e.g., a component of a pathway whose end product is a sulfated macromolecule. The screening assays are designed to identify agents that are useful as therapeutic agents for treating mycobacterial infections. Both cell-based and cell-free assays are provided.
In some embodiments, the screening assays are cell-free screening assays. In these embodiments, the methods generally involve contacting a mycobacterial sulfation pathway component with a test agent, and determining the effect, if any, on an activity, e.g., an enzymatic activity, of the pathway component. Sulfation pathway components that are suitable for use in a cell-free screening assay include, but are not limited to, mycobacterial sulfotransferases; mycobacterial ATP-sulfurylases; mycobacterial APS kinases; mycobacterial PAS and PAPS reductases; and mycobacterial sulfatases. For example, recombinant M-ST polypeptide can be combined with ³⁵S-labeled sulfate donor such as [³⁵S]-PAPS, candidate inhibitor compound, and an acceptor molecule.
In other embodiments, the methods provide cell-based assays. In these embodiments, the methods generally involve contacting a host cell which produces an M-ST polypeptide with a labeled sulfate, e.g. ³⁵S-labeled sulfate and a candidate agent, and determining the effect, if any, on the amount of sulfate incorporation into a substrate for the M-ST in the presence and absence of a candidate agent.
Suitable sulfate acceptor molecules include, but are not limited to, glycopeptidolipids (GPL), including, but not limited to, a GPL containing a 3,4,-di-O-methylrhamnose, and a GPL containing a 6-deoxy-talose; trehalose-containing glycolipids; and glycolipids or glycoproteins of mammalian origin.
A variety of different candidate agents (“test agents”) may be screened by the screening methods of the invention. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, and may be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups. The candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents, also referred to herein as “test agents”) are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
An agent of interest which modulates a sulfotransferase activity of a subject polypeptide decreases the activity at least about 10%, at least about 15%, at least about 20%, at least about 25%, more preferably at least about 50%, more preferably at least about 100%, or 2-fold, more preferably at least about 5-fold, more preferably at least about 10-fold or more when compared to a suitable control.
Agents that decrease a sulfotransferase or other activity of a subject polypeptide to the desired extent may be selected for further study, and assessed for cellular availability, cytotoxicity, biocompatibility, etc. For example, a candidate agent is assessed for any cytotoxic activity it may exhibit toward a eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]) assay, and the like. Agents that do not exhibit cytotoxic activity toward eukaryotic cells are considered candidate agents for use in therapeutic methods for treating a mycobacterial infection.

Cell-Free Assays

Cell-free assay methods generally comprise:
a) contacting a test agent with a sample containing a mycobacterial sulfation pathway polypeptide; and
b) assaying an activity of the mycobacterial sulfation pathway polypeptide in the presence of the substance. An increase or a decrease in the measured activity in comparison to the activity in a suitable control (e.g., a sample comprising a mycobacterial sulfation pathway polypeptide in the absence of the substance being tested) is an indication that the substance modulates an activity of the mycobacterial sulfation pathway polypeptide.
Cell-free assays may be designed in a number of ways. In some embodiments, a mycobacterial sulfation pathway polypeptide (e.g. M-ST) is combined with ³⁵S-labeled sulfate donor such as [³⁵S]-PAPS, a candidate inhibitor compound (“a test agent”), and an acceptor molecule, which may be a natural or synthetic GL, GPL, or a simple nucleophile capable of accepting sulfate (such as phenolic compounds, and the like). The amount of [³⁵S]-sulfate transferred to the acceptor by the candidate agent is then determined by counting the acceptor-associated radioactivity or product quantitation with an antibody specific for the sulfated acceptor, or in a suitable scintillation proximity assay format.
An “agent which inhibits a sulfotransferase activity of a mycobacterial sulfotransferase polypeptide”, as used herein, describes any molecule, e.g. synthetic or natural organic or inorganic compound, protein or pharmaceutical, with the capability of altering a sulfotransferase activity of a sulfotransferase polypeptide, as described herein. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection. Sulfotransferase activity can be measured using any assay known in the art.
A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-ligand binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used.
The above screening methods may be designed a number of different ways, where a variety of assay configurations and protocols may be employed, as are known in the art. For example, one of the components may be bound to a solid support, and the remaining components contacted with the support bound component. The above components of the method may be combined at substantially the same time or at different times. Incubations are performed at any suitable temperature, typically between 4° and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient. Following the contact and incubation steps, the subject methods will generally, though not necessarily, further include a washing step to remove unbound components, where such a washing step is generally employed when required to remove label that would give rise to a background signal during detection, such as radioactive or fluorescently labeled non-specifically bound components. Following the optional washing step, the amount of incorporated sulfate will then be detected.

Cell-Based Assays

Cell-based assay generally involve contacting a cell that produces a mycobacterial sulfation pathway polypeptide with a test agent, and determining the effect, if any, on an activity of the peptide.
In some embodiments, a cell is a mycobacterial cell that produces the mycobacterial sulfation pathway polypeptide endogenously, or a cell, such as a mycobacterial cell, that is transformed with nucleic acid molecule that comprises a nucleotide sequence encoding a mycobacterial sulfation pathway polypeptide. The cell is grown in a culture medium in the presence of a labeled sulfate (e.g., ³⁵SO₄) and the test agent. After a period of time, such as 30 minutes, 1 hour, 2 hours, 4 hours, or 12 hours, an extract of the cells is prepared, and the amount of radioactivity in a sulfated GL or GPL is measured, e.g., using thin-layer chromatography or other technique.
Genetic Complementation Assay
In some embodiments, a genetic complementation assay is provided. In these embodiments, a mutant bacterial cell that does not express a sulfation pathway gene (e.g., by virtue of being knocked out) is used. In some embodiments, a bacterial cell other than a mycobacterium is used. The mutant bacterium serves as a control, and is kept alive by providing necessary nutrients, and the like. A test bacterium is the mutant bacterium that has been genetically transformed with a nucleic acid that includes a sequence that encodes a functional mycobacterial sulfation pathway protein that the bacterium (e.g., by virtue of the knock-out, i.e., a genetic defect) lacks, thereby complementing the defect. The test bacterium and the control bacterium are individually contacted (e.g., in separate cultures) with a test agent. A test agent that kills the test bacterium, but not the control bacterium, is a candidate anti-mycobacterial agent. Viability of the bacterium is determined using standard methods, e.g., measuring the optical density of a culture grown in a liquid medium.
Thus, in some embodiments, the invention provides a method for identifying an agent that inhibits a mycobacterial sulfation pathway gene (e.g., inhibits transcription of the gene or translation of a corresponding mRNA) or gene product. The method generally involves contacting a test mutant bacterium and a control mutant bacterium with a test agent. The mutant bacterium does not produce a polypeptide encoded by the mycobacterial sulfation pathway gene by virtue of a genetic defect and that has been genetically transformed with a construct that includes a nucleotide sequence that encodes the mycobacterial sulfation pathway gene product, thereby genetically complementing the genetic defect. The control mutant bacterium includes the same mutation as the test mutant bacterium, but is not genetically complemented. The control mutant bacterium is maintained in medium that provides a component that keeps the bacterium alive despite the genetic defect. The effect of the test agent on the viability of the test mutant bacterium and the control mutant bacterium is determined. A decrease in the viability of the test mutant bacterium, and no decrease in the viability of the control mutant bacterium, indicates that the test agent is a candidate anti-mycobacterial agent.
This screening method can be generally applied to any mycobacterial sulfation pathway gene for which a knockout strain of another organism can be found and that satisfies three conditions: (1) The knockout or mutant organism is unable to survive under some or all conditions; (2) The knockout organism may be kept alive by genetic complementation with a gene supplied from another organism, the organism of interest (usually, but not necessarily, on a plasmid); and (3) The knockout organism may be kept alive through the administration of or supplementation by some external agent or agents.
External agents may include a substrate or compound that the knockout cell may be able to utilize to restore function; but may also include a second complementation gene that may work by a method unrelated to that of the first complementation gene to keep the knockout organism alive. The condition given in (3) functions as the control and the condition given in (2) functions as the experimental organism.
Thus, in some embodiments, the invention provides a method of identifying an agent that inhibits an activity of a mycobacterial sulfation pathway enzyme. The method generally involves culturing a first and a second bacterial cell in separate cultures in the presence of a test agent. The first and second bacterial cells contain a defect in a sulfation pathway enzyme, and the second bacterial cell has been transfected with a polynucleotide comprising a nucleotide sequence that encodes a mycobacterial sulfation pathway enzyme that complements the defect. After a suitable period of time, the growth of the first bacterial cell and the growth of the second bacterial cell are compared, e.g., the number of bacteria in the first culture is compared with the number of bacteria in the second culture (e.g., by measuring optical density of the cultures). A slower rate of growth in the second culture, compared with the growth rate of the first culture, indicates that the agent specifically inhibits the mycobacterial sulfation pathway enzyme.
A suitable period of time for growing the bacteria is generally from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 16 hours, from about 16 hours to about 24 hours, from about 24 hours to about 36 hours, from about 36 hours to about 48 hours, or from about 48 hours to about 72 hours. Typically, the bacteria are grown (cultured) at a temperature of about 37° C.
A reduction in growth of the second culture, relative to the first culture, indicates that the agent specifically inhibits the mycobacterial sulfation pathway enzyme. Generally, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, of the second culture, compared to the growth of the first culture, indicates that the test agent inhibits the mycobacterial sulfation pathway enzyme and is therefore a candidate agent for treating a mycobacterial infection. For example, after a suitable time in culture, if the A₆₀₀of the second culture is reduced by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the A₆₀₀of the first culture, then the test agent is of interest as a candidate agent for treating mycobacterial infection.
The following is one non-limiting example of such an assay. To discover inhibitors of mycobacterial APS reductase and APS kinase, the genetic complementation system described in Example 4 is used. The screening method is shown schematically in FIG. 22.
Survival or death of these E. coli mutant strains grown in minimal media is used in a real-time assay system. Specifically, the complementation plasmids bearing the CysH and CysC genes described above allows E. coli JM81A to survive in minimal media using sulfate as the sole sulfur source through complementing the defective pathway in this strain. The knockout strain may be used as a control, being kept alive by the administration of either cysteine or methionine, thereby bypassing the defective pathway. Test compounds are administered to each, namely the complemented strain and the control strain, and the strains monitored for survival by measuring their cell density (usually absorbance measured on a spectrophotometer at 600 nm wavelength). An example of such an assay is shown in FIG. 23.
There are four possible outcomes.
(1) Both the complemented strain and the control strain survive,
(2) both strains die;
(3) the complemented strain dies and the control strain lives; or
(4) the complemented strain lives while the control strain dies.
In case (1) the compound has no activity. In case (2) the compound is not selective in its activity. In case (4) the compound has no activity against the gene borne on the complementation plasmid. However, in case (3), whatever factor the compound is acting upon in the complemented strain differs from that in the control strain. In this case it is likely that the compound is actually acting to inhibit the gene or gene product borne on the complementation plasmid. Thus, compounds that give a response corresponding to outcome (3) represent lead compounds that are likely to be inhibitors of APS kinase or APS reductase. These compounds should have the desirable properties of selectivity (being active against only the gene in question among all of the other essential genes in E. coli, and also of being bioavailable, that is they are able to enter the cell (in this case E. coli) and to act on the desired target.

Therapeutic Methods

Methods of Treating a Mycobacterial Infection

The invention further provides methods of treating a mycobacterial infection in an individual. The methods generally involve administering to an individual a therapeutically effective amount of an agent that reduces a level and/or an activity of a mycobacterial sulfation pathway polypeptide, wherein the agent contacts a mycobacterium in the individual and reduces viability and/or virulence of the mycobacterium.
An agent that reduces a level and/or activity of a mycobacterial sulfation pathway polypeptide is administered to an individual in a therapeutically effective amount. As used herein, a “therapeutically effective amount” of an agent that reduces an activity and/or a level of a mycobacterial sulfation pathway polypeptide is an amount that is sufficient to reduce viability and/or virulence by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more, compared to the viability and/or virulence of the mycobacterium not contacted with the agent.
Whether an agent reduces the viability and/or virulence of a mycobacterium can be readily determined by those skilled in the art using standard methods. The term “virulence” encompasses two features of a pathogenic organism: its infectivity (i.e., the ability to colonize a host) and the severity of the disease produced. Virulence can be expressed as the LD₅₀, i.e., the dose that will kill 50% of inoculated animals within a given time. Virulence can also be expressed as transmissibility, i.e., the ability of a bacterium to cause a demonstrable infection in a given animal host. Transmissibility is usually detected by culture methods. The dose required is the ID₅₀, the infection dose in 50% of animals. Virulence can also be expressed as communicability. Virulence can be tested using any known assay, including, but not limited to, mouse colony formation assay, in which the number of mycobacterial colonies in the lung of infected mice is counted at various time points after infection; and macrophage infectivity assays. Other laboratory animals such as rabbits and guinea pigs can also be used. Virulence can also be determined in a cell culture assay using macrophages. Bacteria are incubated with cultured macrophages and the number of bacteria that enter the macrophages determined by washing the macrophages, lysing them, culturing their contents on plates, and counting “colony forming units.”

Methods of Increasing an Immune Response to a Mycobacterium

The invention further provides methods of eliciting an immune response to a pathogenic mycobacterium (e.g., a wild-type, virulent mycobacterium) in a host. The methods generally involve administering to a mammalian host a subject genetically altered mycobacterium (e.g., a subject genetically altered mycobacterium that is avirulent, that exhibits reduced virulence, or that is attenuated). The host mounts an immune response to the genetically altered mycobacterium. In embodiments of particular interest, the immune response provides protection against a virulent strain of mycobacterium.
In some embodiments, a subject avirulent mycobacterium that is administered is of the same species as the virulent mycobacterium, and an immune response is generated to both the avirulent and the virulent mycobacterium. In other embodiments, the avirulent mycobacterium is a different species than the virulent mycobacterium, and an immune response is generated to both the avirulent and the virulent mycobacterium. In some embodiments, administration of a subject avirulent mycobacterium elicits an immune response to more than one species of virulent mycobacterium.
A subject genetically altered mycobacterium is administered to a host. The term “virulent” in the context of mycobacteria refers to a bacterium or strain of bacteria that replicates within a host cell or animal at a rate that is detrimental to the cell or animal within its host range. More particularly virulent mycobacteria persist longer in a host than avirulent mycobacteria. Virulent mycobacteria are typically disease producing and infection leads to various disease states including fulminant disease in the lung, disseminated systemic military tuberculosis, tuberculosis meningitis, and tuberculosis abscesses of various tissues. Infection by virulent mycobacteria often results in death of the host organism. Typically, infection of guinea pigs is used as an assay for mycobacterial virulence. In contrast, the term “avirulent” or “attenuated” refers to a bacterium or strain of bacteria that either does not replicate within a host cell or animal within its host range, or replicates at a rate that is not significantly detrimental to the cell or animal.
Acceptable routes of administration include, but are not limited to, intramuscular, subcutaneous, intradermal, oral, inhalational (e.g., intranasal, oral, intratracheal), and the like. Typically, an immunogenic composition as described below is administered in a pharmaceutically acceptable formulation, using conventional routes of administration. Additional acceptable routes of administration are as discussed below for therapeutic agents.
In response to administration of a subject genetically altered mycobacterium, a host mounts an immune response to the genetically altered mycobacterium, and, in many embodiments, to virulent strains of mycobacterium. An immune response includes, but is not limited to, a humoral immune response, wherein mycobacteria-specific antibodies are produced; and a cellular immune response, in which mycobacteria-specific cytotoxic T lymphocytes (CTLs) are produced. Whether mycobacteria-specific antibodies and/or CTLs are produced can be determined using any known assay. Such assays are standard in the art.
In many embodiments, an immune response to a genetically altered mycobacterium provides immunoprotection against one or more virulent strains of mycobacteria. Whether an immune response is immunoprotective can be determined, e.g., in an experimental animal, by counting the number of virulent mycobacteria in the animal at various time points (e.g., 7 days, 2 weeks, 1 month, 2 months, and 6 months or longer) after challenge with a virulent strain of mycobacterium. An immune response is immunoprotective if the number of virulent mycobacterium in the animal is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, when compared with an animal that was not administered with the genetically altered mycobacterium before challenge with a virulent mycobacterial strain, where the comparison is made at the same time point after challenge.

Intracellular Pathogen Infections Amenable to Treatment

The methods and compositions described herein can be used in the treatment or prevention of any of a variety of infections by a mycobacterial species. Of particular interest is the treatment and/or prevention of infection or disease by M. tuberculosis, M. avium (or M. avium-intracellulare), M. leprae (particularly M. leprae infection leading to tuberculoid leprosy), M. kansasii, M. fortuitum, M. chelonae, and M. absecessus. While treatment of humans is of particular interest, the methods of the invention can also be used to prevent intracellular pathogen infection or disease in non-human subjects. For example, M. avium causes lymphadenitis in slaughter pigs; M. paratuberculosis infection causes paratuberculosis, a tuberculosis-like disease that can result in great production losses in cattle, sheep and goats; and M. bovis is carried by cattle and can cause a tuberculin-like infection in humans.
Individuals amenable to treatment with an agent of the invention include any individual diagnosed with an active mycobacterial infection. Individuals amenable to treatment also include individuals deemed to be at risk of having an active mycobacterial infection. At risk individuals include, but are not limited to, individuals infected with human immunodeficiency virus.
Individuals to be vaccinated include individuals that have never been infected with a mycobacterium; and individuals who have a latent mycobacterial infection.

Therapeutic Agents

The invention further provides an agent identified using a screening method of the invention. In many embodiments, an agent identified by a screening method of the invention reduces viability and/or virulence of a pathogenic mycobacterium. Whether an agent has activity in reducing viability and/or virulence of a pathogenic mycobacterium can be determined using any known assay.
In vitro cell cultures are accepted by those skilled in the art as assays for determining the susceptibility of M. tuberculosis and other mycobacteria to inhibitory compounds. See, e.g., Mor et al. (Antimicrobial Agents and Chemotherapy 39:2073-2077, (1975)). A variety of assays are known to mimic physiological conditions and these include, but are not limited to Mor, et al. (supra) and Mor et al., Antimicrobial Agents and Chemotherapy 38:1161-1164, 1994. In these assays, cells susceptible to infection by M. tuberculosis, other mycobacteria are placed in culture in vitro. There are a number of different cell types that can be used that are susceptible to intracellular pathogens, including, but not limited to, macrophages and monocytes. Mononuclear phagocytes can be obtained as established cells lines or as primary cells taken from a patient, where the patient cells are placed into culture and used within several months. Primary human monocytes, tissue monocyte-derived macrophages (MDMs) or myeloid cell lines including HL60, U937 or THP-1 cells can be used. Myeloid cell lines are known in the art and are readily available from the ATCC (American Type Culture Collection, Rockville Md.).
Peripheral blood mononuclear cells (PBMC) can be used to generate primary monocytes and MDMs. These cells are readily isolated from heparinized blood on Ficoll-sodium diatrizoate gradients (Pharmacia Fine Chemical, Piscataway, N.J.) or the like. PBMC are cultured in wells at about 1.5 to about 2.0×10⁶mononuclear cells/ml and the monocytes or MDMs subsequently purified by adherence to glass or plastic.
Isolated alveolar macrophages can be obtained using lung lavage collection methods well known in the art. For lavage methods and the isolation of alveolar macrophages from the bronchial lavage fluid see McGowan, et al. Lung 169:215-226, 1991 and McGowan, et al. Am. Rev. Respir. Dis. 127:449-455, 1983 respectively.
Suspensions of bacterial pathogen can be tested in broth culture initially, if necessary or desired, to determine whether or not a compound or compounds directly inhibit the growth of the pathogen in suspension culture. There are a number of suspension culture methods known in the art.
A test agent can also be tested for its ability to inhibit intracellular pathogens in tissue culture assays. In general, in these assays, the macrophages are placed in culture and incubated with the intracellular pathogen at an approximate cell to pathogen ratio of preferably at least 1:1 to about 1:5 cells:pathogen. For assays assessing M. tuberculosis infection, freshly adherent monocytes, 12 day-old adherent MDMs, or freshly adherent alveolar macrophages are incubated with M. tuberculosis or other pathogenic mycobacterium at a ratio of about 1:1 to about 1:5 (phagocyte:bacterium). For M. tuberculosis, e.g., the bacteria are incubated with the phagocyte for 2 hr at 37° C. in RPMI/HEPES media with 2.5% serum or human serum albumin (serum-free).
The cells are washed to remove non-adherent bacteria and monolayers are replated with RPMI containing 1% autologous serum (to maintain phagocyte viability but not to sustain extracellular growth of bacteria). A test agent is added about 24 hours later and mycobacterial growth in cell lysates is then assessed over the next several days either by the radiometric BACTEC system or by colony-forming units on agarose plates. In each experiment, growth is assessed relative to control monolayers where no drug has been added.
Those skilled in the art will recognize that there are other assays that could be used to assess growth inhibition including assays to differentiate between pathogen stasis or pathogen death by plating cell lysates onto or into media known to support growth of the particular pathogen.
Whether an agent reduces virulence can be determined using any known assay for virulence.
In some embodiments, an active agent of the invention inhibits a mycobacterial APS kinase and/or a mycobacterial APS reductase. In some embodiments, the subject compounds and compositions thereof comprise a secondary amine having a first, hetero-aromatic group and a second, aromatic or cyclic ester group. The first, hetero-aromatic group may comprise any substituted or unsubstituted carbon and nitrogen containing heteroaromatic group, any substituted or unsubstituted carbon, nitrogen and oxygen containing heteroaromatic group, or any substituted or unsubstituted carbon, nitrogen and sulfur containing heteroaromatic group. The second group may comprise any substituted or unsubstituted phenyl or other aromatic group, or any substituted or unsubstituted cyclic ester.
More specifically, the subject compounds may comprise a secondary amine having the structure:
wherein A comprises a hetero-aromatic group, B comprises an aromatic group or a cyclic ester, and Z comprises a bi-functional moiety that links group B to the secondary amine nitrogen. The group Z may be omitted in certain embodiments.
By way of example, and not necessarily of limitation, the group A may comprise
wherein D₁through D₇each may independently comprise either carbon or nitrogen, and X₁-X₅each may independently comprise hydrogen or any functionality. The groups X₁-X₅thus may each comprise, for example, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, azido, alkylamino, halo, carboxyl, or other functional group, and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In other embodiments, the group A may comprise:
wherein Y is either oxygen or sulfur, and wherein X₁and X₂each may comprise hydrogen or any other functionality such as, for example, an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, halo, carboxyl, or other group, and stereoisomers, solvates, and pharmaceutically acceptable salts thereof. In some of the specific embodiments discussed below, the group A comprises:
and wherein the groups X₁-X₅each more specifically may comprise hydrogen, hydroxyl, methyl and/or alkylamino groups.
The group B may comprise, by way of example:
wherein X₁-X₃each may comprise hydrogen or any functionality such as alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, azido, alkylamino, halo, carboxyl, or other functional group, and stereoisomers, solvates, and pharmaceutically acceptable salts thereof. In specific embodiments described below, the group B comprises a 4-aminophenyl, 4-azidophenyl, 3-hydroxyphenly, and a 2-carboxyphenyl group.
In still other embodiments, the group B may comprise:
wherein X₁-X₂each may independently comprise hydrogen or any functionality such as, for example, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, carboxyl, or other functional group, and stereoisomers, solvates, and pharmaceutically acceptable salts thereof. In a specific embodiment discussed below, the group B may comprise:
The group Z may comprise a methylene, aryl methylene, ethelyene, arylmethylene, ethelyene oxide, propylyene, propylene oxide, sulfone (—SO₂—), imido, keto, ether, thioether, ester, or any other group capable of linking the group B to the secondary amine functionality. In certain embodiments, the group Z may be omitted such that group B is directly joined or bonded to the secondary amine functionality.
More specifically, in certain embodiments a subject compound may comprise the following general formula:
where X₁and X₂are each independently an ether (—O—), thioether (—S—), sulfone (—SO₂—), —NH—, or —CH₂—, and where each of R₁-R₉is independently a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, carboxyl, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In other embodiments, a subject compound may comprise the general formula:
where each of X₁and X₂may independently comprise an ether (—O—), thioether (—S—), sulfone (—SO₂—), —NH—, or —CH₂—; and where each of R₁, R₂, R₃, R⁴, R₅, and R₆is independently a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, carboxyl, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In still other embodiments, a subject compound may have the generic formula:
where each of X₁-X₇may independently comprise an ether (—O—), thioether (—S—), sulfone (—SO₂—), N, —NH—, or —CH₂—; and where each of R₁-R₉is independently a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, carboxyl, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In further embodiments, a subject compound may have the general formula:
where each of X₁and X₂may independently comprise an ether (—O—), thioether (—S—), sulfone (—SO₂—), N, —NH—, or —CH₂—; and where each of R₁-R₁₃is independently a hydrogen, carboxyl, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In other embodiments, a subject compound may have the general formula:
where X comprises N, C, O or S; and where each of R₁-R₇is independently a hydrogen, carboxyl, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In other embodiments, a subject compound may have the general formula:
where each of X₁, X₂, and X₃independently comprise C, N, O or S; and where each of R₁-R₉is independently a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, keto, aryl, hetero-aryl, hydroxyl, alkoxyl, aryloxyl, amino, alkylamino, azido, halo, carboxyl, or other functional group; and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
“Acyl” is a specie of heteroalkyl wherein a terminal carbon of the heteroalkyl group is in the form of a carbonyl group, i.e., (alkyl or heteroalkyl)-C═O, where examples include acetyl (CH₃—(C═O)—).
“Acyloxy” refers to a heteroalkylene group of the formula —C(═O)—O— bonded to “X” so as to form —C(═O)—O—X wherein X may be any of alkyl, aryl, heteroalkyl, or heteroaryl.
“Alkenyl” is a specie of alkyl group, where an alkenyl group has at least one carbon-carbon double bond.
“Alkenylene” is a specie of alkylene group where the alkylene group has at least one double bond.
“Alkyl” is a monovalent, saturated or unsaturated, straight, branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In various embodiments, the alkyl group has 1-20 carbon atoms, i.e., is a C₁-C₂₀(or C₁-C₂₀) group, or is a C₁-C₁₈group, a C₁-C₁₂group, a C₁-C₆group, or a C₁-C₄group. Independently, in various embodiments, the alkyl group: has zero branches (i.e., is a straight chain), one branch, two branches, or more than two branches; is saturated; is unsaturated (where an unsaturated alkyl group may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than three triple bonds); is, or includes, a cyclic structure; is acyclic. Exemplary alkyl groups include C₁alkyl (i.e., —CH₃(methyl)), C₂alkyl (i.e., —CH₂CH₃(ethyl), —CH═CH₂(ethenyl) and —C≡CH (ethynyl)) and C₃alkyl (i.e., —CH₂CH₂CH₃(n-propyl), —CH(CH₃)₂(i-propyl), —CH═CH—CH₃(1-propenyl), —C≡C—CH₃(1-propynyl), —CH₂—CH═CH₂(2-propenyl), —CH₂—C≡CH (2-propynyl), —C(CH₃)═CH₂(1-methylethenyl), and —CH(CH₂)₂(cyclopropyl)).
“Alkylene” is a polyvalent, saturated or unsaturated, straight, branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In various embodiments, the alkylene group has 1-20 carbon atoms, i.e., is a C₁-C₂₀group, or is a C₁-C₁₈group, a C₁-C₁₂group, a C₁-C₆group, or a C₁-C₄group. Independently, in various embodiments, the alkylene group: has zero branches (i.e., is a straight chain), one branch, two branches, or more than two branches; is saturated; is unsaturated (where an unsaturated alkylene group may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than three triple bonds); is or contains a cyclic group; is acyclic; is divalent, i.e., has two open sites that each bond to a non-alkylene group; is trivalent, i.e., has three open sites that each bond to a non-alkylene group; has more than three open sites. Exemplary alkylene groups include C₁alkylene (i.e., —CH₂—) and C₂alkylene (i.e., —CH₂CH₂—, —CH═CH—, —C≡C—, —C(═CH₂)—, and —CH(CH₃)—).
“Aralkenyl” is another name for arylalkenylene, wherein at least one of the open bonding sites of an alkenylene group is bonded to an aryl group.
“Aralkyl” is another name for arylalkylene, wherein at least one of the open bonding sites of an alkylene group is bonded to an aryl group, where benzyl is an example.
“Aryl” is a monovalent, aromatic, hydrocarbon, ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.). In various embodiments, the monocyclic aryl ring is C5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system. A C6 ring system, i.e., a phenyl ring, is an exemplary aryl group. In various embodiments, the polycyclic ring is a bicyclic aryl group, where exemplary bicyclic aryl groups are C8-C12, or C9-C10. A naphthyl ring, which has 10 carbon atoms, is an exemplary polycyclic aryl group.
“Arylene” is a polyvalent, aromatic hydrocarbon, ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.). In various embodiments, the monocyclic arylene group is C5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system. A C6 ring system, i.e., a phenylene ring, is an exemplary aryl group. In various embodiments, the polycyclic ring is a bicyclic arylene group, where exemplary bicyclic arylene groups are C8-C12, or C9-C10. A naphthylene ring, which has 10 carbon atoms, is an exemplary polycyclic aryl group. The arylene group may be divalent, i.e., it has two open sites that each bond to another group; or trivalent, i.e., it has three open sites that each bond to another group; or it may have more than three open sites.
“Cycloalkenyl” is a specie of alkyl group where a cycloalkenyl group is a cyclic hydrocarbon group with at least one double bond.
“Cycloalkenylene” is a specie of alkylene group which is a cyclic hydrocarbon with at least one double bond and with at least two bonding sites.
“Cycloalkyl” is a specie of alkyl group, where a cycloalkyl is a cyclic hydrocarbon group.
“Cycloalkylalkylene” is a species of alkyl group wherein at least one open bonding site of an alkylene group is joined to a cycloalkyl group.
“Cycloalkylene” is a specie of alkylene group which is a cyclic hydrocarbon group with at least two open bonding sites.
“Cycloalkylenealkylene” is a specie of alkylene group wherein a cycloalkylene group is bonded to a non-cyclic alkylene group, and each of the cycloalkylene and non-cyclic alkylene group have at least one open bonding site.
Haloalkyl is a specie of heteroalkyl wherein at least one carbon of an alkyl group is bonded to at least one halogen.
“Halogen” refers to fluorine, chlorine, bromine and iodide. Fluorine and chlorine are exemplary halogens in compounds and compositions of the present invention.
Heteroalkylenearyl is a heteroalkylene group with at least one of its open bonding sites joined to an aryl group, where benzoyl (—C(═O)—Ph) is an example.
“Heteroalkyl” is an alkyl group (as defined herein) wherein at least one of the carbon atoms is replaced with a heteroatom. Exemplary heteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may, but typically does not, have the same number of valence sites as carbon. Accordingly, when a carbon is replaced with a heteroatom, the number of hydrogens bonded to the heteroatom may need to be increased or decreased to match the number of valence sites of the heteroatom. For instance, if carbon (valence of four) is replaced with nitrogen (valence of three), then one of the hydrogens formerly attached to the replaced carbon must be deleted. Likewise, if carbon is replaced with halogen (valence of one), then three (i.e., all) of the hydrogens formerly bonded to the replaced carbon must be deleted.
“Heteroalkylene” is an alkylene group (as defined herein) wherein at least one of the carbon atoms is replaced with a heteroatom. Exemplary heteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may, but typically does not, have the same number of valence sites as carbon. Accordingly, when a carbon is replaced with a heteroatom, the number of hydrogens bonded to the heteroatom may need to be increased or decreased to match the number of valence sites of the heteroatom, as explained elsewhere herein.
“Heteroaralkenyl” is another name for heteroarylalkenylene, wherein at least one of the open bonding sites of an alkenylene group is bonded to a heteroaryl group.
“Heteroaralkyl” is another name for heteroarylalkylene, wherein at least one of the open bonding sites of an alkylene group is bonded to a heteroalkyl group.
“Heteroaryl” is a monovalent aromatic ring system containing carbon and at least one heteroatom in the ring. The heteroaryl group may, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may be monocyclic or polycyclic, where the polycyclic ring may contained fused, spiro or bridged ring junctions. In one embodiment, the heteroaryl is selected from monocyclic and bicyclic. Monocyclic heteroaryl rings may contain from about 5 to about 10 member atoms (carbon and heteroatoms), e.g., from 5-7, or from 5-6 member atoms in the ring. Bicyclic heteroaryl rings may contain from about 8-12 member atoms, or 9-10 member atoms in the ring. The heteroaryl ring may be unsubstituted or substituted. In one embodiment, the heteroaryl ring is unsubstituted. In another embodiment, the heteroaryl ring is substituted. Exemplary heteroaryl groups include benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole and thiophene.
“Heteroarylene” is a polyvalent aromatic ring system containing carbon and at least one heteroatom in the ring. In other words, a heteroarylene group is a heteroaryl group that has more than one open site for bonding to other groups. The heteroarylene group may, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroarylene rings may be monocyclic or polycyclic, where the polycyclic ring may contained fused, spiro or bridged ring junctions. In one embodiment, the heteroaryl is selected from monocyclic and bicyclic. Monocyclic heteroarylene rings may contain from about 5 to about 10 member atoms (carbon and heteroatoms), e.g., from 5-7, or from 5-6 member atoms in the ring. Bicyclic heteroarylene rings may contain from about 8-12 member atoms, or 9-10 member atoms in the ring.
“Heteroatom” is a halogen, nitrogen, oxygen, silicon or sulfur atom. Groups containing more than one heteroatom may contain different heteroatoms.
“Pharmaceutically acceptable salt” and “salts thereof” in the compounds of the present invention refers to acid addition salts and base addition salts.
Acid addition salts refer to those salts formed from compounds of the present invention and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and/or organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
Base addition salts refer to those salts formed from compounds of the present invention and inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Suitable salts include the ammonium, potassium, sodium, calcium and magnesium salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and the like.
Non-limiting examples of biologically active compounds are found in Example 5.

Formulations, Dosages, and Routes of Administration

Formulations

In the subject methods, the active agent(s) may be administered to a host using any convenient means capable of resulting in treatment of a mycobacterial infection.
Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
Other modes of administration will also find use with the subject invention. For instance, an agent of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), generally about 1% to about 2%.
Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
An agent of the invention can be administered as injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Dosages

Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 pg to about 1,000 μg or about 10,000 μg of an agent that treats a mycobacterial infection. Alternatively, a target dosage of a subject agent can be considered to be about in the range of about 0.1-1000 μM, about 0.5-500 μM, about 1-100 μM, or about 5-50 μM in a sample of host blood drawn within the first 24-48 hours after administration of the agent.
Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

Routes of Administration

A therapeutic agent is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.
The agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
The agent can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.
Methods of administration of the agent through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. Iontophoretic transmission may be accomplished using commercially available “patches” which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
Kits with unit doses of the active agent, e.g. in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Preferred compounds and unit doses are those described herein above.

Combination Therapies

In some embodiments, a therapeutic agent of the invention is administered in combination with a conventional anti-pathogenic agent in treatment of a mycobacterial infection. The additional anti-pathogenic agent may be any agent (e.g., chemotherapeutic agent) identified as having activity against the intracellular pathogen of interest (e.g., in inhibition of extracellular or intracellular growth stages of the intracellular pathogen (e.g., mycobacteria), enhancement of intracellular pathogen clearance (e.g., mycobacteria), etc.). Exemplary anti-pathogenic agents include, but are not necessarily limited to, antibiotics, including antimicrobial agents, (e.g., bacteriostatic and bacteriocidal agents (e.g., aminoglycosides, β-lactam antibiotics, cephalosporins, macrolides, penicillins, tetracyclines, quinolones, and the like), antivirals (e.g., amprenavirs, acyclovirs, amantadines, virus penciclovirs, and the like), and the like), antifungals, (e.g., imidazoles, triazoles, allylamines, polyenes, and the like), as well as anti-parasitic agents (e.g., atovaquones, chloroquines, pyrimethamines, ivermectins, mefloquines, pentamidines, primaquines, and the like). Where the subject being treated is particularly susceptible to infection by intracellular pathogens, including opportunistic pathogens, it may be desirable to administer a subject therapeutic agent in a combination therapeutic regimen with chemotherapeutic agents that exhibit activity against microbial and/or parasitic pathogens, e.g., antimicrobial agents, antiviral agents, antifungal agents, anti-parasitic agents, etc. Such combination therapies can involve simultaneous or consecutive administration of an anti-mycobacterial agent of the invention and such a chemotherapeutic agent(s).
Specific exemplary conventional anti-pathogenic/chemotherapeutic agents and combinatory therapies, particularly anti-mycobacterial agents and combinatory therapies, include, but are not necessarily limited to, clarithromycin (e.g., by oral administration or injection); capreomycin sulfate (e.g., by intramuscular injection or intravenous infusion, e.g., CAPASTAT®); ethambutol HCl (e.g., by oral administration of tablets or capsules, e.g., MYAMBUTOL®); isoniazid (e.g., by intramuscular injection or oral administration, e.g., NYDRAZID®); aminosalicylic acid (e.g., aminosalicyclic acid granules for oral administration, e.g., PASER® GRANULES); rifapentine (e.g., by oral administration; e.g., PRIFTIN®); PYRAZINAMIDE (e.g., by oral administration); rifampin (e.g., by oral administration, e.g., RIFADIN®, or by intravenous administration, e.g., RIFADIN IV®); rifampin and isoniazid combination therapy (e.g., by oral administration, e.g., RIFAMATE®); rifampin, isoniazid, and pyrazinamide combination therapy (e.g., by oral administration, e.g., RIFATER®); cycloserine (e.g., by oral administration, e.g., SEROMYCIN®); streptomycin sulfate (e.g., by injection or oral administration); ethionamide (e.g., by oral administration, e.g., TRECATOR®-SC), and the like.
The anti-pathogenic/chemotherapeutic agent and therapeutic agent of the invention can be administered within the same or different formulation; by the same or different routes; or concurrently, simultaneously, or consecutively. The therapeutic agent can be delivered according to a regimen (e.g., frequency during a selected interval (e.g., number of times per day), delivery route, etc.) that is the same as, similar to, or different from that of the anti-pathogenic agent. When administered in combination, a therapeutic agent of the invention and an anti-pathogenic agent are generally administered within about 96 hours, about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 8 hours, about 4 hours, about 2 hours, about 1 hour, or about 30 minutes or less, of each other. Thus, although it may be desirable to do so in some situations, it is not necessarily required that the therapeutic agent of the invention and an anti-pathogenic agent (e.g., antibacterial agent) be delivered simultaneously.

Vaccines

As discussed above, a subject genetically altered mycobacterium (e.g., a genetically modified mycobacterium that is avirulent, that has reduced virulence, or that is attenuated) finds use in immunogenic compositions, to elicit an immune response to a pathogenic mycobacterium. In many embodiments, a subject genetically altered mycobacterium elicits an immune response to a pathogenic mycobacterium, thereby providing immunoprotection against a pathogenic mycobacterium. Formulations, dosages, and routes of administration for the subject genetically altered mycobacteria are any conventional formulations, dosages, and routes of administration currently in use in mycobacteria (e.g., BCG) vaccines. Whether a subject genetically altered mycobacterium is effective in eliciting an immunoprotective immune response can be determined by administering the subject mycobacterium to a test animal, and, after a period of time, challenging the animal with a pathogenic strain of mycobacterium.
The invention provides immunogenic compositions comprising a genetically altered mycobacterium of the invention. When they are used to induce or enhance an immune response, the genetically modified mycobacteria of the present invention are administered to an individual using known methods. They will generally be administered by the same routes by which conventional (presently available) vaccines are administered and/or by routes which mimic the route by which infection by the pathogen of interest occurs. They can be administered in a composition which includes, in addition to the mutant mycobacterium, a physiologically acceptable carrier. The composition may also include an immunostimulating agent or adjuvant, flavoring agent, or stabilizer.
A subject immunogenic composition is administered in an “effective amount” that is, an amount of genetically altered mycobacterium that is effective in a selected route of administration to elicit or induce an immune response to the mycobacterium.
In some embodiments, an effective dose or a unit dose of immunogenic composition is in a range of from about 10²to about 10⁷, from about 10³to about 10⁶, or from about 10⁴to about 10⁵genetically altered mycobacteria. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. The levels of immunity provided by the immunogenic composition can be monitored to determine the need, if any, for boosters. For example, following an assessment of antibody titers in the serum and/or counting the number of mycobacterium in a sample from the individual, optional booster immunizations may be desired. The immune response to a subject genetically modified mycobacterium may be enhanced by the use of adjuvant and or an immunostimulant.
In some embodiments, a composition comprising the genetically altered mycobacterium is administered using conventional devices including but not limited to syringes, devices for intranasal administration of compositions, and vaccine guns. Thus, one embodiment of the present invention is a device comprising a member which receives the genetically altered mycobacterium (or composition comprising the genetically altered mycobacterium) in communication with a mechanism for delivering the immunogenic composition to the subject.
Compositions comprising a genetically modified mycobacterium of the invention may include a buffer, which is selected according to the desired use of the attenuated mycobacterium, and may also include other substances appropriate to the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use. In some instances, the composition can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, Remington's Pharmaceutical Sciences, A. R. Gennaro editor (latest edition) Mack Publishing Company; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.
When used as an immunogenic composition, a genetically altered mycobacterium of the invention can be formulated in a variety of ways. In general, an immunogenic composition of the invention is formulated according to methods well known in the art using suitable pharmaceutical carrier(s) and/or vehicle(s). A suitable vehicle is sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
Optionally, an immunogenic composition of the invention may be formulated to contain other components, including, e.g., adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those of skill in the vaccine art. Adjuvants include, but are not limited to, aluminum salt adjuvants (Nicklas (1992) Res. Immunol. 143:489-493); saponin adjuvants; Ribi's adjuvants (Ribi ImmunoChem Research Inc., Hamilton, Mont.); Montanide ISA adjuvants (Seppic, Paris, France); Hunter's TiterMax adjuvants (CytRx Corp., Norcross, Ga.); Gerbu adjuvants (Gerbu Biotechnik GmbH, Gaiberg, Germany); and nitrocellulose (Nilsson and Larsson (1992) Res. Immunol. 143:553-557). In addition, other components that may modulate an immune response may be included in the formulation, including, but not limited to, cytokines, such as interleukins; colony-stimulating factors (e.g., GM-CSF, CSF, and the like); and tumor necrosis factor.
The invention further provides kits comprising a subject immunogenic composition in a pharmaceutically acceptable formulation, packaged in a sterile container or a sterile delivery device. In some embodiments, a sterile vial containing lyophilized subject genetically altered mycobacteria is provided. A separate vial containing a suspension base for reconstituting lyophilized genetically altered mycobacteria may also be provided. Typically, a kit contains a sterile vial containing a unit dosage form, e.g., an amount of genetically modified mycobacteria suitable for a single dose. The sterile vial may be a syringe. Additional components include needles. Package inserts containing information on the use of a subject kit may also be provided.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations are used, e.g., min (minutes); h (hours); s (seconds); U (Units); and the like.

Example 1

Protocol for Generating M. tuberculosis Targeted Knockouts

Using H37Rv genomic DNA as a template and a polymerase chain reaction (PCR), amplify ˜2 kilobase DNA sequences that directly flank the gene of interest. Subclone these fragments into the pNIL vector multiple cloning site. Using a restriction site present in pNIL that is placed between the two inserted flanking genomic fragments, subclone the hyg^rmarker. This marker can be amplified by PCR from a hyg^rvector with primers that add the unique site used for insertion into pNIL. Next, digest the pNIL vector that is now carrying a marked interrupted allele of the gene, with Pac I. This digestion will linearize the plasmid. In parallel, digest the pGOAL vector with Pac I. Purify the ˜6 kB product resulting from this digestion. This fragment contains the counter selectable marker, SacB, as well as another marker, β-galactosidase. Subclone this cassette into the Pac I-linearized pNIL. The knockout plasmid is now complete.
In order to increase recombination efficiency, irradiate ˜2 μg of the knockout plasmid with ultraviolet light. Immediately transform, by electroporation, an aliquot of competent H37Rv with the irradiated DNA. For the first selection, plate the transformation on hygromycin+kanamycin. Colonies that appear on this plate should be single crossover homologous recombinants. A small percentage of these colonies may be the result of illegitimate recombination. Remove colonies from this plate and plate again on media containing sucrose, hygromycin and X-gal. White colonies that grow on this plate are primarily double crossover homologous recombinants and thus the interrupted allele of the gene should have replaced the wild type allele. See, e.g., Parish and Stoker (2000) Microbiology 146(8): 1969-75.

Example 2

Phage Method for Generating M. tuberculosis Targeted Knockouts

Another method has recently become available for generating site-specific mutations in M. tuberculosis. This new method uses a genetically modified version of the mycobacterial phage, TM4, to introduce an interrupted allele of the target gene at a much higher frequency than that obtainable by electroporation. The phage transduction efficiency is high enough to directly screen for double crossover homologous recombinants.
Genomic DNA is isolated from colonies harvested at the last stage of selection. The presence of the interrupted allele can be screened using PCR or by southern blot analysis. See, e.g., Glickman et al. (2000) Molecular Cell 5(4):717-27.

Example 3

Identification of Mycobacterial Sulfate Assimilation Proteins

Materials and Methods

Pfu DNA polymerase was obtained from Stratagene. Restriction enzymes were from NEB (New England Biolabs) or Amersham-Pharmacia Biotech. Calf intestinal alkaline phosphatase (CIAP) was from Amersham-Pharmacia Biotech. Plasmid miniprep kits and the QIAquick kit for DNA extraction from agarose gel were from Qiagen. T4 DNA ligase was from NEB. Plasmids used in this work are shown in Table 1.

TABLE 1

Bacterial strains and plasmids used in this study.

Strain or plasmid	Relevant characteristics

E. coli strains
JM81A	cysC92, tfr-8
JM96	thr-1, leuB6, lacY1, glnV44(AS), gal-6,
	λ⁻, trp-1, hisG1(Fs), rfbD1, cysH56,
	galP63, Δ(gltB-gltF)500, rpsL9,
	malT1(λ^R), xylA7, mtlA2, ΔargH1, thi-1
BL21(DE3)	F⁻ompT r_B ⁻m_B ⁻
DH5α	supE44, thi-1 ΔlacU169(f80lacZΔM15)
	endA1 recA1 hsdR17 gyrA96 relA1
M. smegmatis
strains
mc²155::cysH	cysH deletion strain, Hyg^r
mc²155::cysC	cysC deletion strain, Hyg
Plasmids
pUC18/RBS	E. coli expression vector, Amp^r
pUC18/RBS/BioB	pUC18/RBS containing E. coli bioB gene
pUC18/RBS/CysH	pUC18/RBS containing M. tuberculosis cysH gene
pUC18/RBS/CysC	pUC18/RBS containing putative cysC
	C-terminal fragment of M. tuberculosis cysNC gene

Amp^rand Kan^rdenote resistance to ampicillin and kanamycin, respectively.

Oligonucleotide primers are shown in Table 2. Sequences in bold indicate restriction sites.

TABLE 2

5′-GATATACATATGAGCGGCGAGACAACCAGGC-3′	(SEQ ID NO:33)	MTCYSHF2

5′-GTGGTGCTCGAGCGAGGCGTGCAACCCG-3′	(SEQ ID NO:34)	MTCYSHR2

5′-AAGGGGCATATGAGCCCGAACACGGTGC-3′	(SEQ ID NO:35)	MTCYSCF

5′-AAGGGGCTCGAGTTAAGACGATGACTCCAACAGGTC-3′	(SEQ ID NO:36)	MTCYSCR

5′-GGGGCCATGGGTAGCGGCGAGACAACCAGG-3′	(SEQ ID NO:37)	CYSHPUCF

5′-GGGGGGATCCCTCGAGTTACGAGGCGTGCAACCCG-3′	(SEQ ID NO:38)	CYSHPUCR

5′-GGGGCCATGGGTAGCCCGAACACGGTGC-3′	(SEQ ID NO:39)	CYSCPUCF

5′-GGGCCATGGGGACCGACGTGACGACGTCAACG-3′	(SEQ ID NO:40)	pUCMsHFor

5′-GGGCTCGAGTCACGAGACGTGCAGCCCGC-3′	(SEQ ID NO:41)	pUCMsHRev

5′-GGGGAACCATGGGTTTAACGTATGATAATTGGGAAG-3′	(SEQ ID NO:42)	pUCCYSHBsF

5′-GGGGAACTCGAGTTATTCATGCAGTCCGC-3′	(SEQ ID NO:43)	pUCCYSHBsR

5′-GTGCTGGTGCCCGCGATCGGGCCCCTTGCTGAGCACCGT-3′	(SEQ ID NO:44)	MTKONC5MF

5′-ACGGTGCTCAGCAAGGGGCCCGATCGCGGGCACCAGCAC-3′	(SEQ ID NO:45)	MTKONC5MR

5′-TATTCTATCAAGCTTCACGAGATCGGCACCGATCAG-3′	(SEQ ID NO:46)	CysHKO #1

5′-AGATCATAGGTACCGATCAACCCGATCGCGGCGTGG-3′	(SEQ ID NO:47)	CysHKO #2

5′-CTTATTATGGTACCCTCGTCGGTCCAGCGCAGCAGC-3′	(SEQ ID NO:48)	CysHKO #3

5′-TAGATAATGCGGCCGCCGGTGTGTAGGTGTTGAAGTC-3′	(SEQ ID NO:49)	CysHKO #4

5′-GGGGTTAATTAACATGAGCGGCGAGACAACCAGG-3′	(SEQ ID NO:50)	CYSHPMSF

5′-GGGGGGATCCCGAGGCGTGCAACCCG-3′	(SEQ ID NO:51)	CYSHPMSR

Preliminary sequence data for M. smegmatis and M. avium were obtained from The Institute for Genomic Research website. E. coli JM81A and JM96 were obtained from the E. coli genetic stock center (CGSC), Yale University, USA.
Cloning of cysH and cysC Genes from Genomic DNA

Preparation of Pet Vectors

The gene encoding CysH (cysH) was amplified by the polymerase chain reaction (PCR) and subcloned into pCR4Blunt-TOPO. The PCR mixture contained 10 μM oligonucleotide primers (MYCYSHF2 and MTCYSHR2), 0.25 mM concentrations of the four deoxynucleotide triphosphates in 50 μl of Pfu polymerase buffer, 10% dimethylsulfoxide, and 100 ng of M. tuberculosis genomic DNA. After heating to 95° C., the reaction was initiated by adding 5 Units (U) of Pfu DNA polymerase. The PCR was performed in a thermal cycler (Perkin Elmer, GeneAmp PCR System 2400). The following PCR program was used: 25 cycles (20 seconds (s) at 94° C., 30 s at 50° C., and 55 at 72° C.) and then incubation for 7 min at 72° C. Agarose gel electrophoresis of the PCR mixture revealed a single DNA fragment of approximately 500 bp. This fragment was cut from the gel and purified using the QIAquick kit.
The product was ligated into pCR4Blunt-TOPO according to the manufacturer's instructions (Invitrogen). Isolated colonies were grown overnight in liquid media and plasmid DNA isolated by miniprep. Plasmids containing insert were identified by restriction digest and confirmed by sequencing. The insert was excised by digestion with NdeI/XhoI, separated by agarose gel electrophoresis and purified using the QIAquick kit. The product was ligated into the NdeI/XhoI digested pET24b(+) vector (treated with CIAP) using T4 DNA ligase. After incubation at 16° C. for 2 hours (h), 8 μl of the reaction mixture was used to transform 100 μl of E. coli DH50α. After growth on LB amp, colonies were selected and grown overnight. Plasmid DNA minipreps were screened by restriction digest to afford pET24b(+)CysH.
The C-terminal portion of the cysNC gene was amplified from genomic DNA using primers MTCYSCF and MTCYSCR and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised from this vector by digestion with NdeI/XhoI and ligated into CIAP treated NdeI/XhoI digested pET28b(+) to afford pET28b(+)CysC.

Preparation of Complementation Vectors

The gene encoding CysH (cysH) was amplified from the pET24b(+)CysH vector described above using primers CYSHPUCF and CYSHPUCR and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/BamHI and ligated into CIAP treated NcoI/BamHI digested pUC18/RBS, to generate pUC18/RBS/MtCysH.
The gene encoding CysC (cysC) was amplified as above using primers CYSCPUCF and MTCYSCR and cloned into pCR4Blunt-TOPO as above. After sequencing the insert was excised from this vector by digestion with NcoI/XhoI and the two fragments generated were separated by gel electrophoresis and purified as above. The longer NcoI/XhoI fragment was ligated into NcoI/XhoI digested pUC18/RBS/MtCysH from above and transformed into E. coli DH5α. Colonies containing the correct insert were verified by restriction digest. This vector was digested with NcoI and treated with calf intestinal alkaline phosphatase and ligated to the second NcoI/NcoI fragment. After transformation and plasmid isolation, the plasmid minipreps were screened for correctly oriented insert with EagI/XhoI, affording pUC18/RBS/MtCysC.
The gene encoding the M. smegmatis CysH (cysH) was amplified from M. smegmatis mc²155 genomic DNA using primers pUCMsHFor and pUCMsHRev and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/XhoI and ligated into CIAP treated NcoI/XhoI digested pUC18/RBS to yield pUC18/RBS/MsCysH.
The gene encoding the B. subtilis CysH (cysH) was amplified from pBS170 using primers pUCCYSHBsF and pUCCYSHBsR and cloned into pCR4Blunt-TOPO as above. After sequencing, the insert was excised by digestion with NcoI/XhoI and ligated into CIAP treated NcoI/XhoI digested pUC18/RBS to yield pUC18/RBS/BsCysH.
The S103G mutant of CysC in pUC18/RBS/CysC was generated using the QuikChange protocol from Stratagene. Briefly, two mutagenic primers MTKONC5MF and MTKONC5MR were used to amplify the template. Agarose gel electrophoresis was used to confirm that the reaction was successful. After the amplification reaction, DpnI was added to the reaction mixture and the mixture incubated at 37° C. for 1 h. 1 μl of the reaction mixture was used to transform 50 μl of super-competent E. coli XL1-Blue (Stratagene). The cells were grown on LB amp and, after miniprep of plasmid DNA, restriction digest with BanI (the mutagenic primers introduce a silent mutation creating a BanI restriction site) was used to identify mutants. These were sequenced to confirm the desired insert, affording pUC18/RBS/CysCS103G.

Genetic Complementation

E. coli JM81A and JM96 were grown in Oxoid CM1 media (1 g Oxoid Lab Lemco powder, 2 g yeast extract, 5 g peptone, 5 g NaCl per liter). Plasmid DNA was transformed into cells by electroporation (Bio-Rad Gene-Pulser, following the manufacturers protocol). Transformants were grown on CM1 agarose containing 100 mg/l ampicillin before transfer to M9 minimal media supplemented with thiamin (0.0005%), mannitol (0.2%), glucose (0.2%), and 18 amino acids excluding cysteine and methione (each 25 mg/L) and containing MgSO₄(0.01%) as sole sulfur source. SDS-PAGE of crude, whole-cell extracts was used to confirm the constitutive expression of CysC, CysC S103G, and CysC PS103G from their respective plasmids in E. coli JM81A.
Construction of CysH M. smegmatis Deletion Mutant
The cysH deletion mutant was constructed using the allelic replacement method of Parish and Stoker ((2000) Microbiol. 146:1969-1975). Oligonucleotide primers were used to amplify 2 kB regions upstream and downstream of the cysH gene. The upstream region was generated using primers CysHKO#3 and CysHKO#4, which generate a NotI/KpnI fragment and the downstream region was generated using primers CysHKO#1 and CysHKO#2, which generate a KpnI/HindIII fragment. The PCR products were gel purified and digested with the relevant restriction enzymes and ligated into a similarly digested p2NIL vector that was pre-treated with calf intestinal alkaline phosphatase. A hygromycin resistance marker was inserted between the two fragments into the KpnI restriction site. The final delivery vector, p2NIL MsCysH was generated by adding the PacI cassette (_PAg85-lacZ _Phsp60-sacB) from pGOAL17 to the vector bearing the mutated allele. This cassette contains the lacZ reporter gene and the sacB negative selection marker. sacB, encoding levan sucrase, confers toxicity to the cell when grown on sucrose containing media. The delivery vector was pretreated with UV light (120 mJ cm⁻²and used to electroporate M. smegmatis mc²155.
Transformants were selected on Middlebrook 7H11 media containing 20 mg L⁻¹kanamycin and 50 mg L⁻¹hygromycin. After 5 days colonies were tested for the presence of the lacZ gene and positive colonies were grown in 7H9 media containing 50 mg L⁻ hygromycin overnight. Serial dilutions were plated onto 7H11 plates containing 2% sucrose, 50 mg L⁻¹, and X-gal 50 mg L⁻¹. Colonies that did not turn blue were tested for kanamycin sensitivity and were then subjected to genotypic analysis. The construction of the cysC deletion mutant has been described elsewhere.

Genotypic Analysis

DNA was prepared from colonies by standard methods. Southern blotting analysis was carried out by generating two probes, one specific for the upstream region of the gene and one specific for the downstream region. Genomic DNA was digested with restriction enzymes that generated unique bands for the wildtype and mutant strains.

Construction of Mycobacterial Complementation Vectors

Complementation of the mutant strain was performed using the vector pMS3GS. This vector was constructed by inserting a 400 bp region containing the M. tuberculosis glutamine synthetase promoter into pMS3. A cloning site was introduced that allowed the cloning of a PacI-BamHI fragment. The M. tuberculosis cysH gene was amplified from genomic DNA using CYSHPMSF and CYSHPMSR and cloned into pCR4-TOPO as described above. After sequencing, the insert was excised from this vector by digestion with PacI/BamHI and ligated into CIAP treated PacI/BamHI digested pMS3GS to afford pMS3GSMtCysH.

Growth Curves

The growth rates of cultures of wildtype and mutant strains of M. smegmatis were determined in 7H9 Middlebrook media that contained 0.05 % Tween 80, 20 mg L⁻¹kanamycin and 2 mM methionine. Cultures were inoculated at 0.05 OD₆₀₀and were grown with shaking (250 rpm) at 37° C.
Expression of M. tuberculosis CysC
pET28b(+)CysC was transformed into BL21 STAR and grown on LB agarose containing 50 mg/ml kanamycin. An isolated colony was picked and grown in 2 ml of LB media containing 50 mg/ml kanamycin. When this culture had reached an A₆₀₀=0.5, 1 ml was used to inoculate 500 ml of 2YT media containing 50 mg/ml kanamycin. The culture was grown at 37° C. with shaking until an A₆₀₀=0.5, then the suspension was cooled to 20° C. and IPTG added to a final concentration of 0.4 mM. The culture was allowed to grow overnight. Cells were collected by centrifugation (10 min at 4000 rpm), and suspended in lysis buffer (20 mM Tris buffer containing 100 mM NaCl and 10 mM imidazole) before disruption by ultrasonication. The cell lysate was cleared by centrifugation (10 min at 10000 rpm), and the supernatant applied to a column of NiNTA agarose resin and eluted with 20 mM Tris buffer (pH 7.8) containing 100 mM NaCl and a gradient of imidazole up to 250 mM. The fractions containing protein were concentrated and stored in the same buffer. Total yield was approximately 25 mg of protein per liter of culture. Further characterization was performed using a Perkin-Elmer Sciex API III electrospray mass spectrometer that gave a mass of 23192 Da. This compares satisfactorily with the calculated mass predicted for the protein with the loss of the N-terminal methionine (calc. 23168). Protein concentrations were measured using the Pierce Micro BCA analysis kit.

Assay of MtCysC

Kinetic parameters were measured at 25° C. using a 50 mM Tris buffer (pH 8.0) containing 1 mM KC1 and 0.1% bovine serum albumin. Each sample contained 700 μL of buffer, 25 U of lactate dehydrogenase and 35 U of pyruvate kinase (from rabbit muscle, 50% suspension in glycerol), 25 U of P1 nuclease, 100 μL of 50 mM ATP, 5 mM MgCl₂and 100 mM Tris base and varying amounts of APS. Prior to the addition of APS kinase, the background rate was measured and typically was 0.001 A₃₄₀units per min. Measurements were started by addition of MtCysC. Measurements of the decrease of absorption at 340 nm per min in a continuous assay yielded reaction rates using an extinction coefficient for NADH of 6.22 mM⁻¹min⁻¹. The decrease was linear during all measurements. The concentration of APS was determined by measuring the total change in absorbance at 340 nm in a reaction catalyzed by APS kinase catalyzed but omitting P1 nuclease. Michaelis parameters (v_maxand km) were extracted from this data by best fit to the Michaelis-Menten equation using the program Grafit (Leatherbarrow, R. J., Erithacus Software, Staines). K_mand V_max/E₀values were obtained by measuring rates in a series of cells at a range of substrate concentrations (6-10 concentrations) which encompassed the K_mvalue ultimately determined, generally from 0.2×K_mto 5×K_m.

Results

The M. tuberculosis H37Rv gene sequence annotated as CysH(Rv2392) was identified in the original publication of the genome. The amino acid sequence of the protein encoded by this gene is provided in FIG. 14. We have discovered homologs of this gene in the genomes of other Mycobacterial species, namely M. avium and M. smegmatis m²155. The amino acid sequence of the protein encoded by the CysH gene in M. smegmatis is provided in FIG. 15. The amino acid sequences of the protein encoded by the CysH gene in M. avium is provided in FIG. 16. An alignment of the amino acid sequences of the protein encoded by the CysH gene in M. tuberculosis (“Myctub”), M. avium (“Mycavi”), and M. smegmatis (“Mycsme”) is shown in FIG. 17. Additionally, sequences identical to that seen in M. tuberculosis H37Rv were seen in other members of the M. tuberculosis complex including M. tuberculosis CDC1551 and M. bovis BCG. These sequences were identified by the use of the BLAST algorithm. Our searches for homologs have thus far been confined to organisms for which a genomic sequencing project is underway or has already been completed.
We used genetic complementation in specific E. coli knockout strains to define the substrate specificity of two of these CysH homologs. In this approach, E. coli strains defective in known places in their pathway for sulfate assimilation may be used as an experimental organism. The two E. coli strains used in this study were JM96 and JM81A and were obtained from the E. coli Genetic Stock Center (CGSC). The complete phenotypes of these organisms are shown in Table 1, above.
The genes for CysH of M. tuberculosis H37Rv and M. smegmatis m²155 were amplified from genomic DNA using the polymerase chain reaction and ligated into a pUC18-based plasmid with a lac promoter that allowed constitutive expression in these knockout strains. The plasmid was introduced into the two knockout strains by transformation and selection on media containing ampicillin. Resistance to ampicillin is conferred by the complementation plasmid. The complementation assay and results are shown in FIG. 18. The complemented strains were grown on minimal media containing sulfate as sole sulfur source. The original E. coli mutant strains are unable to grow on such media. In both cases, the CysH genes from M. tuberculosis H37Rv and M. smegmatis mc²155 allowed the two mutant strains to survive, thereby confirming that these genes encode for APS reductases. High levels of identity between these two genes and the corresponding gene for CysH that we identified in M. avium, in particular the presence of conserved CCXXXKXXXL (SEQ ID NO:53) and CXXC (SEQ ID NO:54) motifs (see FIG. 17) lead us to the conclusion that the CysH gene of this organism that we have identified also encodes for an APS reductase.
This result allows us to redefine the sulfate assimilation pathway of M. tuberculosis and other Mycobacteria. Accordingly, the Mycobacteria APS reductase gene acts on APS to provide sulfite, which eventually is incorporated into cysteine and methionine. The APS kinase gene, which forms the carboxyl-terminal portion of Rv1286 and which we have demonstrated to be active, also acts on APS to produce PAPS. PAPS is produced for the use of this organism's sulfotransferases. Consequently, inhibition of the APS reductase will prevent the formation of cysteine and methionine. On the other hand, inhibition of APS kinase will prevent the formation of PAPS and, it follows, the formation of sulfated metabolites through the action of the sulfotransferases of this organism.
In order to confirm the presence of a functional APS kinase in M. tuberculosis, the carboxyl-terminal domain of Rv1286 (CysN/CysC) was amplified by PCR and subcloned into the complementation vector described above. This plasmid was transformed into the E. coli knockout strain JM81A (which contains a knockout of APS kinase). When grown on minimal media containing sulfate as sole sulfur source, the complementation plasmid bearing this portion of the CysN/CysC gene enabled the survival of this strain. This result confirms that M. tuberculosis possesses a functional APS kinase and shows that it is encoded for by the carboxyl-terminal domain of this protein. We searched for homologs of CysC in other, sequenced members of the Mycobacteria, namely M. smegmatis and M. avium and in both cases identified genes with high levels of homology that we expect to contain APS kinases.
The amino acid sequence of the protein encoded by the CysN/CysC gene of M. smegmatis is shown in FIG. 19. The amino acid sequence of the protein encoded by the CysN/CysC gene of M. avium is shown in FIG. 20.
Identification of cysH, cysC and cysN Homologs in M. tuberculosis and M. smegmatis
cysH and cysC Homologs were identified in M. tuberculosis and M. smegmatis by BLAST analysis and in the former case correspond to the annotated sequences in the published genome. Interestingly, amino acid sequence comparison of these CysH proteins shows that they align well with both PAPS and APS reductases from a variety of organisms. However, the mycobacterial CysH proteins each contain two pairs of cysteine residues in the C-terminal half of the sequence. These two pairs of cysteine residues are common to all the known APS reductases and are absent in all but one of the proven PAPS reductases (that of Bacillus subtilis, vide infra).
The M. tuberculosis CysC gene is fused to the C-terminus of CysN, the GTPase that forms a heterodimer with CysD. This is not uncommon, for example, similar fusions are found in the functionally equivalent NodQ genes of S. meliloti and in CysN/CysC of Pseudomonas aeruginosa. Unlike these organisms, M. tuberculosis contains only single copies of each domain of cysH, cysC, and cysN, thereby representing a much simpler sulfate assimilation system than that of many other bacteria. The CysN/CysC protein overlaps the CysD protein by 4 nucleotides and appears to be part of the same operon. A putative RBS upstream of the start of CysN/CysC was located that lies within the C-terminus of the preceding gene, CysD.
Functional Complementation of E. coli CysC and CysH Knockout Strains
Given the large degree of sequence similarity between the PAPS and APS reductases, we chose to confirm the function of the M. tuberculosis and M. smegmatis cysH genes using genetic complementation in E. coli. The cysH genes were amplified by PCR from genomic DNA using primers complementary to the N- and C-termini. The PCR products were ligated into a pUC18-based vector containing a ribosomal binding site (RBS) upstream of the insertion point. Owing to the high copy number of the pUC18 plasmid in E. coli (>100 copies per cell) and the low copy number of the lac repressor protein (approximately 10 per cell), this plasmid allows the constitutive expression of proteins in the absence of a chemical inducer. The plasmids bearing the M. tuberculosis and M. smegmatis cysH genes were separately transformed into E. coli JM81A (a mutant strain lacking APS kinase) and JM96 (a mutant strain lacking PAPS reductase), and grown on ampicillin containing CM1 medium (a rich medium able to support the growth of these knockout E. coli strains). Isolated colonies were plated onto M9 minimal media supplemented with 18 amino acids (not cysteine or methionine), containing sulfate as the sole metabolizable sulfur source.
Complementation of JM96, an E. coli strain capable of the synthesis of PAPS but not its reduction, confirms that the gene product either has PAPS or APS reductase activity. Complementation of JM81A, an E. coli strain capable of the synthesis of APS, but not PAPS, shows that the gene must encode an APS reductase. pUC18/RBS/MtCysH and pUC18/RBS/MsCysH were able to complement both E. coli JM81A and JM96 strains to cysteine prototrophy. This result is consistent with the M. tuberculosis and M. smegmatis cysH encoding APS reductases. The assignment of APS reductase activity to both the M. tuberculosis and M. smegmatis CysH enzymes is in agreement with the observation that all proven APS reductases contain two pairs of conserved cysteine residues.
However, as noted above, there is one CysH, that from B. subtilis, that has been assigned PAPS reductase activity that contains these same two pairs of cysteines. We were concerned with the assignment of this gene product as a PAPS reductase, which was made on the basis of its ability to complement the E. coli mutant JM96, a strain lacking in PAPS reductase. While the ability to restore this strain to cysteine prototrophy is consistent with the gene product being a phosphosulfate reductase, it does not show whether the enzyme is an APS or PAPS reductase. Consequently, we obtained the plasmid used in the original study by Mendoza and colleagues, pBS170, and confirmed its ability to complement JM96, but also tested its ability to complement JM81A. Interestingly, we were able to repeat the original result of Mendoza and coworkers with JM96 but found that the plasmid did not restore prototrophy to JM81A. However, of particular concern here was the low growth rate seen with JM96. While colonies could be seen on plates with JM96 16 transformed with pUC18/RBS/MtCysH after 24 h growth, similar sized colonies with JM96 transformed with pBS170 took around 48 h to appear. It was thought that the expression of the B. subtilis gene could be limiting from the pBluescript SKII(+) vector, particularly as this construct used the native B. subtilis RBS (−14 to −8, AGGAGAA) (Mansilla and deMendoza (1997) J. Bacteriol 179:976-981). Consequently, we subcloned the B. subtilis cysH into the pUC18/RBS vector (which uses a typical E. coli RBS, −13 to −8, AGGAGG) and tested it for its ability to complement JM96 and JM81A. Both mutant cell lines were transformed to cysteine prototrophy, thereby confirming an APS reductase activity of the B. subtilis enzyme.

Identification of an Active CysC Domain

We recently showed that M. tuberculosis possesses three open reading frames with high levels of homology to the sulfotransferase gene family. In this work we have shown that in M. tuberculosis APS is used directly for the production of sulfite; it appears that PAPS is produced for the sole use of these putative sulfotransferases. Given this newly defined sulfate assimilation pathway for M. tuberculosis, we were interested in generating a functional knockout of PAPS biosynthesis in this organism, and thereby of all the sulfotransferases. Consequently, we identified APS kinase, CysC, as a possible target for generating a functional knockout of PAPS biosynthesis and therefore of all sulfotransferase activity in M. tuberculosis. As discussed above, CysC is fused to CysN, thereby complicating the generation of a knockout.
In order to construct a defined knockout of CysC, the APS kinase domain of CysN/CysC needed to be identified. We decided to confirm the identification of the CysC domain by using genetic complementation. The C-terminal domain of CysN/CysC was identified by alignment to the CysN and CysC proteins of E. coli. According to our analysis, the CysN and CysC domains of M. tuberculosis are separated by a short linker with the sequence TPST. The C-terminal domain of CysN/CysC was amplified from genomic DNA and the product was subcloned into pUC18/RBS and tested for its ability to complement the E. coli strain JM81A. Transformation of this strain with the plasmid restored it to cysteine prototrophy. With a complementation system in hand for detecting alterations in function of CysC, we focused on generating a single point mutant incapable of complementing E. coli JM81A with the aim of establishing a method for the generation of a CysC knockout, without disrupting CysN. Our approach was inspired by the results of Satischandran et al. Satischandran et al. (1989) J. Biol. Chem. 264:15012-15021; and Satischandran et al. (1992) Biochem. 31:11684-11688. These workers found that upon incubation of the E. coli CysC with γ³²P-ATP in the absence of APS, the enzyme was radiolabeled. Upon proteolysis, the radiolabelled peptide was isolated, and sequenced, indicating the presence of a phosphorylated serine, S109. On the basis of this result, these workers suggested that the enzyme mechanism of phosphoryl transfer proceeds through a covalent phosphoserine intermediate.
Consequently, we mutated the corresponding residue in the M. tuberculosis CysC, S103, to glycine. However, the plasmid bearing this mutation, pUC18/RBS/CysCS103G, still restored cysteine prototrophy to JM81A when grown on minimal media containing sulfate as sole sulfur source. This result, while surprising, was not entirely unexpected. Segel and coworkers found during studies with the closely related CysC from Penicillium chrysogenum that mutation of the corresponding serine residue (S107) in this enzyme to alanine gave a mutant with kinetic characteristics similar to the wild-type. MacRae et al. (1998) J. Biol. Chem. 273:28583-28589. While these workers identified other mutations in the phosphate binding loop of the P. chrysogenum CysC that abolished enzyme activity. P. chrysogenum contains a second APS-like protein, with strong homology to CysC, that binds APS but has no kinase activity. This protein lacks both the conserved serine of APS kinases and contains several differences in the phosphate binding loop. Segel and coworkers showed that mutation of the P. chrysogenum CysC in the phosphate binding loop to the corresponding residues of the APS binding protein resulted in elimination of enzyme activity. In our case mutation of the phosphate binding loop of the M. tuberculosis S103G CysC mutant to the same residues as found in the P. chrysogenum APS binding protein generated a mutant protein that was unable to complement E. coli JM81A.
Construction of a CysH and CysC Deletion Mutant of M. smegmatis
To confirm the proposed route for sulfate assimilation of M. smegmatis we constructed two deletion mutants. The cysH and cysC genes were interrupted using the allelic replacement method of Parish and Stoker. Briefly, a delivery vector containing the interrupted allele was constructed in the suicide plasmid p2NIL by replacing the middle portion of the gene with a hygromycin resistance marker. After irradiation with UV light, a pre-treatment that has been shown to promote homologous recombination, the delivery vectors were transformed into M. smegmatis mc²155 and kanamycin/hygromycin resistant colonies obtained. These colonies should be single crossovers that have integrated the suicide plasmid.
Putative single crossover colonies were grown in liquid media and then grown on media containing hygromycin, sucrose and X-gal. Sucrose acts as a negative selection marker and allows only those cells that have lost the sacB gene to grow. The loss of this gene is confirmed by the absence of the lacZ gene. Colonies that afforded colorless colonies represent potential knockouts and were confirmed by Southern analysis. The preparation of the cysC knockout was performed by a similar approach and will be reported elsewhere. These mutants were tested for auxotrophy in liquid media containing 2 mM cysteine or 2 mM methionine. In the case of the cysH deletion mutant, this strain was found to be both a cysteine and methionine auxotroph, thereby confirming the essentiality of the cysH gene for sulfate assimilation. However, the cysC mutant was not a methionine or cysteine auxotroph, a result consistent with our hypothesis that this gene is not required for the assimilation of sulfur into the sulfur containing amino acids. Further, complementation of the cysH knockout strain with the complementation plasmid pMS3GSMtCysH restored this strain to cysteine and methionine prototrophy, confirming the role of the M. tuberculosis cysH gene in the reduction of APS.
In vitro Assay of M. tuberculosis CysC
CysC was cloned into pET28b(+) and over-expressed in E. coli as an N-terminal Hi_s6-fusion. Purification was achieved through affinity chromatography on NiNTA resin. The purified protein had an apparent molecular weight of 23 kDa by SDS-PAGE and a MW as determined by ESI-MS of 23192 Da. This compares favorably with the predicted molecular weight of 23 168 Da for the protein with the loss of the N-terminal methionine, presumably effected by the endogenous methionyl aminopeptidase of the expression host. The enzyme was tested for its ability to phosphorylate APS using the coupled assay of Burnell and Whatley ((1975) Anal. Biochem. 68:281-288), with the modifications of Renosto et al. ((1984) J. Biol. Chem. 259:2113-2123). This assay allows the direct monitoring of rates by coupling the production of ADP (from ATP) to pyruvate kinase, and the lactate dehydrogenase catalyzed reduction of pyruvate by NADH. The decrease in the concentration of NADH may be continuously monitored at 340 nm. P1 3′-nuclease is also included to regenerate APS from the product, PAPS, thereby enabling the measurement of very low K_mvalues.
Using this assay, we measured an apparent K_mvalue of 0.64±0.10 μM and an apparent V_maxE₀value of 0.85±0.04 s⁻¹(for an apparent V_maxE₀/K_m1334 mM⁻¹s⁻¹) for APS in the presence of saturating ATP (5 mM). In the presence of 75 mM sulfate, the apparent K_mvalue increased nearly four-fold to 1.7±0.1 μM with little change in the V_maxE₀value (1.05 s⁻¹) leading to a two-fold decrease in V_maxE₀(611 mM⁻¹s⁻¹). The higher K_mvalue for the enzyme in 75 mM sulfate simplifies the kinetics somewhat and provides closer mimicry of the intracellular ionic strength. To confirm that the assay was being run at a saturating concentration of ATP, the kinetic parameters were measured in the presence of saturating APS (10 μM) in buffer containing 75 mM sulfate. The kinetic parameters confirmed that the concentration of ATP was saturating (apparent K_m=0.91±0.10 mM, apparent V_maxE₀=0.95±0.03s⁻¹, apparent (V_maxE₀)/K_m=1.04 mM⁻¹s⁻¹). This K_mvalue is similar to that seen for the APS kinase of E. coli (K_m=0.25 μM). However, the M. tuberculosis enzyme differs from that of E. coli in that the former does not appear to suffer from the potent substrate inhibition seen for the latter (Satishchandran and Markham (2000) Archiv. Biochem. Biophys 378:210-215).

Example 4

Screening Method to Identify Inhibitors of Mycobacterial APS Reductase and APS Kinase

A sulfation assimilation pathway used by M. tuberculosis, M. smegmatis, and M. avium is shown in FIG. 21 a. Sulfate assimilation pathways in plants and bacteria are shown in FIG. 21 b. Common enzyme designations are given below each arrow in FIG. 21 b.
To discover inhibitors of mycobacterial APS reductase and APS kinase, the above-described genetic complementation system is used. The screening method is shown schematically in FIG. 22.
Survival or death of these E. coli mutant strains grown in minimal media is used in a real-time assay system. Specifically, the complementation plasmids bearing the CysH and CysC genes described above allows E. coli JM81A to survive in minimal media using sulfate as the sole sulfur source through complementing the defective pathway in this strain. The knockout strain may be used as a control, being kept alive by the administration of either cysteine or methionine, thereby bypassing the defective pathway. Test compounds are administered to each, namely the complemented strain and the control strain, and the strains monitored for survival by measuring their cell density (usually absorbance measured on a spectrophotometer at 600 nm wavelength). An example of such an assay is shown in FIG. 23.
There are four possible outcomes.
(1) Both the complemented strain and the control strain survive,
(2) both strains die;
(3) the complemented strain dies and the control strain lives; or
(4) the complemented strain lives while the control strain dies.
These outcomes are depicted in Table 3.

TABLE 3

Complemented strain	Control strain	Activity of test compound

Survive	Survive	No activity
Die	Die	Activity not selective
Die	Survive	Candidate selective inhibitor
Survive	Die	No activity against
		complementing gene or
		gene product

In case (1) the compound has no activity. In case (2) the compound is not selective in its activity. In case (4) the compound has no activity against the gene borne on the complementation plasmid. However, in case (3), whatever factor the compound is acting upon in the complemented strain differs from that in the control strain. In this case it is likely that the compound is actually acting to inhibit the gene or gene product borne on the complementation plasmid. Thus, compounds that give a response corresponding to outcome (3) represent lead compounds that are likely to be inhibitors of APS kinase or APS reductase. These compounds should have the desirable properties of selectivity (being active against only the gene in question among all of the other essential genes in E. coli, and also of being bioavailable, that is they are able to enter the cell (in this case E. coli) and to act on the desired target.
This method is suitable as a first level screen as compounds that are identified may be causing outcome (3) by acting on other genes in the pathway, including the first step, production of APS that is catalyzed by ATP sulfurylase, or later steps such as the reduction of sulfite to sulfide, and the incorporation of sulfide into O-acetylserine to generate cysteine.
To determine the specificity of the inhibitor's action, a second complementation system may be used that operates in a different way to the first, enabling the determination of the compound's true site of action. In the case of the discovery of inhibitors of APS kinase and APS reductase, as these genes act in the same pathway, they can be used to determine the exact mode of action of an inhibitor. Thus, if an inhibitor acts only on one complemented strain, then this shows that it must act solely on that enzyme. Compounds that act on both strains must act on other enzymes in the pathway and themselves give a valuable indication of possible lead compounds for future screening efforts. As can be seen, this screen has many advantages for high-throughput screening given its simplicity and ease of scale-up.

Example 5

Discovery of Inhibitors of APS Reductase and APS Kinase

In order to discover inhibitors of these enzymes, we have made use of the genetic complementation system described in Example 4 to use survival or death of these E. coli mutant strains grown in minimal media as a real-time assay system. Specifically, the complementation plasmids bearing the CysH and CysC genes described above allows E. coli JM81A to survive in minimal media using sulfate as the sole sulfur source through complementing the defective pathway in this strain. The knockout strain itself may be used as a control, being kept alive by supplementation with cysteine, thereby bypassing the defective pathway. Compounds from libraries may be administered to each strain, namely the complemented strain and the control strain, and the strains monitored for survival by measuring their cell density (usually absorbance measured on a spectrophotometer at 600 nm wavelength) (FIG. 23).
We used this complementation based screening approach to search for inhibitors of mycobacterial APS kinase and APS reductase. Strains bearing complementation plasmids were grown in M9 minimal media in 384 well plates. Using the High-throughput screening facility at the Institute of Chemistry and Chemical Biology at Harvard University, 18000 compounds were added to each of the two complemented strains and the control for a total of 54000 experiments. Compounds were transferred using robotic pin-transfer into each of the 384 well plates for a final concentration of 12-25 mg L⁻¹. Cells were then grown at 37° C. for two days before measuring their absorbance at 650 nm on a 384 well plate reader. Absorbance values for the experimental strains were converted into percentage inhibition relative to the reference strain. 50 compounds were found that gave a 40% or greater inhibition of growth on one or other experimental strain, but not on the control strain. These 50 compounds were cherry picked and the inhibition assay repeated on a larger scale to confirm the observed phenotype. Shown below are six of the most potent compounds detected so far.




E. coli CysC	94	95	47	93
E. coli CysH	29	0	0	25
E. coli BioB	77	85	0	1



E. coli CysC	98	78
E. coli CysH	97	90
E. coli BioB	0	0

The values under each compound indicate the percent inhibition of growth of each complemented E. coli strain when grown in the presence of 25 μg/mL of each compound. E. coli CysC is JM81A complemented with M. tuberculosis CysC; E. coli CysH is JM81A complemented with M. smegmatis CysH; E. coli BioB is JM81A control strain containing the BioB gene. 0% represents attenuation of growth or no inhibition of growth.

Example 6

Sulfotransferase Knockout M. tuberculosis Strains

Individual M. tuberculosis mutant strains have been constructed that lack each of the sulfotransferases we identified: Rv2267c, Rv3529c, and Rv1373. The strains were generated following the method of Parish and Stoker. Parish, T. and N. G. Stoker (2000). “Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement.” Microbiology 146 (Pt 8): 1969-75. Briefly, PCR was used to amplify approximately 2 kB fragments of H37Rv genomic DNA flanking the sulfotransferase gene to be deleted. After digestion with appropriate restriction enzymes, these fragments were ligated into a vector with an antibiotic resistance marker insertion between them. This vector, carrying the interrupted allele of the sulfotransferase was transformed into M. tuberculosis H37Rv. After transformation, the cells were selected for homologous recombination between the plasmid and the genomic DNA. Sulfotransferase mutant strains were screened by southern blot hybridization analysis. We observed the expected pattern and molecular weight of bands in the Southern blot, thus confirming that Rv2267c had been deleted from the genome of M. tuberculosis H37Rv.

Example 7

Assay for the Identification of Sulfated Molecules in Mycobacteria

An assay to search for sulfated compounds absent from the sulfotransferase mutant strains has been developed. The assay uses stable sulfur isotopic labeling and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) to quickly identify sulfur-containing compounds from crude lipid extracts of wild type M. tuberculosis. First, wild type M. tuberculosis is grown in minimal media containing either Na₂ ³²SO₄or Na₂ ³⁴SO₄as the sole sulfur source. In the cells grown with Na₂ ³⁴SO₄-containing media, compounds containing either sulfur or sulfate shift by 2.0 m/z x n, where n is the number of sulfur atoms. Comparison of these isotopically-labeled extracts drastically lowers the spectra complexity and facilitates the rapid identification of compounds containing sulfur. Once these few sulfur-containing compounds are identified, their presence or absence in the sulfotransferase mutant strains is determined. We have found empirically that the initial step using a stable isotope of sulfur to find sulfur-containing compounds is indispensable, although in only about 50% of the cases are these compounds found to be sulfated.

Example 8

Genetic and Biochemical Evidence that Rv2267c is a Sulfotransferase

The assay described in Example 7 was used to search identified for a sulfated compound absent from the M. tuberculosis strain carrying the interrupted, nonfunctional Rv2267c allele. An ion carrying a single negative charge at m/z 881.6 was found shifted by 2.0 mass units when M. tuberculosis was incubated in ³⁴S-containing medium. This same compound was found absent from the M. tuberculosis strain lacking Rv2267c (FIG. 24). Expression of the Rv2267c gene in the Rv2267c mutant returned the m/z 881 ion. This experiment, in combination with our bioinformatics analysis, shows that the Rv2267c gene product is a sulfotransferase responsible for sulfating the m/z 881.6 ion. Structural information is not yet available for the 881.6 compound; however preliminary data suggests it is a novel sulfated compound.

Example 9

The CysH gene (APS reductase) in M. tuberculosis was deleted, and the effect of this deletion on the virulence of the commonly used H37Rv laboratory strain of M. tuberculosis was tested.
The cysH deletion mutant was constructed using the allelic replacement method of Parish and Stoker ((2000) Microbiology 146(8): 1969-75). Oligonucleotide primers were used to amplify 2 kB regions upstream and downstream of the cysH gene. The upstream region was generated using the primer pair MTKOH5F and MTKOH5R, which generates NotI/KpnI and KhindII/PmII fragments, respectively. The sequence of MTKOH5F is 5′ TATTCTATCAAGCTTCACGAGATCGGCACCGATCAG 3′ (SEQ ID NO:55). The sequence of MTKOH5R is 5′ AGATCATAGGTACCGATCAACCCGATCGCGGCGTGG 3′ (SEQ ID NO:56). The downstream region was generated using primers MTKOH3F and MTKOH3R, which generate HindIII/ScaI and KpnI/NotI fragments. The sequence of MTKOH3F is CTTATTATGGTACCCTCGTCGGTCCAGCGCAGCAGC 3′ (SEQ ID NO:57). The sequence of MTKOH3R is 5′ TAGATAATGCGGCCGCCGGTGTGTAGGTGTTGAAGTC 3′ (SEQ ID NO:58). The PCR products were gel purified and digested with the relevant restriction enzymes and ligated into a similarly digested p2NIL vector that was pre-treated with calf intestinal alkaline phosphatase (CIAP). A hygromycin resistance marker was inserted between the two fragments into the KpnI restriction site. The final delivery vectors, p2NIL_MtCysH and p2NIL_MtCysC were generated by adding the PacI cassette (P_Ag85-lacZP_hsp60-sacB) from pGOAL17 to the vector bearing the mutated allele. This cassette contains the lacZ reporter gene and the sacB negative selection marker. sacB, which encodes levan sucrase, confers toxicity to the cell when grown on sucrose containing media.
The delivery vector was pretreated with UV light (120 mJ cm⁻²) and used to electroporate M. tuberculosis H37Rv. Transformants were selected on Middlebrook 7H11 medium containing 20 mg/l kanamycin and 50 mg/l hygromycin. After 3 weeks, colonies were tested for the presence of the lacZ gene and positive colonies were grown overnight in 7H9 medium containing 50 mg/l hygromycin. Serial dilutions were plated onto 7H11 plates containing 2% sucrose, 50 mg/l hygromycin, 2 mM methionine, and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; 50 mg/l). Colonies that did not turn blue were tested for kanamycin sensitivity and were then subjected to genotypic analysis.
Eight-week-old BALB/c mice were injected with either H37RvΔCysH or Mtb H37Rv (wild-type M. tuberculosis) (12 mice per strain). The bacteria were injected into the tail vein. Within 21 weeks, all of the mice infected with Mtb H37Rv succumbed to the infection. In contrast, all mice infected with Mtb H37RvΔCysH survived for at least 29 weeks following infection. The data are shown in FIG. 25. The appearance and behavior of the surviving H37RvΔCysH-infected mice were similar to those of uninfected mice. The sizes of the injections in colony forming units (CFU) were as follows: Mtb H37Rv: 1.21×10⁶; Mtb H37RvΔCysH: 1.3×10⁶. These results indicate that in vivo survival and growth of M. tuberculosis depends on intact sulfate assimilation.
A comparison of lungs of Mtb H37Rv-infected and H37RvΔCysH-infected mice at 13 weeks following infection was made. The Mtb H37Rv-infected mice showed extensive granuloma formation. H37RvΔCysH-infected mice showed few, if any, granulomas.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. A genetically modified mycobacterium, wherein said genetically modified mycobacterium comprises a functionally disabled sulfation pathway gene, such that said sulfation pathway gene does not direct expression of a functional sulfation pathway polypeptide, wherein said genetically modified mycobacterium is avirulent.

2. The mycobacterium of claim 1, wherein the mycobacterium has a functionally disabled APS reductase gene.

3. The mycobacterium of claim 1, wherein the mycobacterium has a functionally disabled APS kinase gene.

4. The mycobacterium of claim 1, wherein the mycobacterium has a functionally disabled sulfotransferase gene.

5. The mycobacterium of claim 1, wherein the mycobacterium is M. tuberculosis.

6. The mycobacterium of claim 1, wherein the LD₅₀is at least about 10-fold higher than a wild-type mycobacterium of the same strain.

7. The mycobacterium of claim 1, wherein the LD₅₀is at least about 50-fold higher than a wild-type mycobacterium of the same strain.

8. An immunogenic composition comprising a genetically modified mycobacterium according to claim 1; and a buffer.

9. A pharmaceutical composition comprising a genetically modified mycobacterium according to claim 1; and a pharmaceutically acceptable excipient.

10. The composition of claim 9, further comprising an adjuvant.

11. A kit comprising a sterile container comprising a genetically modified mycobacterium according to claim 1.

12. The kit according to claim 11, wherein the sterile container comprises a unit dose of mycobacterium of from about 10²to about 10⁷mycobacterium.

13. The kit according to claim 11, wherein the mycobacteria are lyophilized.

14. The kit according to claim 11, wherein the sterile container further comprises a pharmaceutically acceptable excipient.

15. The kit according to claim 11, wherein the sterile container further comprises an adjuvant.

16. A method of increasing an immune response to a pathogenic mycobacterium in a host, comprising administering to the host an immunogenic composition according to claim 8.

17. The method of claim 16, wherein said administering is intramuscular.

18. The method of claim 16, wherein a protective immune response to a wild-type, virulent mycobacterium is induced.

19. The method of claim 18, wherein the virulent mycobacterium is of the same species as the genetically modified mycobacterium.

20. The method of claim 18, wherein the virulent mycobacterium is of a different species than the genetically modified mycobacterium.

21. The method of claim 16, wherein cytotoxic T lymphocytes specific for mycobacteria are induced.

22. A genetically modified mycobacterium, wherein said genetically modified mycobacterium comprises a modified sulfation pathway gene, such that said sulfation pathway gene does not direct expression of a sulfation pathway polypeptide, wherein said genetically modified mycobacterium is avirulent.