EP1095133A1 - Ftsz multimeric proteins and their uses - Google Patents

Ftsz multimeric proteins and their uses

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Publication number
EP1095133A1
EP1095133A1 EP99935377A EP99935377A EP1095133A1 EP 1095133 A1 EP1095133 A1 EP 1095133A1 EP 99935377 A EP99935377 A EP 99935377A EP 99935377 A EP99935377 A EP 99935377A EP 1095133 A1 EP1095133 A1 EP 1095133A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
ftsz
multimenc
polypeptides
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99935377A
Other languages
German (de)
French (fr)
Other versions
EP1095133A4 (en
Inventor
Curtis Preston Hensley
Kenneth H. Pearce
Thomas M. Sossong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
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Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP1095133A1 publication Critical patent/EP1095133A1/en
Publication of EP1095133A4 publication Critical patent/EP1095133A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified higher order structures of FtsZ polypeptides, such as drmers. trimer. tetramers and larger aggregations, and their production and uses, as well as their vanants. their agonists and antagonists, and their uses
  • the invention relates to multimenc polypeptides compnsing FtsZ subunits and their use in screening for antimicrobial compounds
  • FtsZ filamentation temperature sensitive protein functions early in the septation process FtsZ polymerizes in a dynamic ring structure and localizes to the midpoint of the dividing cell This polymeric structure is believed to act as a scaffold for other important cell division proteins In addition to its structural role, FtsZ is also a GTPase Prior to the Applicants findings provided herein, the precise relationship between GTPase activity of FtsZ and the polyme ⁇ zation-depolyme ⁇ zation cycle is poorly understood
  • FtzZ is essential for cell division Mutants in E coh fail to form division septae and filament (Lutkenhaus and A Mukherjee Cell Division in Eschenchia co and Salmonella. Cellular and Molecular Biology. F C Neidhardt ed Washington D C . ASM Press 1996, pp 1615-1626)
  • the present invention relates to FtsZ multimeric and higher order forms of FtsZ, particularly FtsZ from Streptococcus pneumomae and Staphylococcus aurens, among others
  • the invention relates to methods for using such polypeptides. including the treatment of microbial diseases, amongst others
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting the presence or activity of multimeric FtsZ polypeptides
  • Figure 1 illustrates data from a malachite green assay that indicates the hydrolysis of GTP by FtsZ
  • Figure 2 illustrates data from a centnfugation experiment demonstrating the existence of FtsZ drmers and tetramers
  • Figure 3 illustrates data showing a temperature dependence relationship in FtsZ drmers and tetramer association
  • FIG. 4 illustrates data showing concentration dependence of FtsZ self-association
  • Figure 5 illustrates data showing FtsZ self-association in the presence of GDP
  • Figure 6 illustrates data showing FtsZ self-association in the presence of gS-GTP
  • phosphate release assays such as the malachite green or MEG coupled enzyme assay, among other assays provided herein, will be used to identify' compounds that interfere with FtsZ self-recognition, particularly those compounds that prevent GTP hydrolysis
  • Such compounds are believed by the Applicants to be broad spectrum antibacterial agents since it is known that FtsZ is an essential bacterial cell division protein that is present in all known bacterial species
  • the invention relates to multimeric FtsZ polypeptides as descnbed in greater detail below
  • the invention relates to polypeptides of a multimenc FtsZ, particularly of Streptococcus pneumomae, each monomenc subunit of which is related by arnrno acid sequence homology to
  • Lactococcus lactis FtsZ U74322 polypeptide The invention relates especially to multimenc FtsZ each subunit compnsing the amino acid sequences set out in Table 1 as SEQ ID NO 2 or 4 TABLE 1 Monomeric FtsZ Polypeptide Sequences
  • a deposit containing a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve. Aberdeen AB2 1RY, Scotland on 11 Apnl 1996 and assigned deposit number 40794
  • the deposit was descnbed as Streptococcus pneumomae 0100993 on deposit On 17 Apnl 1996 a Streptococcus pneumomae 0100993 DNA library in E coh was similarly deposited with the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumomae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain "
  • the deposited strain contains the full length FtsZ gene
  • the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein
  • a license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumomae 0100993 strain, which polypeptide is contained in the deposited strain
  • FtsZ polynucleotide sequences in the deposited strain such as DNA and RNA
  • amino acid sequences encoded thereby Also provided by the mvention are multimenc FtsZ pol ⁇ pept ⁇ des compnsing the FtsZ polypeptide sequences isolated from the deposited strain and the polynucleotides that encode such sequences Polypeptides
  • the multimenc FtsZ polypeptides of the invention is substantially phylogenetically related to other proteins of the FtsZ family
  • polypeptides particularly of Streptococcus pneumomae multimenc FtsZ polypeptides as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
  • vanants of multimenc FtsZ polypeptide each subunit of which is encoded by naturally occurring alleles of the FtsZ gene
  • the present invention further provides for an isolated polypeptide which
  • (a) comprises or consists of a multimenc FtsZ polypeptide each subunit of which is an amino acid sequence which has at least 75% identity, preferably at least 80%> identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 or 4 over the entire length of SEQ ID NO 2 or 4 respectively,
  • a multimenc FtsZ polypeptide each subunit of which is a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 2 or 4 over the entire length of SEQ ID NO 2 or 4 respectively,
  • a multimenc FtsZ polypeptide each subunit of which is a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 75% identify, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95%> identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2 or 4, over the entire length of SEQ ID NO 2 or 4 respectively
  • the multimenc FtsZ polypeptide include multimers each subunit of which compnses a polypeptide of Table 1 [SEQ ID NO 2 or 4] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FtsZ and constitute a multimenc FtsZ having the biological activity of multimenc FtsZ, and also those which have at least 75% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids
  • the invention also includes a multimenc FtsZ polypeptide each subunit of which is a polypeptide consisting of or compnsing a polypeptide of the formula
  • X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides. and at the carboxyl terminus.
  • Ri and R3 are any amino acid residue or modified amino acid residue
  • m is an integer between 1 and 1000 or zero
  • n is an integer between 1 and 1000 or zero
  • R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified fon ⁇ s thereof In the formula above, R 2 is onented so that its amino terminal amino acid residue is at the left, covalently bound to Ri and its carboxy terminal amino acid residue is at the nght. covalently bound to R3
  • Any stretch of amino acid residues denoted by either Ri or R3, where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer.
  • a polypeptide of the invention is denved from Streptococcus pneumomae. however, it may preferably be obtained from other orgamsms of the same taxonomic genus A polypeptide of the invention may also be obtained, for example, from orgamsms of the same taxonomic family or order
  • a fragment is a vanant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
  • Preferred fragments compnsing a multimenc FtsZ include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2 or 4], or of vanants thereof, such as a continuous senes of residues that includes an amino- and/or carboxyl-te ⁇ runal amino acid sequence
  • Degradation forms of the polypeptides of the invention produced by or m a host cell, particularly a Streptococcus pneumomae are also preferred
  • fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobe regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigemc index regions
  • Further preferred fragments include an isolated polypeptide comprising an amino
  • biologically active fragments which are those fragments that mediate activities of multimenc FtsZ once combined in multimenc form, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • those fragments that are antigemc or immunogemc in an animal especially m a human
  • fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumomae or the ability to initiate, or maintain cause Disease m an individual, particularly a human
  • Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the mvention Vectors, Host Cells, Expression Systems
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention.
  • host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the invention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known in those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide mto the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1 86) and Sambrook. et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. micromjection, cationic lipid-mediated transfection, electroporation. transduction. scrape loading, ballistic introduction and infection
  • bactenal cells such as cells of streptococci, staphylococci, enterococci E coh. streptomyces, cyanobactena, Bacillus subti s. and Streptococcus pneumomae.
  • fungal cells such as cells of a yeast, Kluveromyces . Saccharomyces . a basidiomycete.
  • insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9, animal cells such as CHO, COS. HeLa. C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a g ⁇ nnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bactenal plasmids. from bactenophage. from transposons. from yeast episomes from insertion elements, from yeast chromosomal elements, from viruses such as baculovrruses. papova viruses, such as SV40, vaccinia viruses, adenoviruses.
  • the expression system constructs may contain control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as. for example, those set forth in Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography. affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification
  • Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication
  • This mvention is also related to the use of multimenc FtsZ polypeptides of the mvention for use as diagnostic reagents Detection of multimenc FtsZ polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the multimenc FtsZ protem, may be detected by a vanety of well known techniques, for example, by non-denaturing protem gels, as well as by methods provided herein
  • Polypeptides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals hi another embodiment, an arraj of antibodies agamst multimenc FtsZ can be constructed to conduct efficient screening of. for example, genetic mutations, serotype. taxonomic classification or identification
  • the present invention relates to a diagnostic kit which comprises (a) a multimenc FtsZ polypeptide of the present invention, preferably a multimenc FtsZ polypeptide comprising at least one polypeptide of SEQ ID NO 2 or 4 or a fragment thereof, or (b) an antibody to a multimenc FtsZ polypeptide of the present invention, preferably a multimenc FtsZ polypeptide comprising at least one polypeptide of SEQ ID NO 2 or 4
  • any such kit may comprise a substantial component
  • Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
  • Cells from an organism carrying mutations or polymorphisms (allelic vanations) in a multimenc FtsZ polypeptide of the mvention may also be detected at the polypeptide level by a vanety of techniques
  • a diagnostic assay m accordance with the mvention for detecting over- expression of multimenc FtsZ polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, among other diseases
  • Assay techniques that can be used to determine levels of a multimenc FtsZ polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Multimenc FtsZ polypeptides of the mvention may also be used to assess the binding of small molecule substrates and ligands m.
  • substrates and ligands for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocol's w Immunology 1(2) Chapter 5 (1991)
  • Mutlimenc FtsZ polypeptides of the present mvention are responsible for or mvolved many biological functions, such as bactenal cell division and bactenal cell survival In view of the fact that such mutlimenc FtsZ polypeptides carry important bactenal biological functions, they are therefore responsible for the maintenance of many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which mhibit the function of a mutlimenc FtsZ polypeptide and lead to slowing or stoppmg of bactenal growth or bactenal killing Accordingly, m a further aspect, the present mvention provides for a metliod of screening compounds to identify those which stimulate or which inhibit the function of a mutlimenc FtsZ polypeptide of the mvention.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical branes. and natural product mixtures
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands. receptors, enzymes, etc . as the case may be, of multimenc FtsZ polypeptides. or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))
  • the screening methods may simply measure the binding of a candidate compound to a mutlimenc FtsZ polypeptide or polynucleotide. or to cells or membranes bearing such polypeptide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of a mutlimenc FtsZ polypeptide, using detection systems appropriate to the cells comprising such polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active mutlimenc FtsZ polypeptide and/or constitutively expressed polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of mutlimenc Ft
  • activity means any functional attribute of a FtsZ polypeptide or multimenc FtsZ polypeptide, including, for example, enzymatic activity (such as GTPase activity), binding of ligands, substrates (such as GTP) or subumts (such as FtsZ polypeptide), the formation of multimers. the disassociation of multimers.
  • Mutlimenc FtsZ polypeptides and antibodies that bind to and/or interact with such pol pept ⁇ des of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production mutlimenc FtsZ polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • Methods of measuring the ability of a candidate compound to alter the assembly state of FtsZ are also preferred as screening methods of the invention
  • the mvention also provides a method of screening compounds to identify those which enhance
  • the method of screening may mvolve high-throughput techniques
  • a synthetic reaction mix for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg multimenc FtsZ polypeptide and a labeled substrate or ligand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a multimenc FtsZ agonist or antagonist
  • the ability of the candidate molecule to agomze or antagonize the multimenc FtsZ polypeptide is reflected m decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i , without mducmg the effects of multimenc FtsZ polypeptide are most likely to
  • reporter system Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m multimenc FtsZ polypeptide activity, and binding assays known m the art
  • Mutlimenc FtsZ polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to. ligand binding and cross nkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated) or fused to a peptide sequence suitable for detection or purification, and mcubated with a source of the putative receptor (e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify' agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s).
  • a radioactive isotope for instance, ⁇ 1
  • chemically modified for instance, biotin
  • a preferred assay to screen for antimicrobial compounds using multimenc FtsZ polypeptide is to add a test compound to multimenc FtsZ and determine compounds that antagonist the FtsZ FtsZ interaction by measuring the amount of multimenc FtsZ over after the addition of a test compound.
  • Compounds that antagonist the FtsZ FtsZ interaction are believed to block the turning on of FtsZ GTPase activity Moreover, compounds that affect or inhibit the FtsZ FtsZ interaction are believed to affect the rate of GTP hydrolysis
  • Preferred assay conditions for achieving GTPase activity and/or the formation of multimenc FtsZ FtsZ monomers or lower order forms are 50mM Hepes, 50mM KC1, 5mM MgCl, pH 7 4
  • a number of assays can be used to characterize compounds that block the FtsZ self- association and or compounds that promote FtsZ self-association
  • the interaction between FtsZ monomers can be monitored with physical techniques such as analytical ultracentrifugation. fluorescence polarization, fluorescence energy transfer, surface plasmon resonance, scintillation proximity assay and the use of ion channel switch (ICS) biosensors, among other well known detection techniques
  • ICS ion channel switch
  • Analytical ultracentrifugation is a practical and robust method for measuring molecular mass in solution It may be used to demonstrate that FtsZ undergoes a monomer-dimer-tetramer or assembly process in the absence of FtsZ active site ligands or compounds that otherwise bind or interact with FtsZ While not wanting to be limited to theoretical models, the Applicants believe that the midpoints of both assembly processes are at about 10 micromolar FtsZ.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate
  • Large protem complexes, such as multimenc FtsZ, labeled to comprise a fluorescently-labelled molecule will have higher polarization values than a fluorescently labelled monomeric protein It is preferred that this method be used to characterize small molecules that disrupt FtsZ ohgomers
  • Fluorescence energy transfer may be used characterize small molecules that interfere with the formation of FtsZ dimers. t ⁇ mers.
  • FtsZ can be labelled with both a donor and acceptor fluorophore Upon mixing of the two labelled species and excitation of the donor fluorophore fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer
  • a scintillation proximity assay may be used to characterize the interaction between FtsZ monomers
  • FtsZ can be coupled to a scintillation-filled bead
  • Addition of radio-labelled FsZ results in binding where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon FtZ binding and compounds that prevent FsZ self-association will dimmish signal
  • ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidm-facihtated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale high through-put screening of small molecule combinatorial libraries.
  • methods for identifying compounds which bind to or otherwise mteract with and inhibit or activate an activity or expression of a mutlimenc FtsZ polypeptideof the mvention compnsmg contacting a polypeptide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction
  • a preferred assay embodiment compnses the step of contacting an organism with a test compound and detecting if FtsZ forms a septal ⁇ ng usmg an antibody of the mvention Compounds that are associated with disagregation of such nng are preferred as antimicrobial compounds
  • an assay for multimenc FtsZ agonists is a competitive assay that combmes multimenc FtsZ and a potential agonist with multimenc FtsZ-binding molecules, recombinant multimenc FtsZ bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay
  • the multimenc FtsZ molecule can be labeled, such as by ra ⁇ oactivity or a colonmetnc compound, such that the number of multimenc FtsZ molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • Rayleigh light scattering is an absolute method for determining the molecular weight of macromolecules in aqueous solution Changes m light scattering that result from addition of an external compound reflect changes is the weight average molecular weight and hence m the assembly state, le the relative amounts of monomers, dimers, tetramers and higher order assemblies
  • This technique could be used to identify compounds which reduce the weight average molecular weight, shifting the molecular distribution towards monomer, and hence acting as an inhibitor of FtsZ Potential antagonists mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmd g molecule, without mducmg multimenc FtsZ-
  • Potential antagonists m include a small molecule that b ds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules
  • Other potential antagonists m clude antisense molecules (see Okano. J Neurochem 56 560 (1991). OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION. CRC Press. Boca Raton.
  • Prefened potential antagonists mclude compounds related to and vanants of multimenc FtsZ
  • Other examples of potential polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands. substrates, receptors, enzymes, etc . as the case may be. of the poh/peptide. e g . a fragment of the ligands. substrates, receptors, enzymes, etc . or small molecules winch bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • Certain of the polypeptides of the mvention are biomimetics.
  • Functional mimetics of the natural multimenc FtsZ polypeptide These functional mimetics ma)' be used for. among other things, antagonizing the activity of multimenc FtsZ pohpeptide or as a antigen or lmmunogen m a manner descnbed elsewhere herem
  • Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides
  • preferred functional mimetics m clude. a multmienc FtsZ each subumt compnsmg a polypeptide compnsmg the polypeptide sequence set forth m SEQ ID NO 2 or 4 lacking 20. 30, 40.
  • polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened that these cassettes compnse 5' and 3' restnction sites to allow for a convement means to gate the cassettes together when desired It is further preferred that these cassettes compnse gene expression signals known m the art or descnbed elsewhere herem
  • the present mvention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of a mutlimenc FtsZ polypeptide, by
  • the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of. an elevated activity of, or a decreased activity of multimenc FtsZ polypeptide
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of lmmunoglobulms of various subclasses (IgG.
  • IgM, IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therap ⁇
  • a further aspect of the mvention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914
  • expression of the gene encoding endogenous multimenc FtsZ polypeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example. O'Connor, J Neurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton FL (1988))
  • o gonucleotides which form triple helices with the gene can be supplied (see. for example, Lee et al , Nucleic Acids Res (1979) 6 3073. Cooney et al , Science (1988) 241 456. Dervan et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • multimenc FtsZ polypeptide agonists and antagonists preferably bactenostatic or bactenocidal agonists and antagonists
  • the antagonists and agonists of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • H pylori Helicobacler pylori bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis
  • International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylon International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylon and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the mvention agonists and antagonists of multimenc FtsZ polypeptides and/or polynucleotides found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylon infection Such treatment should decrease the advent of H pylon- m
  • Another aspect of the mvention relates to a method for inducing an lmrnunological response m an individual, particularly a mammal which comprises inoculating the individual with multimenc FtsZ polypeptide. or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumomae infection Also provided are methods whereby such inmiunological response slows bacterial replication
  • Yet another aspect of the invention relates to a method of inducing lmrnunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ⁇ bozyme to direct expression of multmienc FtsZ polypeptide, or a fragment or a variant thereof, for expressing multimenc FtsZ polypeptide.
  • nucleic acid vector may comprise DNA.
  • a further aspect of the invention relates to an lmrnunological composition that when introduced mto an individual, preferably a human, capable of having induced within it an lmrnunological response, induces an lmrnunological response in such individual to a multimenc FtsZ polypeptide, wherein the composition comprises a recombinant multimenc FtsZ polypeptide
  • the lmrnunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity a ⁇ smg from CTL or CD4+ T cells
  • a multimenc FtsZ polypeptide or a fragment thereof may be fused with co-protem or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protem and producmg a fused or modified protem which will have antigemc and/or lmmunogenic properties, and preferably protective properties
  • fused recombinant protem preferably further comprises an antigemc co-protem, such as hpoprotein D from Hemoph ⁇ us mfluenzae.
  • Glutathione-S-transferase GST
  • beta-galactosidase any other relatively large co-protein which solubilizes the protem and facilitates production and purification thereof
  • the co-protein may act as an adjuvant m the sense of providing a generalized stimulation of the immune system of the organism receivmg the protem
  • the co-protein may be attached to either the amino- or carboxy-termmus of the first protem
  • compositions particularly vaccine compositions, and methods comprising the polypeptides of the mvention and immunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
  • polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bactenal cell surface proteins, in po nucleotide constructs used m such genetic immunization experiments in animal models of infection with Streptococcus pneumomae Such experiments will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumomae infection, in mammals, particularly humans
  • a polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue
  • tissue damage include wounds m skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina
  • the invention also includes a vaccine formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down m the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile mjection solutions which may
  • compositions for purposes of compositions, kits and administration
  • compositions compnsmg a multimenc FtsZ polypeptide for administration to a cell or to a multicellular organism
  • the mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agonists or antagonists
  • the polypeptides and polynucleotides of the mvention may be employed m combmation with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an individual
  • Such compositions compnse for instance, a media additive or a therapeutically effective amount of a polypeptide and or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient
  • Such earners may mclude, but are not limited to. saline, buffered salme.
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the mgredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • compositions may be administered m any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, mtravenous, mtrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably lsotomc
  • composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropnate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams
  • topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient
  • a pharmaceutically acceptable earner or excipient Such earners mclude. but are not limited to. salme. buffered saline, dextrose, water, glycerol.
  • the mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the mgredients of the aforementioned compositions of the mvention
  • Polypeptides polynucleotides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • composition will be adapted to the route of admimstration, for instance by a systemic or an oral route
  • Preferred forms of systemic administration include mjection. typically by intravenous mjection
  • Other mjection routes such as subcutaneous, mtramuscular, or intrapentoneal.
  • In-dwelling devices mclude surgical implants, prosthetic devices and catheters, I e . devices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters
  • composition of the mvention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an m-dwelling device Treatment may be continued after surgery during the m-body time of the device
  • composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumomae wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
  • composition of the mvention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • a vaccine composition is conveniently m mjectable form
  • Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1 -3 times and with an interval of 1 -3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
  • All publications and references, including but not limited to patents and patent applications, cited m this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as bemg fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety m the manner described above
  • Activit 1 means any functional attribute of a FtsZ polypeptide or multimenc FtsZ polypeptide, including, for example, enzymatic activity (such as GTPase activity), bmdmg of ligands. substrates (such as GTP) or subunits (such as FtsZ polypeptide), the formation of multimers. the disassociation of multimers, cell division, cell growth ring formation and cell wall invagination, among others "Ant ⁇ body( ⁇ es) '" as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c.
  • Antigenically equivalent denvat ⁇ ve(s) encompasses a polypeptide polynucleotide. or the equivalent of either which will be specifically recognized by certain antibodies which when raised to the protem polypeptide or polynucleotide according to the invention interferes with the immediate physical mteraction between pathogen and mammalian host
  • Bispecific ant ⁇ body( ⁇ es) means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope
  • Bodily mate ⁇ al(s) means any matenal denved from an individual or from an organism infecting, lnfestmg or inhabiting an individual, including but not limited to. cells, tissues and waste, such as. bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skm, unne, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, including , for example, otitis media, conjunctivitis, pneumoma. bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid
  • FtsZ means, as the case may be, FtsZ polypeptides, FtsZ polynucleotides, each including, for example, those from any organism defined herem
  • Fusion protem(s) refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example.
  • EP-A-0464 discloses fusion proteins comprising vanous portions of constant region of immunoglobulm molecules together with another human protein or part thereof In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232262]
  • Host cell(s) is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology. Lesk. A M , ed , Oxford University Press, New York, 1988. Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press. New York. 1993, Computer Analysis of Sequence Data. Part I.
  • Polynucleotide embodiments further include an isolated polynucleotide compnsing a polynucleotide sequence having at least a 50, 60, 70. 80, 85, 90, 95, 97 or 100% identify to the reference sequence of SEQ ID NO 1 or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or 3 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 or
  • n n is the number of nucleotide alterations.
  • x n is the total number of nucleotides in SEQ ID NO 1 or 3
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%. 0 85 for 85%.
  • any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 or 4 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further mclude an isolated polypeptide compnsing a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2 or 4, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or 4 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 or 4 by the integer defining the percent identity divided by 100 and then subtract
  • n a is the number of amino acid alterations
  • x a is the total number of ammo acids in SEQ ID NO 2 or 4
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%. 0 90 for 90%. 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • “Immunologically equivalent denvat ⁇ ve(s)” as used herein encompasses a polypeptide, polynucleotide. or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host '"Immunospecific" means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides m the pnor art
  • “Ind ⁇ v ⁇ dual(s)” means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its ongmal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said orgamsm, which orgamsm may be living or non-living
  • Multimenc FtsZ means a dimenc protein, t ⁇ meric protem, tetrame ⁇ c protem or higher order structure protem compnsmg FtsZ protem subunits
  • Mutlimenc FtsZ may be comprised of FtsZ proteins of different species or amino acid sequences as provided by the invention and described herein
  • Organ ⁇ sm(s) means a (l) prokaryote. including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neisseria, Haemoph us, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersmia, Fancisella Pasturella Moraxella Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Lrsteria, Caly matobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleib iella, Vibrio, Proteus, Erwima, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legwnella, Pseudomonas,
  • mcludmg. but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumomae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vag nahs, Mycobactenum tuberculosis, Mycobacterium bovis, Mycobactenum ulcerans, Mycobacterium leprae, Actinomyctes israehi
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleot ⁇ de(s)" mclude.
  • smgle- and double-stranded DNA DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions
  • hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA
  • the strands m such regions may be from the same molecule or from different molecules
  • the regions may mclude all of one or more of the molecules, but more typically involve only a region of some of the molecules
  • DNAs or RNAs compnsmg unusual bases, such as mosme, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that sen e many useful purposes known to those of skill m the art
  • polynucleot ⁇ de(s) as it is herem embraces such chemically, enzymatically or metabo cally modified forms of polynucleotides. as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg. for example, simple and complex cells
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often referred to as ohgonucleot ⁇ de(s)
  • Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypept ⁇ de(s)” refers to both short chains, commonly refened to as peptides.
  • Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypept ⁇ de(s)" mclude those modified either by natural processes, such as processmg and other post- translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contam many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation.
  • ADP-nbosylation amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a pid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation.
  • gamma- carboxylation glycosylation, GPI anchor formation, hydroxylation, lodination, methylatiort, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, pid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to protems such as arginylation, and ubiquitmation See, for instance. PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES.
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
  • “Vanant(s)" as the tenn is used herem. is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
  • the polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1 and 3] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumomae in E coh
  • the sequencing data from two or more clones containing overlapping Streptococcus pneumomae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 or 3 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Streptococcus pneumomae 0100993 according to standard procedures and size-fractionated by either of two methods
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E co infected with the packaged library
  • the library is amplified by standard procedures Method 2
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall. Alul. Bshl235I). and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh infected with the packaged library The library is amplified by standard procedures Example 2 Functional oligomerization states of FtsZ

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Abstract

The invention provides multimeric FtsZ polypeptides and polynucleotides encoding multimeric FtsZ polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing multimeric FtsZ polypeptides to screen for antibacterial compounds.

Description

FTSZ MULTIMERIC PROTEINS AND THEIR USES
RELATED APPLICATIONS
This application claims benefit of US Provisional Patent Application Number 60/091.680 filed July 2, 1998
FIELD OF THE INVENTION
This invention relates to newly identified higher order structures of FtsZ polypeptides, such as drmers. trimer. tetramers and larger aggregations, and their production and uses, as well as their vanants. their agonists and antagonists, and their uses In particular, the invention relates to multimenc polypeptides compnsing FtsZ subunits and their use in screening for antimicrobial compounds
BACKGROUND OF THE INVENTION In the prokaryotic cell cycle, the FtsZ (filamentation temperature sensitive) protein functions early in the septation process FtsZ polymerizes in a dynamic ring structure and localizes to the midpoint of the dividing cell This polymeric structure is believed to act as a scaffold for other important cell division proteins In addition to its structural role, FtsZ is also a GTPase Prior to the Applicants findings provided herein, the precise relationship between GTPase activity of FtsZ and the polymeπzation-depolymeπzation cycle is poorly understood
FtzZ is essential for cell division Mutants in E coh fail to form division septae and filament (Lutkenhaus and A Mukherjee Cell Division in Eschenchia co and Salmonella. Cellular and Molecular Biology. F C Neidhardt ed Washington D C . ASM Press 1996, pp 1615-1626)
Clearly, there is a need for factors, such as the compounds of the invention that have a present benefit of being useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and charactenzation of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases
SUMMARY OF THE INVENTION
The present invention relates to FtsZ multimeric and higher order forms of FtsZ, particularly FtsZ from Streptococcus pneumomae and Staphylococcus aurens, among others In another aspect, the invention relates to methods for using such polypeptides. including the treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting the presence or activity of multimeric FtsZ polypeptides
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates data from a malachite green assay that indicates the hydrolysis of GTP by FtsZ Figure 2 illustrates data from a centnfugation experiment demonstrating the existence of FtsZ drmers and tetramers
Figure 3 illustrates data showing a temperature dependence relationship in FtsZ drmers and tetramer association
Figure 4 illustrates data showing concentration dependence of FtsZ self-association Figure 5 illustrates data showing FtsZ self-association in the presence of GDP
Figure 6 illustrates data showing FtsZ self-association in the presence of gS-GTP
DETAILED DESCRIPTION OF THE INVENTION
The Applicants have demonstrated, utilizing both analytical ultracentπfugation and enzymatic assays, an important relationship between FtsZ GTPase activity and oligomeric state of FtsZ protein, by showing that FtsZ drmers, trrmers and/or tetramers are required for expression of GTP hydrolysis activity In view of this discovery, phosphate release assays, such as the malachite green or MEG coupled enzyme assay, among other assays provided herein, will be used to identify' compounds that interfere with FtsZ self-recognition, particularly those compounds that prevent GTP hydrolysis Such compounds are believed by the Applicants to be broad spectrum antibacterial agents since it is known that FtsZ is an essential bacterial cell division protein that is present in all known bacterial species
The invention relates to multimeric FtsZ polypeptides as descnbed in greater detail below In particular, the invention relates to polypeptides of a multimenc FtsZ, particularly of Streptococcus pneumomae, each monomenc subunit of which is related by arnrno acid sequence homology to
Lactococcus lactis FtsZ U74322 polypeptide The invention relates especially to multimenc FtsZ each subunit compnsing the amino acid sequences set out in Table 1 as SEQ ID NO 2 or 4 TABLE 1 Monomeric FtsZ Polypeptide Sequences
(A) Streptococcus pneumomae monomeric FtsZ polypeptide [SEQ ID NO 2] MLEFEQGFNHL TLKVIGVGGGGNNAλΛJRMIDHGMNNVEFIAINTDGQALNLSKAESKIQIGEKLTRGLG AGANPEIGKK
AAEES REQI EDAI QGADMVFVT S GMGGGTGTGAAPWAKI AKEMGALTVG WTRP FS FEGRKRQTQAAAG VEAMKAAVDT
LIVIPNDRLLDIVDKSTPMMEAFKEADNVLRQGVQGISDLIAVSGEVNLDFADVKTIMSNQGSALMGIGV S SGENRAVEA
AKKAI S S PLLETSIVGAQGVLMNITGGESLSLFEAQEAADIVQDAADEDVNMI FGTVINPELQDEIWTV
IATGFDDKPT
SHGRKSGSTGFGTSVNTS SNATSKDES FTSNS SNAQATDSVSERTHTTKEDDI PS FI RNREERRSRRTRR
(B) Streptococcus pneumomae polynucleotide encoding the monomeric FtsZ polypeptide of SEQ ID NO 2 [SEQ ID NO 1]
ATGTTAGAATTTGAACAAGGATTTAATCATTTAGCGACTTTAAAGGTCATTGGTGTAGGTGGTGGCGGTA
ACAACGCCGT
AAACCGAATGATTGACCACGGAATGAATAATGTTGAATTTATCGCTATCAACACAGACGGTCAAGCTTTA
AACTTATCTA
AAGCTGAATCTAAAATCCAAATCGGTGAAAAATTAACACGTGGTTTAGGAGCAGGAGCTAATCCTGAAAT CGGTAAAAAA
GCTGCAGAGGAATCTCGTGAACAAATTGAAGATGCAATCCAAGGTGCAGACATGGTATTTGTTACTTCTG
GTATGGGTGG
CGGAACTGGTACTGGTGCAGCACCAGTCGTTGCTAAAATTGCAAAAGAAATGGGCGCATTAACTGTTGGT
GTTGTAACTC GTCCATTTAGTTTTGAAGGACGTAAACGTCAAACTCAAGCTGCTGCTGGAGTAGAAGCTATGAAAGCTGC
AGTAGATACA
TTAATCGTTATACCAAATGACCGTTTATTAGATATCGTTGACAAATCTACGCCAATGATGGAAGCATTTA
AAGAAGCTGA
CAACGTGTTACGCCAAGGTGTACAAGGTATCTCAGACTTAATCGCTGTTTCTGGTGAAGTAAACTTAGAC TTTGCAGACG
TTAAGACAATTATGTCTAACCAAGGTTCTGCATTAATGGGTATTGGTGTTTCTTCTGGTGAAAATAGAGC
GGTAGAAGCT
GCTAAAAAAGCAATCTCTTCTCCATTACTTGAAACATCTATCGTTGGTGCACAAGGTGTGCTTATGAATA
TTACTGGTGG CGAGTCATTGTCATTATTTGAAGCACAAGAGGCTGCTGATATTGTCCAAGATGCTGCAGATGAAGACGTT
AATATGATTT
TCGGTACAGTTATTAATCCTGAATTACAAGATGAGATTGTTGTAACAGTTATTGCAACTGGTTTTGATGA
CAAACCAACA
TCACATGGTCGTAAATCTGGTAGCACTGGATTCGGAACAAGCGTAAATACTTCTAGCAATGCAACTTCTA AAGATGAATC
ATTCACTTCAAATTCATCAAATGCACAAGCAACTGATAGTGTAAGTGAAAGAACACATACAACTAAAGAA
GATGATATTC
CTAGCTTCATTAGAAATAGAGAAGAAAGACGTTCAAGAAGAACAAGACGTTAA Monomeric FtsZ Polypeptide Sequences
(C) Staphylococcus aureus monomeric FtsZ polypeptide [SEQ ID NO:4].
MTFSFDTAAAQGAVIKVIGVGGGGGNAINRMVDEGVTGVEFIAANTDVQALSSTKAETVIQLGPKLTRGL
GAGGQPEVGR
KAAEESEETLTEAISGADMVFITAGMGGGSGTGAAPVIARIAKDLGALTVGV TRPFGFEGSKRGQFAVE
GINQLREHVD T LIISNNNLLEIVDKKTPL EALSEADNVLRQGVQGITDLITNPGLINLDFADVKTVMANKGNALMGIG
IGSGEERWE
AARKAIYS PLLETTI DGAEDVI'VNλ/TGGLDLTLI EAEEASQIVNQAAGQGλ/NI LGTSI DESMRDEIRλn' λA ATGVRQDR
VEKλA APQARSATNYRETVKPAHSHGFDRHFDMAETVELPKQNPRRLEPTQASAFGD DLRRES IVRTTD SWS PVERFE
API SQDEDELDTPPFFKNR
(D) Staphylococcus aureus polynucleotide encoding the monomeric FtsZ polypeptide of SEQ ID NO:4 [SEQ ID NO:3]. ATGACATTTTCATTTGATACAGCTGCTGCTCAAGGGGCAGTGATTAAAGTAATTGGTGTCGGTGGAGGTG
GTGGCAATGC
CATCAACCGTATGGTCGACGAAGGTGTTACAGGCGTAGAATTTATCGCAGCAAACACAGATGTACAAGCA
TTGAGTAGTA
CAAAAGCTGAGACTGTTATTCAGTTGGGACCTAAATTGACTCGTGGTTTGGGTGCAGGAGGTCAACCTGA GGTTGGTCGT
AAAGCCGCTGAAGAAAGCGAAGAAACACTGACGGAAGCTATTAGTGGTGCCGATATGGTCTTCATCACTG
CTGGTATGGG
AGGAGGCTCTGGAACTGGAGCTGCTCCTGTTATTGCTCGTATCGCCAAAGATTTAGGTGCGCTTACAGTT
GGTGTTGTAA CACGTCCCTTTGGTTTTGAAGGAAGTAAGCGTGGACAATTTGCTGTAGAAGGAATCAATCAACTTCGTGA
GCATGTAGAC
ACTCTATTGATTATCTCAAACAACAATTTGCTTGAAATTGTTGATAAGAAAACACCGCTTTTGGAGGCTC
TTAGCGAAGC
GGATAACGTTCTTCGTCAAGGTGTTCAAGGGATTACCGATTTGATTACCAATCCAGGATTGATTAACCTT GACTTTGCCG
ATGTGAAAACGGTAATGGCAAACAAAGGGAATGCTCTTATGGGTATTGGTATCGGTAGTGGAGAAGAACG
TGTGGTAGAA
GCGGCACGTAAGGCAATCTATTCACCACTTCTTGAAACAACTATTGACGGTGCTGAGGATGTTATCGTCA
ACGTTACTGG TGGTCTTGACTTAACCTTGATTGAGGCAGAAGAGGCTTCACAAATTGTGAACCAGGCAGCAGGTCAAGGA
GTGAACATCT
GGCTCGGTACTTCAATTGATGAAAGTATGCGTGATGAAATTCGTGTAACAGTTGTTGCAACGGGTGTTCG
TCAAGACCGC
GTAGAAAAGGTTGTGGCTCCACAAGCTAGATCTGCTACTAACTACCGTGAGACAGTGAAACCAGCTCATT CACATGGCTT
TGATCGTCATTTTGATATGGCAGAAACAGTTGAATTGCCAAAACAAAATCCACGTCGTTTGGAACCAACT
CAGGCATCTG
CTTTTGGTGATTGGGATCTTCGCCGTGAATCGATTGTTCGTACAACAGATTCAGTCGTTTCTCCAGTCGA
GCGCTTTGAA GCCCCAATTTCACAAGATGAAGATGAATTGGATACACCTCCATTTTTCAAAAATCGTTAA Deposited materials
A deposit containing a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve. Aberdeen AB2 1RY, Scotland on 11 Apnl 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumomae 0100993 on deposit On 17 Apnl 1996 a Streptococcus pneumomae 0100993 DNA library in E coh was similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumomae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain "
The deposited strain contains the full length FtsZ gene The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein
The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The strain will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C §112
A license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumomae 0100993 strain, which polypeptide is contained in the deposited strain Further provided by the invention are FtsZ polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby Also provided by the mvention are multimenc FtsZ pol}peptιdes compnsing the FtsZ polypeptide sequences isolated from the deposited strain and the polynucleotides that encode such sequences Polypeptides
The multimenc FtsZ polypeptides of the invention is substantially phylogenetically related to other proteins of the FtsZ family In one aspect of the invention there are provided polypeptides, particularly of Streptococcus pneumomae multimenc FtsZ polypeptides as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same Among the particularly preferred embodiments of the invention are vanants of multimenc FtsZ polypeptide each subunit of which is encoded by naturally occurring alleles of the FtsZ gene The present invention further provides for an isolated polypeptide which
(a) comprises or consists of a multimenc FtsZ polypeptide each subunit of which is an amino acid sequence which has at least 75% identity, preferably at least 80%> identity, more preferably at least 90%) identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 or 4 over the entire length of SEQ ID NO 2 or 4 respectively,
(b) a multimenc FtsZ polypeptide each subunit of which is a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 2 or 4 over the entire length of SEQ ID NO 2 or 4 respectively,
(c) a multimenc FtsZ polypeptide each subunit of which is a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 75% identify, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95%> identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2 or 4, over the entire length of SEQ ID NO 2 or 4 respectively
The multimenc FtsZ polypeptide include multimers each subunit of which compnses a polypeptide of Table 1 [SEQ ID NO 2 or 4] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FtsZ and constitute a multimenc FtsZ having the biological activity of multimenc FtsZ, and also those which have at least 75% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids
The invention also includes a multimenc FtsZ polypeptide each subunit of which is a polypeptide consisting of or compnsing a polypeptide of the formula
X-(Rι)m-(R2)-(R3)n-Y wherein, at the amino terminus. X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides. and at the carboxyl terminus. Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides Ri and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified fonτιs thereof In the formula above, R2 is onented so that its amino terminal amino acid residue is at the left, covalently bound to Ri and its carboxy terminal amino acid residue is at the nght. covalently bound to R3 Any stretch of amino acid residues denoted by either Ri or R3, where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer. preferably a heteropolymer Other preferred embodiments of the invention are provided where m is an integer between 1 and 50. 100 or 500. and n is an integer between 1 and 50, 100, or 500 It is most preferred that a polypeptide of the invention is denved from Streptococcus pneumomae. however, it may preferably be obtained from other orgamsms of the same taxonomic genus A polypeptide of the invention may also be obtained, for example, from orgamsms of the same taxonomic family or order
A fragment is a vanant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
Preferred fragments compnsing a multimenc FtsZ include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2 or 4], or of vanants thereof, such as a continuous senes of residues that includes an amino- and/or carboxyl-teπrunal amino acid sequence Degradation forms of the polypeptides of the invention produced by or m a host cell, particularly a Streptococcus pneumomae, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobe regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigemc index regions Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2 or 4, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO. 2 or 4 Also preferred are biologically active fragments which are those fragments that mediate activities of multimenc FtsZ once combined in multimenc form, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those fragments that are antigemc or immunogemc in an animal, especially m a human Particularly preferred are fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumomae or the ability to initiate, or maintain cause Disease m an individual, particularly a human
Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the mvention Vectors, Host Cells, Expression Systems
The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention. host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the mvention
Recombinant polypeptides of the present mvention may be prepared by processes well known in those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques
For recombinant production of the polypeptides of the mvention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1 86) and Sambrook. et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. micromjection, cationic lipid-mediated transfection, electroporation. transduction. scrape loading, ballistic introduction and infection
Representative examples of appropnate hosts mclude bactenal cells, such as cells of streptococci, staphylococci, enterococci E coh. streptomyces, cyanobactena, Bacillus subti s. and Streptococcus pneumomae. fungal cells, such as cells of a yeast, Kluveromyces . Saccharomyces . a basidiomycete. Candida albicans and Aspergύlus. insect cells such as cells of Drosophύa S2 and Spodoptera Sf9, animal cells such as CHO, COS. HeLa. C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a g τnnosperm or angiosperm
A great vanety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bactenal plasmids. from bactenophage. from transposons. from yeast episomes from insertion elements, from yeast chromosomal elements, from viruses such as baculovrruses. papova viruses, such as SV40, vaccinia viruses, adenoviruses. fowl pox viruses, pseudorabies viruses, picomaviruses and retroviruses, and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as. for example, those set forth in Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
In recombinant expression systems in eukaryotes. for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the penplasnnc space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography. affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication
Diagnostic, Prognostic, Serofyping and Mutation Assays
This mvention is also related to the use of multimenc FtsZ polypeptides of the mvention for use as diagnostic reagents Detection of multimenc FtsZ polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the multimenc FtsZ protem, may be detected by a vanety of well known techniques, for example, by non-denaturing protem gels, as well as by methods provided herein
Polypeptides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals hi another embodiment, an arraj of antibodies agamst multimenc FtsZ can be constructed to conduct efficient screening of. for example, genetic mutations, serotype. taxonomic classification or identification
Thus in another aspect, the present invention relates to a diagnostic kit which comprises (a) a multimenc FtsZ polypeptide of the present invention, preferably a multimenc FtsZ polypeptide comprising at least one polypeptide of SEQ ID NO 2 or 4 or a fragment thereof, or (b) an antibody to a multimenc FtsZ polypeptide of the present invention, preferably a multimenc FtsZ polypeptide comprising at least one polypeptide of SEQ ID NO 2 or 4
It will be appreciated that m any such kit, (a) or (b) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
Cells from an organism carrying mutations or polymorphisms (allelic vanations) in a multimenc FtsZ polypeptide of the mvention may also be detected at the polypeptide level by a vanety of techniques For example, a diagnostic assay m accordance with the mvention for detecting over- expression of multimenc FtsZ polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, among other diseases Assay techniques that can be used to determine levels of a multimenc FtsZ polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
Antagonists and Agonists - Assays and Molecules
Multimenc FtsZ polypeptides of the mvention may also be used to assess the binding of small molecule substrates and ligands m. for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocol's w Immunology 1(2) Chapter 5 (1991)
Mutlimenc FtsZ polypeptides of the present mvention are responsible for or mvolved many biological functions, such as bactenal cell division and bactenal cell survival In view of the fact that such mutlimenc FtsZ polypeptides carry important bactenal biological functions, they are therefore responsible for the maintenance of many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which mhibit the function of a mutlimenc FtsZ polypeptide and lead to slowing or stoppmg of bactenal growth or bactenal killing Accordingly, m a further aspect, the present mvention provides for a metliod of screening compounds to identify those which stimulate or which inhibit the function of a mutlimenc FtsZ polypeptide of the mvention. as well as related polypeptides In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical branes. and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands. receptors, enzymes, etc . as the case may be, of multimenc FtsZ polypeptides. or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screening methods may simply measure the binding of a candidate compound to a mutlimenc FtsZ polypeptide or polynucleotide. or to cells or membranes bearing such polypeptide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of a mutlimenc FtsZ polypeptide, using detection systems appropriate to the cells comprising such polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active mutlimenc FtsZ polypeptide and/or constitutively expressed polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of mutlimenc FtsZ polypeptide Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a mutlimenc FtsZ polypeptide of the present invention, to form a mixture, measuring multimenc FtsZ polypeptide activity in the mixture, and comparing the multimeric FtsZ polypeptide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and multimenc FtsZ polypeptide, as hereinbefore described, can also be used for high- throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , 3 Mol Recognition. 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995)) As used herein "activity" means any functional attribute of a FtsZ polypeptide or multimenc FtsZ polypeptide, including, for example, enzymatic activity (such as GTPase activity), binding of ligands, substrates (such as GTP) or subumts (such as FtsZ polypeptide), the formation of multimers. the disassociation of multimers. cell division, cell growth ring formation and cell wall mvagination, among others Mutlimenc FtsZ polypeptides and antibodies that bind to and/or interact with such pol peptιdes of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production mutlimenc FtsZ polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues Methods of measuring the ability of a candidate compound to alter the assembly state of FtsZ are also preferred as screening methods of the invention The mvention also provides a method of screening compounds to identify those which enhance
(agonist) or block (antagonist) the action of multimenc FtsZ polypeptides, particularly those compounds that are bactenostatic and/or bactenocidal The method of screening may mvolve high-throughput techniques For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg multimenc FtsZ polypeptide and a labeled substrate or ligand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a multimenc FtsZ agonist or antagonist The ability of the candidate molecule to agomze or antagonize the multimenc FtsZ polypeptide is reflected m decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i , without mducmg the effects of multimenc FtsZ polypeptide are most likely to be good antagonists Molecules that bmd well and. as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists Detection of the rate or level of. as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m multimenc FtsZ polypeptide activity, and binding assays known m the art
Mutlimenc FtsZ polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to. ligand binding and cross nkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^1), chemically modified (for instance, biotinylated) or fused to a peptide sequence suitable for detection or purification, and mcubated with a source of the putative receptor (e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify' agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s). if any Standard methods for conducting such assays are well understood in the art A preferred assay to screen for antimicrobial compounds using multimenc FtsZ polypeptide is to add a test compound to multimenc FtsZ and determine compounds that antagonist the FtsZ FtsZ interaction by measuring the amount of multimenc FtsZ over after the addition of a test compound Compounds that antagonist the FtsZ FtsZ interaction are believed to block the turning on of FtsZ GTPase activity Moreover, compounds that affect or inhibit the FtsZ FtsZ interaction are believed to affect the rate of GTP hydrolysis
Preferred assay conditions for achieving GTPase activity and/or the formation of multimenc FtsZ FtsZ monomers or lower order forms are 50mM Hepes, 50mM KC1, 5mM MgCl, pH 7 4
A number of assays can be used to characterize compounds that block the FtsZ self- association and or compounds that promote FtsZ self-association The interaction between FtsZ monomers can be monitored with physical techniques such as analytical ultracentrifugation. fluorescence polarization, fluorescence energy transfer, surface plasmon resonance, scintillation proximity assay and the use of ion channel switch (ICS) biosensors, among other well known detection techniques Analytical ultracentrifugation is a practical and robust method for measuring molecular mass in solution It may be used to demonstrate that FtsZ undergoes a monomer-dimer-tetramer or assembly process in the absence of FtsZ active site ligands or compounds that otherwise bind or interact with FtsZ While not wanting to be limited to theoretical models, the Applicants believe that the midpoints of both assembly processes are at about 10 micromolar FtsZ. so the technique is ideally suited to quantitatively monitor changes in assoication that might occur as a result of the addition of small molecule inhibitors The use of interference optics is preferred and allows these experiments to be done, irrespective of the absorption or fluorescence properties of the inhibitors
The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Large protem complexes, such as multimenc FtsZ, labeled to comprise a fluorescently-labelled molecule will have higher polarization values than a fluorescently labelled monomeric protein It is preferred that this method be used to characterize small molecules that disrupt FtsZ ohgomers Fluorescence energy transfer may be used characterize small molecules that interfere with the formation of FtsZ dimers. tπmers. tetramers or higher order structures FtsZ can be labelled with both a donor and acceptor fluorophore Upon mixing of the two labelled species and excitation of the donor fluorophore fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer
Surface plasmon resonance can be used to monitor the effect of small molecules on FtsZ forms by self-association FtsZ can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric Solution protein can then passed over the FtsZ- coated surface and specific binding can be detected m real-time by monitoring the change m resonance angle caused by a change in local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for FtsZ forms by self-association
A scintillation proximity assay may be used to characterize the interaction between FtsZ monomers FtsZ can be coupled to a scintillation-filled bead Addition of radio-labelled FsZ results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon FtZ binding and compounds that prevent FsZ self-association will dimmish signal
ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidm-facihtated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale high through-put screening of small molecule combinatorial libraries In other embodiments of the mvention there are provided methods for identifying compounds which bind to or otherwise mteract with and inhibit or activate an activity or expression of a mutlimenc FtsZ polypeptideof the mvention compnsmg contacting a polypeptide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably be g associated with a second component capable of providmg a detectable signal in response to the bmdmg or mteraction of the polypeptide with the compound, and detennining whether the compound bmds to or othenvise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide
A preferred assay embodiment compnses the step of contacting an organism with a test compound and detecting if FtsZ forms a septal πng usmg an antibody of the mvention Compounds that are associated with disagregation of such nng are preferred as antimicrobial compounds
Another example of an assay for multimenc FtsZ agonists is a competitive assay that combmes multimenc FtsZ and a potential agonist with multimenc FtsZ-binding molecules, recombinant multimenc FtsZ bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay The multimenc FtsZ molecule can be labeled, such as by raώoactivity or a colonmetnc compound, such that the number of multimenc FtsZ molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
Rayleigh light scattering is an absolute method for determining the molecular weight of macromolecules in aqueous solution Changes m light scattering that result from addition of an external compound reflect changes is the weight average molecular weight and hence m the assembly state, le the relative amounts of monomers, dimers, tetramers and higher order assemblies This technique could be used to identify compounds which reduce the weight average molecular weight, shifting the molecular distribution towards monomer, and hence acting as an inhibitor of FtsZ Potential antagonists mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmd g molecule, without mducmg multimenc FtsZ-induced activities, thereby preventmg the action or expression of multimenc FtsZ polypeptides by excluding multimenc FtsZ polypeptides from bmdmg
Potential antagonists mclude a small molecule that b ds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagonists mclude antisense molecules (see Okano. J Neurochem 56 560 (1991). OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION. CRC Press. Boca Raton. FL (1988), for a descnption of these molecules) Prefened potential antagonists mclude compounds related to and vanants of multimenc FtsZ Other examples of potential polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands. substrates, receptors, enzymes, etc . as the case may be. of the poh/peptide. e g . a fragment of the ligands. substrates, receptors, enzymes, etc . or small molecules winch bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented Certain of the polypeptides of the mvention are biomimetics. functional mimetics of the natural multimenc FtsZ polypeptide These functional mimetics ma)' be used for. among other things, antagonizing the activity of multimenc FtsZ pohpeptide or as a antigen or lmmunogen m a manner descnbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, preferred functional mimetics mclude. a multmienc FtsZ each subumt compnsmg a polypeptide compnsmg the polypeptide sequence set forth m SEQ ID NO 2 or 4 lacking 20. 30, 40. 50, 60, 70 or 80 ammo- or carboxy-terminal ammo acid residues, including fusion proteins compnsmg one or more of these truncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened that these cassettes compnse 5' and 3' restnction sites to allow for a convement means to gate the cassettes together when desired It is further preferred that these cassettes compnse gene expression signals known m the art or descnbed elsewhere herem
Thus, m another aspect, the present mvention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises
(a) a mutlimenc FtsZ polypeptide of the present invention,
(b) a recombinant cell expressing a mutlimenc FtsZ polypeptide of the present invention,
(c) a cell membrane expressing a mutlimenc FtsZ polypeptide of the present invention, or
(d) antibody to an mutlimenc FtsZ polypeptide of the present mvention, which polypeptide is preferably that of SEQ ID NO 2 or 4
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component
It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of a mutlimenc FtsZ polypeptide, by
(a) determining in the first instance the three-dimensional structure of the polypeptide. or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s). binding sιte(s) or motιf(s) of an agonist, antagonist or inhibitor.
(c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s). reactive sιte(s), and/or motifts), and (d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
In a further aspect, the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of. an elevated activity of, or a decreased activity of multimenc FtsZ polypeptide
If the expression and or activity of the polypeptide is m excess, several approaches are available One approach compnses administenng to an individual m need thereof an inhibitor compound (antagonist) as herem descnbed. optionally combmation with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression of the polypeptide. such as. for example, by blockmg the bmdmg of ligands, substrates, receptors, enzymes, etc . or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the ligand. substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the multimenc FtsZ polypeptide In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of lmmunoglobulms of various subclasses (IgG. IgM, IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therap} A further aspect of the mvention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914
In still another approach expression of the gene encoding endogenous multimenc FtsZ polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step m gene expression, but is preferably targeted against transcription and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example. O'Connor, J Neurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton FL (1988)) Alternatively, o gonucleotides which form triple helices with the gene can be supplied (see. for example, Lee et al , Nucleic Acids Res (1979) 6 3073. Cooney et al , Science (1988) 241 456. Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
In accordance w th yet another aspect of the mvention. there are provided multimenc FtsZ polypeptide agonists and antagonists, preferably bactenostatic or bactenocidal agonists and antagonists The antagonists and agonists of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
Helicobacler pylori (herein "H pylori") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylon (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylon and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the mvention (agonists and antagonists of multimenc FtsZ polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylon infection Such treatment should decrease the advent of H pylon- mduced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis Vaccines There are provided by the mvention, products, compositions and methods for assessmg multmienc FtsZ expression, treatmg disease, assaying genetic vanation, and administering a multimenc FtsZ polypeptide and or polynucleotide to an organism to raise an lmrnunological response against a bactena especially a Streptococcus pneumomae bactena
Another aspect of the mvention relates to a method for inducing an lmrnunological response m an individual, particularly a mammal which comprises inoculating the individual with multimenc FtsZ polypeptide. or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumomae infection Also provided are methods whereby such inmiunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing lmrnunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or πbozyme to direct expression of multmienc FtsZ polypeptide, or a fragment or a variant thereof, for expressing multimenc FtsZ polypeptide. or a fragment or a variant thereof in vivo in order to induce an lmrnunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not One example of administering the gene is by accelerating it mto the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA. RNA, a πbozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protem complex
A further aspect of the invention relates to an lmrnunological composition that when introduced mto an individual, preferably a human, capable of having induced within it an lmrnunological response, induces an lmrnunological response in such individual to a multimenc FtsZ polypeptide, wherein the composition comprises a recombinant multimenc FtsZ polypeptide The lmrnunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity aπsmg from CTL or CD4+ T cells
A multimenc FtsZ polypeptide or a fragment thereof may be fused with co-protem or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protem and producmg a fused or modified protem which will have antigemc and/or lmmunogenic properties, and preferably protective properties Thus fused recombinant protem. preferably further comprises an antigemc co-protem, such as hpoprotein D from Hemophύus mfluenzae. Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes the protem and facilitates production and purification thereof Moreover, the co-protein may act as an adjuvant m the sense of providing a generalized stimulation of the immune system of the organism receivmg the protem The co-protein may be attached to either the amino- or carboxy-termmus of the first protem
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides of the mvention and immunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bactenal cell surface proteins, in po nucleotide constructs used m such genetic immunization experiments in animal models of infection with Streptococcus pneumomae Such experiments will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumomae infection, in mammals, particularly humans
A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds m skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina The invention also includes a vaccine formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down m the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile mjection solutions which may contain anti-oxidants, buffers, bacteπostatic compounds and solutes which render the formulation lsotomc with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickenmg agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation While the invention has been described with reference to certain multimenc FtsZ polypeptides, it is to be understood that this covers fragments of the naturally occurring polypeptides. and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the lmmunogenic properties of the recombinant polypeptides or polynucleotides
Compositions, kits and administration
In a further aspect of the mvention there are provided compositions compnsmg a multimenc FtsZ polypeptide for administration to a cell or to a multicellular organism
The mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agonists or antagonists The polypeptides and polynucleotides of the mvention may be employed m combmation with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an individual Such compositions compnse, for instance, a media additive or a therapeutically effective amount of a polypeptide and or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude, but are not limited to. saline, buffered salme. dextrose water, glycerol ethanol and combmations thereof The formulation should suit the mode of administration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the mgredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
The pharmaceutical compositions may be administered m any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, mtravenous, mtrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably lsotomc
Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropnate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
In a further aspect, the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient Such earners mclude. but are not limited to. salme. buffered saline, dextrose, water, glycerol. ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the mgredients of the aforementioned compositions of the mvention Polypeptides polynucleotides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
The composition will be adapted to the route of admimstration, for instance by a systemic or an oral route Preferred forms of systemic administration mclude mjection. typically by intravenous mjection Other mjection routes, such as subcutaneous, mtramuscular, or intrapentoneal. can be used Alternative means for systemic admimstration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated fonmilation, oral admimstration may also be possible Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels, and the like For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg. typically around 1 mg/kg The physician in any event w ll determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual The above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention
In-dwelling devices mclude surgical implants, prosthetic devices and catheters, I e . devices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters
The composition of the mvention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an m-dwelling device Treatment may be continued after surgery during the m-body time of the device In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumomae wound infections
Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation
In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
Alternatively, the composition of the mvention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μ g/ml to lOmg/ml for bathing of wounds or indwelling devices A vaccine composition is conveniently m mjectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1 -3 times and with an interval of 1 -3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals All publications and references, including but not limited to patents and patent applications, cited m this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as bemg fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety m the manner described above for publications and references
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herem "Activit 1 means any functional attribute of a FtsZ polypeptide or multimenc FtsZ polypeptide, including, for example, enzymatic activity (such as GTPase activity), bmdmg of ligands. substrates (such as GTP) or subunits (such as FtsZ polypeptide), the formation of multimers. the disassociation of multimers, cell division, cell growth ring formation and cell wall invagination, among others "Antιbody(ιes)'" as used herein includes polyclonal and monoclonal antibodies, chimeπc. single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobu n expression library "Antigenically equivalent denvatιve(s)" as used herein encompasses a polypeptide polynucleotide. or the equivalent of either which will be specifically recognized by certain antibodies which when raised to the protem polypeptide or polynucleotide according to the invention interferes with the immediate physical mteraction between pathogen and mammalian host
"Bispecific antιbody(ιes)" means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope
"Bodily mateπal(s) means any matenal denved from an individual or from an organism infecting, lnfestmg or inhabiting an individual, including but not limited to. cells, tissues and waste, such as. bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skm, unne, stool or autopsy matenals
"Dιsease(s)" means any disease caused by or related to infection by a bactena, including , for example, otitis media, conjunctivitis, pneumoma. bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid "FtsZ" means, as the case may be, FtsZ polypeptides, FtsZ polynucleotides, each including, for example, those from any organism defined herem
"Fusion protem(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example. EP-A-0464 discloses fusion proteins comprising vanous portions of constant region of immunoglobulm molecules together with another human protein or part thereof In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protem has been expressed, detected and purified "Host cell(s)" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
"Identity." as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology. Lesk. A M , ed , Oxford University Press, New York, 1988. Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press. New York. 1993, Computer Analysis of Sequence Data. Part I. Griffin. A M . and Griffin. H G . eds Humana Press. New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje. G . Academic Press, 1987. and Sequence Analysis Primer, Gnbskov. M and Devereux. J , eds . M Stockton Press. New York, 1991. and Carillo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between t o sequences include, but are not limited to, the GCG program package (Devereux, J . et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN. and FASTA (Atschul. S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al . NCBI NLM NIH Bethesda. MD 20894. Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity
Parameters for polypeptide sequence comparison include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group. Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)
Parameters for polynucleotide comparison include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3
Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons A preferred meaning for "identity" for polynucleotides and polypeptides. as the case may be, are provided in (1) and (2) below
( 1 ) Polynucleotide embodiments further include an isolated polynucleotide compnsing a polynucleotide sequence having at least a 50, 60, 70. 80, 85, 90, 95, 97 or 100% identify to the reference sequence of SEQ ID NO 1 or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or 3 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 or 3 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1 or 3, or
nn ≤ xn " (xn * )»
wherein nn is the number of nucleotide alterations. xn is the total number of nucleotides in SEQ ID NO 1 or 3, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%. 0 85 for 85%. 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 or 4 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
(2) Polypeptide embodiments further mclude an isolated polypeptide compnsing a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2 or 4, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or 4 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2 or 4, or
na ≤ xa " (xa * y)>
wherein na is the number of amino acid alterations, xa is the total number of ammo acids in SEQ ID NO 2 or 4, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%. 0 90 for 90%. 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of xa and y is rounded down to the nearest integer prior to subtracting it from xa
"Immunologically equivalent denvatιve(s)" as used herein encompasses a polypeptide, polynucleotide. or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host '"Immunospecific" means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides m the pnor art
"Indιvιdual(s)" means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human
"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its ongmal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said orgamsm, which orgamsm may be living or non-living
"Multimenc FtsZ" means a dimenc protein, tπmeric protem, tetrameπc protem or higher order structure protem compnsmg FtsZ protem subunits Mutlimenc FtsZ may be comprised of FtsZ proteins of different species or amino acid sequences as provided by the invention and described herein
"Organιsm(s)" means a (l) prokaryote. including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neisseria, Haemoph us, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersmia, Fancisella Pasturella Moraxella Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Lrsteria, Caly matobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleib iella, Vibrio, Proteus, Erwima, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legwnella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma. and further mcludmg. but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumomae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vag nahs, Mycobactenum tuberculosis, Mycobacterium bovis, Mycobactenum ulcerans, Mycobacterium leprae, Actinomyctes israehi, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coh, Shigella dysente ae, Haemophύus influenzae, Haemophύus aegyptius, Haemophilus parainfluenzae, Haemophύus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundn, Proteus irabilis, Proteus vulgaris, Yersmia pestis, Kleibsiella pneumomae, Serratia marcessens, Serratia hquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexnen, Pseudomonas aerugmosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostndium tetani, Clostndium botuhnum, Treponema pallidum, Rickettsia rickettsn and Chlamydia trachomitis, (u) an archaeon, mcludmg but not limited to Achaebacter, and (in) a unicellular or filamentous eukaryote, mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lact s, or Candida albicans
"Polynucleotιde(s)" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude. without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically involve only a region of some of the molecules One of the molecules of a tnple- he cal region often is an o gonucleotide As used herem, the term "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that contam one or more modified bases Thus DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as mosme, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that sen e many useful purposes known to those of skill m the art The term "polynucleotιde(s)" as it is herem embraces such chemically, enzymatically or metabo cally modified forms of polynucleotides. as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg. for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s)
"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides. oligopeptides and ohgomers and to longer chains generally referred to as proteins Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(s)" mclude those modified either by natural processes, such as processmg and other post- translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contam many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation. ADP-nbosylation. amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a pid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation. gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylatiort, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, pid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to protems such as arginylation, and ubiquitmation See, for instance. PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES. 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F . Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson. Ed , Academic Press. New York (1983). Seifter et al . Meth Enzymol 182 626-646 (1990) and Rattan et al . Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
"Vanant(s)" as the tenn is used herem. is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with hke charactenstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr. among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vanants which several, 5-10, 1-5. 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
The examples below are earned out usmg standard techniques, which are well known and routine to those of skill m the art. except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing
The polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1 and 3] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumomae in E coh The sequencing data from two or more clones containing overlapping Streptococcus pneumomae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 or 3 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
Total cellular DNA is isolated from Streptococcus pneumomae 0100993 according to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E co infected with the packaged library The library is amplified by standard procedures Method 2
Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall. Alul. Bshl235I). and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh infected with the packaged library The library is amplified by standard procedures Example 2 Functional oligomerization states of FtsZ
A KM of 200 μM and a kcat of 0 155 sec'l was determined for the GTPase activity of E coh FtsZ using a real-time phosphate release assay To correlate this activity with the polymerization state of FtsZ. several biophysical studies were undertaken Analytical ultracentrifugation demonstrated that FtsZ can exist as monomer, dimer. or tetramer m the absence of nucleotide with K, 2 = 1 5 ± 0 96 μM and K24 = 10 0 ± 0 28 μM The oligomeric state of FtsZ in the presence of nucleotides and inhibitors was examined Additionally, circular dichroism is being used to evaluate if structural changes are associated w th the oligomeric and nucleotide- bound states of FtsZ Refer to Figures 1 -6

Claims

What is claimed is: 1 An isolated multimenc FtsZ polypeptide selected from the group consisting of
(1) a multimenc FtsZ polypeptide comprising an isolated polypeptide comprising an amino acid sequence selected from the group having at least
(a) 75% identify,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 or 4 over the entire length of SEQ ID
NO 2 or 4 respectively, (n) a multimeric FtsZ polypeptide comprising isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2 or 4 or (in) a multimenc FtsZ polypeptide comprising isolated polypeptide which is the amino acid sequence of SEQ ID NO 2 or 4
2 An antibody lmmunospecific for the polypeptide of claim 1
3 A method for screening to identify compounds that activate or that inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of
(a) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,
(b) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor,
(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide. using detection systems appropriate to the cells or cell membranes bearing the polypeptide,
(d) mixing a candidate compound with a solution containing a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide m the mixture, and comparing the activity of the mixture to a standard,
(e) detecting the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide in cells, using for instance, an ELISA assay, or (f) (1) contacting a composition compnsmg the polypeptide with the compound to be screened under conditions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providing a detectable signal in response to the mteraction of the polypeptide with the compound, and
(2) detennining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the mteraction of the compound with the polypeptide
4 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell
5 A host cell comprising the expression system of claim 4 or a membrane thereof expressing a multimeric FtsZ polypeptide
6 A process for producing a multimenc FtsZ polypeptide comprising the step of culturmg a host cell of claim 8 under conditions sufficient for the production of said polypeptide
7 A process for producing a host cell comprising a multimenc FtsZ polypeptide comprising the step of transforming or transfecting a cell with the expression system of claim 8 such the host cell, under appropriate culture conditions, produces a a multimenc FtsZ polypeptide polypeptide
8 A host cell produced by the process of claim 7 or a membrane thereof expressing a multimenc FtsZ polypeptide comprising polypeptide
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US6197300B1 (en) * 1997-08-12 2001-03-06 Smithkline Beecham Corporation ftsZ
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EP0899334A2 (en) * 1997-08-12 1999-03-03 Smithkline Beecham Corporation FtsZ polypeptides from Streptococcus pneumoniae

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