JP2002515242A - Target evaluation of genetic aids in antibiotic drug discovery - Google Patents

Abstract

(57) [Summary] Providing a quick and efficient method for establishing whether bacterial genes are essential or not.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

This application is based on Provisional Patent Application No. 60 / 085,593, filed May 15, 1998,
Claiming priority under 5 USC §119, the entire disclosure of this provisional patent application is hereby incorporated by reference in its entirety.

[0002] The present invention provides methods for genetic analysis in pathogenic bacteria that provide a means for discovering and validating novel therapeutic targets.

[0003]

[Prior art]

The increased availability of bacterial genome sequence information has created a need for measurable methods for accessing gene function. Sequence homology should be able to confer potential functions on many gene products. However, functional genetic analysis is necessary to make effective use of genomic data.

[0004] The technology of the present invention allows for the functional classification of genes according to essential or non-essential characteristics with respect to bacterial growth or pathogenicity. Essential for growth or toxicity,
The gene products identified by this method provide a source of novel targets for high-throughput screening for the discovery of small molecules with bactericidal activity.

[0005] Genetic-Assisted Targeted Evaluation; GATE
) Uses homologous recombination between an insertable plasmid with the target gene fragment and the bacterial chromosome to produce a gene disruptive mutation at the specified locus. GATE
Is a fast, reproducible, large-scale, powerful method for the analysis of gene knockout in bacteria, which allows the analysis of very large numbers of genes. A further property of GATE is the possibility of extensive functional genetic analysis of pathogenic bacteria, including both Gram-positive and Gram-negative organisms. As proof of principle, Streptococcu
GATE has been shown to be useful in two less important medically relevant gram-positive pathogens, S. pyogenes and Staphylococcus aureus.

[0006] GATE has several advantages over prior art methods for determining whether a gene is essential for bacterial growth. Typically, the inability to easily obtain transformants using a plasmid construct designed to produce either a gene disruption or a gene replacement mutation at the locus of interest means that the gene is not easily accessible to bacterial growth. Perceived as essential evidence. However, the inability to obtain transformants is itself insufficient for assessing gene function due to the lack of sufficient positive controls for successful transformation and plasmid insertion. Similarly, the evaluation of genes that are essential for the construction of a partial diploid requires undue manipulation and can transport not only full-length clones for the gene of interest, but also complementary gene copies to secondary genomic sites, It requires the development of a gene transfer system for pathogenic bacteria, for example, specific transfer phages.

[0007]

[Problems to be solved by the invention]

Thus, there is a need in the art for a rapid and efficient method for establishing whether a bacterial gene is essential or non-essential.

[0008]

[Means for Solving the Problems]

The present invention provides a method for determining whether a bacterial gene of interest is essential or non-essential, which method is performed by the following steps: (a) using the following in a substantially simultaneous manner Transforming the bacterial culture by: (i) a first plasmid comprising a recombination cassette containing a first selectable marker flanked by sequences at its 5 'and 3' ends from the gene of interest; A plasmid that can be inserted into the genome of the bacterium by homologous recombination; and (ii) derived from genes whose 5 ′ and 3 ′ ends are known not to be essential for bacterial growth. A second plasmid comprising a recombination cassette containing a second selectable marker adjacent to the sequence to be inserted, said recombination cassette being insertable into the bacterial genome by homologous recombination. (B) under selective conditions in which only (i) cells expressing the first selectable marker survive, or (ii) selective conditions in which only cells expressing the second selectable marker survive. Culturing the transform cultures produced in step (a) individually; (c) measuring the number of viable cells in cultures (i) and (ii) of step (b); and (d) Comparing the measured values obtained in step (c), wherein the culture (i)
Lack of detectable colonies in culture and appearance of colonies in culture (ii)
Demonstrating that the gene of interest is essential for the growth of the bacterium.

[0009] As used herein, the term "substantially simultaneous" refers to a stimulus (eg, electrical pulse) in which two DNA preparations used in transformation are used to introduce DNA into bacterial cells. , Heat shock, etc.) before being mixed with the bacteria or otherwise both being contacted with the bacteria. The 5 'and 3' sequences adjacent to each selectable marker are of sufficient length to allow homologous recombination of the gene of interest or a non-essential gene (control gene) into the respective chromosomal copy, ie at least about 100 each. It is understood to be a nucleotide.

[0010] In one embodiment, the selectable marker is an antibiotic resistance gene and the selective condition is an individual culture containing each antibiotic specific for the same type of resistance gene.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION

All patent applications, patents, and references cited herein are hereby incorporated by reference in their entirety. In case of conflict, the present disclosure will prevail.

[0012] Genetic analysis using GATE can be performed by analyzing 300-500 bp of either (i) the target gene whose phenotype is being investigated, or (ii) a non-essential gene whose sudden displacement phenotype is easily recorded. Performed by co-transformation of pathogenic bacteria using two independent insertable plasmids containing internal fragments. Each insertable plasmid was marked with a different antibiotic resistance determinant, and transformants with gene disruptions at the target locus or non-essential control locus were identified as
Be distinguishable by selection on the appropriate antibiotic medium. Gene disruption
Created by a single recombination event between the plasmid-encoding internal gene fragment and the homologous chromosomal sequence, resulting in the disruption of a truncated copy of the target gene. The determination that a gene is essential / non-essential is made by comparing the number of antibiotic resistant transformants generated by the plasmid insertion event that inactivates the target with the number formed using the control gene. Done.

[0013] The use of non-essential recordable genes as internal standards for plasmid transformation and homologous recombination is a key property of this method. This provides a benchmark for assessing the non-essential properties of a given target gene for bacterial growth. If it is possible to obtain a plasmid insert with the target gene construct at a frequency comparable to that obtained with the control gene, it is inferred that the target gene is not essential for growth. In contrast, if the same transformation mixture is easily accessible for gene disruption at the control locus, but no plasmid insert at the target locus is available, the target is essential for growth. Infer that.

[0014] Suitable candidates for non-essential recordable genes are readily identifiable in most pathogenic bacteria. GATE was prepared in a Petri dish containing casein as a protease substrate, using the non-essential gene speB, which encodes an extracellular cysteine protease that can easily assay the activity of bacterial colonies, as an internal control.
Performed on ptococcus pyogenes. Similarly, to apply GATE to genetic analysis in Staphylococcus aureus, a gene disruption mutation in the structural gene for secretory lipase (geh), whose expression can be conveniently recorded in an indicator medium, was used.

GATE is applicable to any pathogenic bacterium that is capable of homologous recombination and for which a gene transfer method (eg, electrical transformation, natural capacity, or conjugation) can be established. GATE is well suited for the analysis of many genes. Since homologous recombination can proceed efficiently in many bacteria using cloned internal gene fragments as short as 300-500 bp, this approach requires a modest amount of DNA sequence information to create a gene disruption. You only need it. To increase the throughput at which mutations can be made, (i) blunt-ended P
Rapid cloning of CR-producing gene fragments, including (ii) a series of antibiotic resistance markers (such as, for example, erythromycin, kanamycin, or spectinomycin resistance genes) selectable in a wide range of Gram-positive bacteria. Have been constructed (eg, such as Stratagene's pCR-Script ^™ Cam derivative). GATE also includes high throughput methods for genotyping mutant strains. A PCR method using a crude lysate of an individual bacterial colony has been developed, thus making it possible to determine whether a gene disruption phenomenon has occurred at an accurate chromosomal location.

GATE has been successfully used in functional genetic analysis performed on 26 predicted genes in the Gram-positive bacterial pathogen S. Pyogenes. These targets are
Significantly contained genes of well-known function in other bacteria, for example, S. pyogenes homologs of genes involved in essential cellular processes such as transcription, translated DNA replication, and cell wall biosynthesis. Representative genes that are suspected to be non-essential for growth on rich media, including the S. pyogenes homologue of a virulence factor that is well known in other bacteria, have also been investigated. In some cases, functional analyzes were performed on genes that are essential in certain bacteria and not in others, so that the results of experiments in S. pyogenes cannot be predicted in advance.

In a co-transformation experiment, an insertable vector having a target gene and an internal fragment of a non-essential recordable control gene, speB, was introduced into S. pyogenes by electroporation and the target or control locus. Gene disruption events were selected on rich antibiotic media. The effects on gene function in vitro were examined based on colony counts as described above.
Essential and non-essential loci could easily be distinguished: 16 of the 26 predicted genes evaluated clearly encoded functions essential for bacterial growth, while the remaining 10 Coded non-essential features. A further advantage of GATE as an approach to functional analysis is that in addition to classifying genes according to essential or non-essential properties for growth, for example, growth under conditions of oxidative stress, modification of osmotic pressure or pH, or iron Mutant strains can later be assayed for alternative phenotypes in vitro, such as depletion of phenotype. This method can be used to identify functions involved in pathogenesis.

The present invention is described below in working examples which are intended to further describe the invention without limiting the scope of the invention.

[0019]

【Example】

Example 1 To test whether a particular gene is essential or not in S. pyogenes, an insertable plasmid vector containing different antibiotic resistance markers was used (Tao et al., 1992). These plasmids cannot replicate in S. pyogenes because they do not contain a replication origin of Streptococcal origin (Tao et al., 1992). The insertable plasmid used in this experiment consisted of a PCR Script vector (Stratagene) that had been modified by the addition of an antibiotic resistance marker.

The kanamycin and erythromycin resistance genes were ligated to Pfu polymerase (Strat
agene) using the plasmids pSF151 and pVA891.1 (Tao et al., 1992).
Was produced by PCR amplification. PCRS conferring kanamycin resistance
The following oligonucleotide primers were used to construct the script-kan: 5'-GATCCCATGGGCGAACCATT-3 'and 5'-GATCCCATGGAATTCCTCGT-3'. The following oligonucleotide primers were used to construct PCRScript-erms that confer erythromycin resistance: 5'-GATCCCATGGCGAAATGATA-3 'and
5'-GATCCCATGGGGCGCTAGGG-3 '. The PCR-producing antibiotic resistance gene was replaced with PCRScr
It was ligated to the NcoI site of the ipt vector. An internal fragment of 300-500 bp corresponding to each gene of interest was ligated to S. pyogenes genomic DNA template (S. pyogenes
Strain 2F-3MB-1512) using PCR amplification with Pfu polymerase (Stratagene), and gene-specific primers were prepared using DNA sequence data (Strep
Tococcus pyogenes genomic sequence database, University of Oklahoma). The PCR reaction mixture contains 300 ng of genomic DNA template, 100
ng of each primer, 0.2 mM dNTPs, and 2.5 units of Pfu polymerase. The reaction was amplified for 30 cycles (94 ° C., 1 minute; 50 ° C., 1 minute; 72 ° C., 3 minutes). Each gene fragment was ligated to the SmaI site of PCRScript-erm in the presence of SrfI to prevent recircularization of the vector. A non-essential control gene (speB) internal gene fragment was
It was ligated to the Smal site of ipt-kan.

S. pyogenes cotransformation was performed to determine whether a particular gene was essential or not. 10 micrograms of each plasmid were combined, ethanol precipitated and dried under vacuum. Transformation of S. pyogenes was performed by electroporation (McLaughlin and Ferretti, 1992). When preparing electrically competent cells, 100 ml of early log phase culture was grown at 4 ° C.
And collected by centrifugation at 3000 g for 10 min. The supernatant was removed and the cell pellet was resuspended in 5 ml spent media. The cell suspension is heat shocked at 42 ° C. for 9 minutes, after which the cells are harvested by centrifugation and 1% of 15% sterile glycerin.
Washed twice with 0 ml. The final pellet was resuspended in 0.6 ml of 15% glycerin. For transformation, 200 ml of electrically competent cells were added directly to the tube containing the dried plasmid DNA pellet. Transfer the transformation mixture to a cold sterilized electroporation cuvette,
A single pulse was applied (1.75 kV, 25 μF capacitance, 400 W resistor). The cell suspension was placed on ice for 30 minutes, then transferred to 10 ml of THY broth and incubated at 30 ° C./5% CO ₂ for 2-3 hours. Cells were harvested by centrifugation and the transformants were resuspended in 1 μl of THY broth. One half of the cell suspension was placed in 100 μl aliquots on a THY agar plate containing kanamycin (100 μg / ml), and the remaining 0.5 ml was placed in Tyr containing erythromycin (1 μg / ml).
Placed on HY agar plate. Plates were incubated at 37 ° C./5% CO ₂ for 48 hours.

Colonies were enumerated to determine whether the test gene was essential or not. If the test gene is not essential, the number of colonies on the test plate (erythromycin) should be comparable to the control plate (kanamycin). If the test gene is essential, the erythromycin plate should have no colonies, while the control kanamycin plate will have a significant number of colonies showing insertions in non-essential control genes (typically 200-300 ).

[0023] Disruption of speB was visually recorded on agar containing 1% casein. Wild type S. p
The yogenes colony is generally surrounded by a clear halo due to the speB-encoding proteolytic activity, while the speB mutant with a deficiency in protease activity produces a significantly smaller halo. Disruption of the test gene is accomplished by direct plasmid transformation of representative transformant colonies using plasmids and gene-specific primers.
Confirmed molecularly by CR amplification.

Using the method described above, 26 S. pyogenes open reading frames (
ORF) was analyzed to determine whether it was required or not. The results are shown in Table 1 below.

[Table 1]

As can be seen from the data in Table 1, 16 genes were identified as essential and 10 genes were not.

References: McLaughlin, RE and Ferretti, J. (1992): Methods in Molecular Biology
, Vol. 47: Electroporation Protocols for Microorganisms Tao, L., LeBlanc, DJ and Ferretti, J. (1992) Gene 120, 105-110.

[Brief description of the drawings]

FIG. 1 is a schematic showing the use of the GATE assay for target validation in the discovery of novel antibiotic drugs.

FIG. 2 shows Streptococcus py in the GATE assay of the present invention.
FIG. 2 illustrates the advantage of using ogenes group A bacteria.

FIG. 3 is a schematic diagram using a gene disruption strategy using GATE according to the present invention.

FIG. 4 shows the use of the speB locus encoding the Streptococcus erythrogenus B toxin gene used as an internal standard to classify test genes in S. pyogenes.

FIG. 5 shows analysis of selected ORFs in S. pyogenes. Colony counts for each co-transformation of control and test genes are shown.

FIG. 6 shows the use of the GATE method of the present invention in the Gram-negative pathogen Staphylococcus aureus. Successful disruption of the structural gene for the α-hemolysin (hla) gene was revealed by loss of hemolysis on blood agar and confirmed by PCR analysis.

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Claims (1)

[Claims] 1. A step of transforming a bacterial culture using in a substantially simultaneous manner the following: (i) its 5 'and 3' ends flanking a sequence derived from the gene of interest. A first plasmid comprising a recombination cassette comprising a first selectable marker, wherein said recombination cassette is insertable into the genome of said bacterium by homologous recombination; and (ii) part 5 A second plasmid comprising a recombination cassette containing a second selectable marker flanked at the 'and 3' ends by sequences derived from genes known not to be essential for bacterial growth, said recombinant cassette comprising: Are plasmids that can be inserted into the genome of the bacterium by homologous recombination; (b) (i) conditions under which only cells expressing the first selectable marker survive, or (ii) Under conditions where only cells expressing the second selection marker will survive,
Individually culturing the transform cultures produced in step (a); (c) measuring the number of viable cells in cultures (i) and (ii) of step (b); and (d) Comparing the measured values obtained in (c), wherein the culture (i)
Lack of detectable colonies in culture and appearance of colonies in culture (ii)
Indicating that the gene of interest is essential for the growth of the bacterium. A method for determining whether a bacterial gene of interest is essential or non-essential.