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WO1999060111A1 - Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens - Google Patents

Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens Download PDF

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Publication number
WO1999060111A1
WO1999060111A1 PCT/US1999/010919 US9910919W WO9960111A1 WO 1999060111 A1 WO1999060111 A1 WO 1999060111A1 US 9910919 W US9910919 W US 9910919W WO 9960111 A1 WO9960111 A1 WO 9960111A1
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Prior art keywords
gene
nonessential
essential
gate
bacteria
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PCT/US1999/010919
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English (en)
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WO1999060111A9 (fr
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Judith Healy
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Anadys Pharmaceuticals, Inc.
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Publication date
Application filed by Anadys Pharmaceuticals, Inc. filed Critical Anadys Pharmaceuticals, Inc.
Priority to AU46720/99A priority Critical patent/AU4672099A/en
Priority to CA002328497A priority patent/CA2328497A1/fr
Priority to JP2000549719A priority patent/JP2002515242A/ja
Priority to EP99930113A priority patent/EP1078049A1/fr
Publication of WO1999060111A1 publication Critical patent/WO1999060111A1/fr
Publication of WO1999060111A9 publication Critical patent/WO1999060111A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention is directed to methods for gene analysis in pathogenic bacteria
  • the technology of the present invention allows for the functional classification of genes
  • GATE Genetics-Assisted Targeted Evaluation
  • GATE is a robust procedure for gene knock-out analysis
  • GATE GATE-specific antigen-binding protein kinase inhibitors
  • a further attribute of GATE is its potential for functional genetic analysis in a broad range of pathogenic bacteria, including both Gram-positive and Gram-negative organisms.
  • Streptococcus pyogenes and Staphylococcus aureus has been demonstrated.
  • GATE holds several advantages over prior art methods for determining whether or not a gene is likely to be essential for bacterial growth. Typically, simple failure to obtain transformants with a plasmid construct designed to produce either gene disruption or gene replacement mutations at a locus of interest is interpreted as evidence that the gene is essential for bacterial growth. However, failure to obtain transformants is not in itself sufficient for assignment of gene function, due to lack of adequate positive controls for successful transformation and plasmid integration.
  • the present invention provides methods for determining the essentiality or non- essentiality of a bacterial gene of interest, which are carried out by the stpes of:
  • recombination cassette is capable of being integrated into the genome of the bacteria by homologous recombination
  • step (b) individually culturing the transformed culture produced in step (a) under selective conditions in which (i) only cells expressing the first selectable marker will survive or (ii) only cells
  • step (c) measuring the number of surviving cells in cultures (i) and (ii) of step (b); and (d) comparing the measurements made in step (c), wherein the lack of detectable colonies in culture (i) and the appearance of colonies in culture (ii) indicates that the gene of interest
  • Substantially simultaneous indicates that the two DNA preparations used in the transformation are mixed or are otherwise both contacted with the bacterial cells prior to the stimulus (e.g., electric pulse, heat shock, and the like) used to introduce the DNA into the bacterial cells. It will be understood that the 5' and 3' sequences that flank each selectable marker are of sufficient length, i.e., at least about 100 nt each, to allow for homologous recombination into the chromosomal copy of the gene of interest or the non-essential (control gene), respectively.
  • the selectable markers are antibiotic resistance genes
  • the selective conditions are individual cultures comprising each antibiotic specified by the cognate
  • Figure 1 is a schematic diagram showing the use of the GATE assay for target
  • Figure 2 depicts the advantages of using Streptococcus pyogenes group A bacteria in
  • FIG. 3 is a schematic diagram showing the gene disruption strategy using GATE
  • Figure 4 shows the use of the speB locus encoding the Streptococcus erythrogenus toxin B gene used as an internal standard for classifying test genes in S. pyogenes.
  • Figure 5 shows the analysis of selected ORFs in S. pyogenes. Colony counts for each
  • Figure 6 shows the use of the GATE technology of the present invention in the Gram- positive pathogen Staphylococcus aureus.
  • Successful disruption of the structural gene for - hemolysin (hla) gene was evidenced by the loss of hemolysis on blood agar and was confirmed by
  • Gene analysis using GATE is performed by cotransformation of pathogenic bacteria with two independent integrative plasmids which contain a 300-500 bp internal fragment of either (i) the target gene, whose mutant phenotype is being investigated, or (ii) a nonessential gene for which there is an easily scored mutant phenotype.
  • Each integrative plasmid is marked with a different antibiotic resistance determinant so that transformants bearing gene disruptions at either the target locus or at the nonessential control locus can be distinguished by selection on appropriate antibiotic media.
  • Gene disruptions are created by a single recombination event between the plasmid-encoded internal gene fragment and the homologous chromosomal sequence, resulting in duplication of truncated copies of the target gene. Determination of gene essentiality/nonessentiality is made by comparing the number of antibiotic-resistant transformants generated by plasmid integration events which inactivate the target with those formed using the control gene.
  • a nonessential scorable gene as an internal standard for plasmid transformation and homologous recombination is a key feature of this method. This provides a benchmark against which to assess the dispensability of a given target gene for bacterial growth. If it is possible to obtain plasmid integration with a target gene construct at a frequency comparable to that obtained with the control gene, it is inferred that the target gene is nonessential for growth. If, on the other hand, it is not possible to obtain plasmid integration at the target locus when in the same transformation mixture gene disruption at the control locus is readily obtained, it is inferred that the target is essential for growth.
  • Suitable candidates for nonessential scorable genes are easily identifiable in most pathogenic bacteria.
  • GATE was performed in Streptococcus pyogenes using as an internal control a nonessential gene, speB, encoding an extracellular cysteine protease whose activity in bacterial colonies is readily assayed on Petri dishes containing casein as a protease substrate.
  • speB nonessential gene
  • a gene disruption mutation in the structural gene for a secreted lipase (gen) whose expression can be scored conveniently on an indicator medium was used.
  • GATE can be applied to any pathogenic bacterium that undergoes homologous recombination and for which gene delivery procedures (e.g., electrotransformation, natural competence, or conjugation) can be established.
  • GATE is well-suited for analysis of large numbers of genes. The approach requires only a modest amount of DNA sequence information in order to create a gene disruption, because homologous recombination can proceed efficiently in many bacteria using cloned internal gene fragments as short as 300-500 bp.
  • a series of integrative vectors such as, e.g.
  • derivatives of Stratagene's pCR-ScriptTMCam which (i) permit rapid cloning of blunt-end, PCR-generated gene fragments, and (ii) contain antibiotic-resistance markers selectable in a wide variety of Gram-positive bacteria (such as, e.g., erythromycin, kanamycin, or spectinomycin resistance genes) has been constructed.
  • GATE also incorporates high through-put methods for verifying the genotypes of mutant strains. PCR procedures have been developed that utilize crude lysates of individual bacterial colonies and thus permit the determination as to whether or not a gene disruption event occurred at the correct chromosomal location.
  • GATE has been successfully implemented in a functional genetic analysis performed on 26 predicted genes in the Gram-positive bacterial pathogen S. pyrogenes. These targets predominantly included S. pyrogenes homologs of genes of known function in other bacteria, e.g., genes involved in essential cellular processes such as transcription, translation, DNA replication, and cell wall biogenesis. Representative genes which are suspected to be nonessential for growth in rich medium, including S. pyrogenes homologs of virulence factors known in other bacteria, have also been examined. In a few cases, functional analysis was performed on genes which were essential in certain bacteria and nonessential in others, such that the outcome of the experiment in S. pyogenes could not be predicted in advance.
  • integrative vectors bearing an internal fragment of the target gene and the speB nonessential scorable control gene were introduced into S. pyogenes by electroporation and gene disruption events at target or control loci were selected for on rich antibiotic media. Inferences as to gene function in vitro were made based on colony counts as described above. It was possible to readily distinguish essential and nonessential loci: 16 of the 26 predicted genes that were evaluated clearly encoded functions essential for bacterial growth, while the remaining 10 encoded functions that were nonessential.
  • GATE as an approach to functional analysis is the fact that, in addition to classifying genes according to their essential or nonessential character for growth, mutant strains can be assayed later for alternative phenotypes in vitro such as, e.g. , growth under conditions of oxidative stress, altered osmolarity or pH, or iron deprivation. This procedure can be used to identify functions involved in pathogenicity.
  • EXAMPLE I In order to test whether a particular gene is essential or nonessential in S. pyogenes, integrative plasmid vectors were used that contain different antibiotic resistance markers (Tao, et al., 1992). These plasmids cannot replicate in S. pyogenes because they do not contain a streptococcal origin of replication (Tao, et al., 1992). Integrative plasmids used in this study consisted of the PCR Script vector (Stratagene) which had been modified by the addition of antibiotic resistance markers.
  • kanamycin and erythromycin resistance genes were generated by PCR amplification of plasmids pSF151 and pVA891.1, respectively (Tao, et al., 1992) using Pfu polymerase (Stratagene).
  • Pfu polymerase Pfu polymerase
  • PCRScript-erm conferring erythromycin-resistance
  • the oligonucleotide primers were: 5 '-GATCCCATGGCGAAATGATA-3 ' and 5 ' -GATCCCATGGGGCGCTAGGG-3 ' .
  • PCR-generated antibiotic resistance genes were ligated into the Nco I site of the PCRScript vector.
  • a 300-500 bp internal fragment corresponding to each gene of interest was generated by PCR amplification with Pfu polymerase (Stratagene) using S. pyogenes genomic DNA template (S.
  • the PCR reaction mixtures contained 300 ng of genomic DNA template, 100 ng of each primer, 0.2mM dNTPs, and 2.5 units of Pfu polymerase. The reactions were amplified for 30 cycles (94°C, 1 ' ; 50°C, 1'; 72°C, 3'). Each gene fragment was ligated into the Sma I site of PCRScript-erm in the presence of Srf ⁇ to prevent vector recircularization. An internal gene fragment of the nonessential control gene (speB) was ligated into the Sma I site of PCRScript-kan
  • Cotransformation of S. pyogenes was performed to determine whether a particular gene is essential or nonessential. Ten micrograms of each plasmid were combined, ethanol precipitated, and dried under vacuum. Transformation of S. pyogenes was by electroporation (McLaughlin and Ferretti, 1992). When preparing electrocompetent cells, 100 ml of early log phase cultures were harvested by centrifugation at 3000 g for 10 minutes at 4°C. The supernatant was saved and cell pellets were resuspended in 5 ml of the spent media.
  • the cell suspension was subjected to heat shock at 42° C for 9 minutes, after which cells were collected by centrifugation and washed 2X in 10 ml of 15 % sterile glycerol. Final pellets were resuspended in 0.6 ml of 15 % glycerol.
  • 200 ml of electrocompetent cells were added directly to the tube containing the dried plasmid DNA pellet.
  • the transformation mixture was transferred to a chilled sterile electroporation cuvet and a single pulse was applied (1.75kV, 25 ⁇ F capacitance, 400W resistance). Cell suspensions were placed on ice for 30 minutes, then transferred to 10 ml THY broth and incubated at 37° C/5 %CO 2 for 2-3 hours.
  • Colonies were counted to determine whether the test gene is essential or nonessential.
  • test gene is nonessential, the number of colonies on the test plates (erythromycin) should be comparable to the control plates (kanamycin). If the test gene is essential, the erythromycin plates should have no colonies, while the control kanamycin plates should contain significant numbers of colonies (typically 200-300) indicating integration into the nonessential control gene.

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  • Chemical & Material Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne un procédé baptisé GATE (Genetics-Assisted Target Evaluation, ou 'estimation de cibles assistée génétiquement'), destiné à l'analyse des gènes dans des bactéries pathogènes. Le procédé GATE permet de classer les gènes de manière fonctionnelle, selon leur caractère essentiel ou non essentiel en rapport avec la croissance bactérienne ou la pathogénie. La technique de l'invention s'appuie sur un gène non essentiel pouvant être soumis à la notation, utilisé en tant que standard interne par rapport auquel on évalue l'aptitude à la distribution d'un gène cible donné par disruption génique au moyen de l'intégration de type Campbell. Une analyse fonctionnelle a été effectuée sur 26 gènes du pathogène gram positif Streptococcus pyogenes. L'application de ce procédé a permis d'identifier clairement les loci essentiels et non essentiels: seize sur les vingt-six gènes évalués chez S. pyogenes codaient clairement les fonctions essentielles pour la croissance bactérienne, les dix autres fonctions codées n'étant pas essentielles. Le succès du procédé GATE a aussi été démontré vis-à-vis de Staphylococcus aureus.
PCT/US1999/010919 1998-05-15 1999-05-13 Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens WO1999060111A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU46720/99A AU4672099A (en) 1998-05-15 1999-05-13 Genetics-assisted target evaluation in antibacterial drug discovery
CA002328497A CA2328497A1 (fr) 1998-05-15 1999-05-13 Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens
JP2000549719A JP2002515242A (ja) 1998-05-15 1999-05-13 抗生物質薬剤の発見における遺伝学的補助の標的評価
EP99930113A EP1078049A1 (fr) 1998-05-15 1999-05-13 Estimation de cibles assistee genetiquement et utilisee dans la conception de medicaments antibacteriens

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8559398P 1998-05-15 1998-05-15
US60/085,593 1998-05-15

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WO1999060111A1 true WO1999060111A1 (fr) 1999-11-25
WO1999060111A9 WO1999060111A9 (fr) 2000-02-24

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020161A1 (fr) * 1996-11-06 1998-05-14 Smithkline Beecham Corporation Procedes d'identification de genes indispensables a la croissance d'un organisme

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020161A1 (fr) * 1996-11-06 1998-05-14 Smithkline Beecham Corporation Procedes d'identification de genes indispensables a la croissance d'un organisme

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARIGONI ET AL.: "A GENOME-BASED APPROACH FOR THE IDENTIFICATION OF ESSENTIAL BACTERIAL GENES", NAT.BIOTECHNOL., vol. 16, no. 9, September 1998 (1998-09-01), pages 851 - 856, XP002119419 *
BALTZ ET AL: "DNA sequence sampling of the Streptococcus pneumoniae genome to identify novel targets for antibiotic development", MICROBIAL DRUG RESISTANCE, vol. 4, no. 1, 21 March 1998 (1998-03-21), pages 1 - 9 9, XP002112153, ISSN: 1076-6294 *
GUEROUT-FLEURY ET AL.: "ANTIBIOTIC-RESISTANCE CASSETTES FOR BACILLUS SUBTILIS", GENE, vol. 167, 1995, pages 335 - 336, XP002119416 *
HAMILTON ET AL.: "NEW METHOD FOR GENERATING DELETIONS AND GENE REPLACEMENT IN ESCHERICHIA COLI", JOURNAL OF BACTERIOLOGY, vol. 171, no. 9, 1989, pages 4617 - 4622, XP002119417 *
MADIGAN ET AL.: "BIOLOGY OF MICROORGANISM", 1997, BROCK, USA, XP002119420 *
TRIAS AND GORDON: "INNOVATIVE APPROACHES TO NOVEL ANTIBACTERIAL DRUG DISCOVERY", CURRENT OPINION IN BIOTECHNOLOGY, vol. 8, 1997, pages 757 - 762, XP002119418 *

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AU4672099A (en) 1999-12-06
EP1078049A1 (fr) 2001-02-28
WO1999060111A9 (fr) 2000-02-24
JP2002515242A (ja) 2002-05-28
CA2328497A1 (fr) 1999-11-25

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