Overview of Antibacterial Target Selection

Michael J. Pucci1

1 Achillion Pharmaceuticals, Inc., New Haven, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.2
DOI:  10.1002/0471141755.ph13a02s31
Online Posting Date:  January, 2006
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Research in microbial genomics has yielded a vast amount of DNA sequence data and a number of new or improved tools for defining the bacterial genome. These findings have dramatically increased the number of potential antibacterial targets, particularly enzymatic sites, and have greatly enhanced their characterization.Potential targets are prioritized according to necessity for survival (essentiality), spectrum, and selectivity using a combination of bioinformatic analyses and experimental results. Experimental methods such as mutagenesis and conditional expression techniques are used to assess essentiality in vitro or virulence in vivo in animal hosts.

Keywords: bacterial genomics; gene essentiality; mutagenesis; pathogenicity; antibacterials

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Table of Contents

  • Determination of Gene Essentiality
  • In Vivo Essential Genes as Targets for Antibacterials
  • Additonal Considerations
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Akerley, B.J., Rubin, E.J., Camilli, A., Lampe, D.J., Robertson, H.M., and Mekalanos, J.J. 1998. Systematic identification of essential genes by in vitro mariner mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 95:8927‐8932.
   Alksne, L. and Projan, S.J. 2000. Bacterial virulence as a target for antimicrobial chemotherapy. Curr. Opin. Biotechnol. 11:625‐638.
   Arigoni, F., Talabot, F., Peitsch, M., Edgerton, M.D., Meldrum, E., Allet, E., Fish, R., Jamotte, T., Curchod, M.L., and Loferer, H. 1998. A genome‐based approach for the identification of essential bacterial genes. Nat. Biotechnol. 3:483‐489.
   Autret, N., Dubail, I., Trieu‐Cuot, P., Berche, P., and Charbit, A. 2001. Identification of new genes involved in the virulence of Listeria monocytogenes by signature‐tagged transposn mutagenesis. Infect. Immun. 69:2054‐2065.
   Berg, C.M., Berg, D.E., and Groisman, E.A. 1989. Transposable elements and the genetic engineering of bacteria. In Mobile DNA (D.E. Berg and M. Howe, eds.) pp. 879‐925. American Society for Microbiology, Washington, D.C.
   Berlyn, M.K.B. 1998. Linkage map of Escherichia coli K‐12, edition 10: The traditional map. Microbiol. Mol. Biol. Rev. 62:814‐984.
   Betz, S.F., Baxter, S.M., and Fetrow, J.S. Function first: A powerful approach to post‐genomic drug discovery. Drug Discov. Today 7:865‐871.
   Bhavsar, A.P., Zhao, X., and Brown, E.D. 2001. Development and characterization of a xylose‐dependent system for expression of cloned genes in Bacillus subtilis: Conditional complementation of a teichoic acid mutant. Appl. Environ. Microbiol. 67:403‐410.
   Biswas, I., Gruss, A., Ehrlich, S.D., and Maguin, E. 1993. High‐efficiency gene inactivation and replacement system for gram‐positive bacteria. J. Bacteriol. 175:3628‐3635.
   Blattner, F.R., Plunkett, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado‐Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.P., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B., and Shao, Y. 1997. The complete genome of Echerichia coli K‐12. Science 277:1453‐1474.
   Blomfield, I.C., Vaughn, V., Rest, R.F., and Eisenstein, B.I. 1991. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon. Mol. Microbiol. 5:1447‐1457.
   Bruccoleri, R.E., Dougherty, T.J., and Davison, D.B. 1998. Concordance analysis of microbial genomes. Nucl. Acids Res. 26:4482‐4486.
   Chalker, A.F. and Lunsford, R.D. 2002. Rational identification of new antibacterial drug targets that are essential for viability using a genomics‐based approach. Pharmacol. Ther. 95:1‐20.
   Claverys, J.P., Dintilhae, A., Pestova, E.V., Martin, B., and Morrison, D.A. 1995. Construction and evaluation of new drug‐resistance cassettes for gene disruption mutagenesis in Streptococcus pneumoniae, using an ami test platform. Gene 164:123‐128.
   Copass, M., Grandi, G., and Rappuoli, R. 1997. Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker. Infect. Immun. 65:1949‐1952.
   DeVito, J.A., Mills, J.A., Liu, V.G., Agarwal, A., Sizemore, C.F., Yao, Z., Stoughton, D.M., Cappiello, M.G., Barbosa, M.D.F.S., Foster, L.A., and Pompliano, D.L. 2002. An array of target‐specific screening strains for antibacterial discovery. Nat. Biotechnol. 20:478‐483.
   Dougherty, B.A. and Smith, H.O. 1999. Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis. Microbiology 145:401‐409.
   Dougherty, T.J., Barrett, J.F., and Pucci, M.J. 2003. Genomics‐based approaches to novel antimicrobial target discovery. In Microbial Genomics and Drug Discovery (T.J. Dougherty. and S.J. Projan, eds.). Marcel Dekker, New York.
   Edelstein, P.H., Edelstein, M.A., Higa, F., and Falkow, S. 1999. Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model. Proc. Natl. Acad. Sci. U.S.A. 96:8190‐8195.
   Eichenbaum, Z., Federle, M.J., Marra, D., DeVos, W.M., Kuipers, O.P., Kleerebezem, M., and Scott, J.R. 1998. Use of the lactococcal nisA promoter to regulate gene expression in Gram‐positive bacteria: Comparison of induction level and promoter strength. Appl. Environ. Microbiol. 64:2763‐2769.
   Fan, F., Lunsford, R.D., Sylvester, D., Fan, J., Celesnik, H., Iordanescu, S., Rosenberg, M., and McDevitt, D. 2001. Regulated ectopic expression and Allelic replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus. Plasmid 46:71‐75.
   Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.F., Dougherty, B.A., Merrick, J.M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J.D., Scott, J., Shirley, R., Liu, L.‐I., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., McDonald, L.A., Small, K.V., Fraser, C.M., and Smith, H.O. 1995. Whole‐genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496‐512.
   Forsyth, R.A., Hasselbeck, R.J., Ohlsen, K.L., Yamamoto, R.T., Xu, H., Trawic, J.D., Wall, D., Wang, L., Brown‐Driver, V., Froelich, J.M., Kedar, G.C., King, P., McCarthy, M., Malone, C., Misiner, B., Robbins, D., Tan, Z., Zhu, Z.‐Y., Carr, G., Mosca, D.A., Zamudio, C., Foulkes, J.G., and Zyskind, J.W. 2002. A genome‐wide strategy for the identification of essential genes in Staphylococus aureus. Mol. Microbiol. 43:1387‐1400.
   Freiberg, C., Wieland, B., Splatmann, F., Ehlert, K., Brotz, H., and Labischinski, H. 2001. Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria. J. Mol. Microbiol. Biotechnol. 3:483‐489.
   Gerdes, S.Y., Scholle, M.D., Campbell, J.W., Balazzi, G., Ravasz, E., Daugherty, M.D., Somera, A.L., Kyrpides, N.C., Anderson, I., Gelfand, M.S., Bhattacharya, S., Kapatral, V., D'Souza, M., Baev, M.V., Grechkin, Y., Mseeh, F., Fonstein, M.Y., Overbeek, R., Barabasi, A.L., Oltvai, Z.N., and Osterman, A.L. 2003. Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J. Bacteriol. 185:5673‐5684.
   Guo, B.P. and Mekalanos, J.J. 2001. Helicobacter pylori mutagenesis by mariner in vitro transposition. FEMS Immumol. Med. Microbiol. 30:87‐93.
   Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. 1995. Tight regulation, modulation, and high‐level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121‐4130.
   Hamer, I., DeZwaan, T.M., Montenegro‐Chamorro, M.V., Frank, S.A., and Hamer, J.E. 2001. Recent advances in large‐scale transposon mutagenesis. Curr. Opin. Chem. Biol. 5:67‐73.
   Haney, S.A., Alkane, L.E., Dunman, P.M., Murphy, E., and Projan, S.J. 2002. Genomics in anti‐infective drug discovery: Getting to endgame. Curr. Pharm. Des. 8:1099‐1118.
   Hensel, M., Shea, J.E., Gleeson, C., Jones, M.D., Dalton, E., and Holden, D.W. 1995. Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400‐403.
   Herring, C.D. and Blattner, F.R. 2004. Conditional lethal amber mutations in essential Escherichia coli genes. J. Bacteriol. 186:2673‐2681.
   Hutchinson, C.A., Peterson, S.N., Gill, S.R., Cline, R.T., White, O., Fraser, C.M., Smith, H.O. and Venter, J.C. 1999. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286:2165‐2169.
   Jana, M., Luong, T.T., Komatsuzawa, H., Shigeta, M., and Lee, C.Y. 2000. A method for demonstrating gene essentiality in Staphylococcus aureus. Plasmid 44:100‐104.
   Ji, Y., Zhang, B., Van Horn, S.F., Warren, P., Woodnutt, G., Burnham, M.K.R., and Rosenberg, M. 2001. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293:2266‐2269.
   Judson, N. and Mekalanos, J.J. 2000. Transposon‐based approaches to identify essential bacterial genes. Nat. Biotechnol. 18:740‐745.
   Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G., Andersen, K.K., Arnaud, M. et al. 2003. Essential Bacillus subtilis genes. Proc. Natl. Acad. Sci. U.S.A. 100:4678‐4683.
   Lampe, D.J., Churchill, M.E., and Robertson, H.M. 1996. A purified mariner transposon is sufficient to mediate transposition in vitro. EMBO J. 15:5470‐5479.
   Lawrence, J.G. 2003. Gene organization, selection, selfishness, and serendipity. Annu. Rev. Microbiol. 57:419‐440.
   Lederberg, J. and Lederberg, E. 1952. Replica plating and the indirect plating of bacterial mutants. J. Bacteriol. 63:399‐406.
   Lee, M.S., Seok, C., and Morrison, D.A. 1998. Insertion‐duplication mutagenesis in Streptococcus pneumoniae: Targeting fragment length is a critical parameter in use as a random insertion tool. Appl. Environ. Microbiol. 64:4796‐4802.
   Leenhouts, K.J., Kok, J., and Venema, G. 1989. Campbell‐like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. Lactis. Appl. Environ. Microbiol. 55:394‐400.
   Link, A.J., Phillips, D., and Church, G.M., 1997. Methods for generating precise deletions and insertions in the genome of wild‐type Escherichia coli: Application to open reading frame characterization. J. Bacteriol. 179:6228‐6237.
   Lowe, A.M., Beattie, D.T., and Deresiewicz, R.L., 1998. Identification of novel staphylococcal virulence genes by in vivo expression technology. Mol. Microbiol. 27:967‐976.
   Maguin, E., Prevost, H., Ehrlich, S.D., and Gruss, A. 1996. Efficient insertional mutagenesis in lactococci and other gram‐positive bacteria. J. Bacteriol. 178:931‐935.
   Mahan, M.J., Tobias, J.W., Slauch, J.M., Hanna, P.C., Collier, R.J., and Mekalanos, J. 1995. Antibiotic‐based selection for bacterial genes that are specifically induced during infection of a host. Proc. Natl. Acad. Sci. U.S.A. 92:669‐673.
   Marra, A., Asundi, J., Bartilson, M., Lawson, S., Fang, F., Christine, J., Wiesne, A., Brigham, D., Schneider, W.P., and Hromockyj, A.E. 2002. Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis. Infect. Immun. 70:1422‐1433.
   McDevitt, D. and Rosenberg, M. 2001. Exploiting genomics to discover new antibiotics. Trends Microbiol. 9:611‐617.
   Mei, J.M., Nourbakhah, F., Ford, C.W., and Holden, D.W. 1997. Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature‐tagged mutagenesis. Mol. Microbiol. 26:399‐407.
   Murphy, K.C. 1998. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180:2063‐2067.
   Pelicic, V., Reyrat, J.M., and Gicquel, B. 1996. Generation of unmarked directed mutations in mycobacteria, using sucrose counter‐selectable suicide vectors. Mol. Microbiol. 20:919‐925.
   Reyrat, J.‐M., Pelicic, V., Gicquel, B., and Rappuoli, R. 1998. Counterselectable markers: Untapped tools for bacterial genetics and pathogenesis. Infect. Immun. 66:4011‐4017.
   Reznikoff, W.S. 1972. The operon revisited. Annu. Rev. Genet. 6:133‐156.
   Schmid, M.B. 2001. New targets and strategies for identification of novels classes of antibiotics. In Antibiotic Development and Resistance (D. Hughes and D.I. Anderson, eds.) pp. 197‐208. Taylor and Francis, New York.
   Schmid, M.B. 2004. Seeing is believing: The impact of structural genomics on antimicrobial drug discovery. Nature Rev. Microbiol. 2:739‐746.
   Schmid, M.B., Kapur, N., Isaacson, D.R., Lindroos, P., and Sharpe, C. 1989. Genetic analysis of temperature‐sensitive lethal mutants of Salmonella typhimurium. Genetics 123:625‐633.
   Schneider, W.P., Ho, S.K., Christine, J., Yao, M., Marra, A., and Hromockyj, A.E. 2002. Virulence gene identification by differential fluorescence induction analysis of Staphylococcus aureus gene expressin during infection‐simulating culture. Infect. Immun. 70:1326‐1333.
   Shea, J.E., Hensel, M., Gleeson, C., and Holden, D.W. 1996. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A. 93:2593‐2597.
   Slauch, J.M., Mahan, M.J., and Mekalanos, J.J. 1994. In vivo expression technology for selection of bacterial genes specifically induced in host tissues. Methods Enzymol. 235:481‐492.
   Thanassi, J.A., Hartman‐Neumann, S.L., Dougherty, T.J., Dougherty, B.A., and Pucci, M.J. 2002. Identification of 113 conserved essential genes using a high‐throughput gene disruption system in Streptococcus pneumoniae. Nucl. Acids Res. 30:3152‐3162.
   Urban, A., Neukirchen, S., and Jaeger, K.E. 1997. A rapid and efficient method for site‐directed mutagenesis using one‐step overlap extension PCR. Nucl. Acids Res. 25:2227‐2228.
   Vagner, V.E., Dervyn, E., and Dusko Ehrlich, S. 1998. A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144:3097‐3104.
   Valdivia, R.H. and Falkow, S. 1996. Bacterial genetics by flow cytometry: Rapid isolation of Salmonella typhimurium acid‐inducible promoters by differential fluorescence induction. Mol. Microbiol. 22:367‐378.
   Valdivia, R.H. and Falkow, S. 1997. Fluorescence‐based isolation of bacterial genes expressed within host cells. Science 277:2007‐2011.
   Vilar, J.M.G., Guet, C.C., and Liebler, S. 2003. Modeling network dynamics: The lac operon, a case study. J. Cell Biol. 161:471‐476.
   Walter, J., Heng, N.C., Hammes, W.F., Leach, D.M., Tannock, G.W., and Hertel, C. 2003. Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Appl. Environ. Microbiol. 69:2044‐2051.
   Winans, S.C., Elledge, S.J., Kreuger, J.H., and Walker, G.C. 1985. Site‐directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J. Bacteriol. 161:1219‐1221.
   Wong, S.M. and Mekalanos, J.J. 2000. Genetic footprinting with mariner‐based transposition in Pseudomonas aeruginosa. Proc. Natl.Acad. Sci. U.S.A. 97:10191‐10196.
   Wu, Y., Lee, S.W., Hillman, J.D., and Progulske‐Fox, A. 2002. Identification and testing of Porphyromonas gingivalis virulence genes with a pPGIVET system. Infect. Immun. 70:928‐937.
   Yansura, D.G. and Henner, D.J. 1984. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci.U.S.A. 81:439‐445.
   Yin, D. and Ji, Y. 2002. Genomic analysis using conditional phenotypes generated by antisense RNA. Curr. Opin. Microbiol. 5:330‐333.
   Yu, D., Ellis, H.M., Lee, E.C., Jenkins, N.A., Copeland, N.G., and Court, D.L. 2000. An efficient recombination system for chromosome engineering Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97:5978‐5983.
   Zhang, L., Fan, F., Palmer, L.M., Lonetto, M.A., Petit, C., Voelker, L.L., St. John, A., Bankosky, B., Rosenberg, M., and McDevitt, D. 2000. Regulated expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action. Gene 255:297‐305.
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