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Philosophical Transactions of the Royal Society B: Biological Sciences logoLink to Philosophical Transactions of the Royal Society B: Biological Sciences
. 2000 May 29;355(1397):613–622. doi: 10.1098/rstb.2000.0602

Identification and analysis of bacterial virulence genes in vivo.

K E Unsworth 1, D W Holden 1
PMCID: PMC1692767  PMID: 10874734

Abstract

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

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Selected References

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  1. Benjamin W. H., Jr, Hall P., Roberts S. J., Briles D. E. The primary effect of the Ity locus is on the rate of growth of Salmonella typhimurium that are relatively protected from killing. J Immunol. 1990 Apr 15;144(8):3143–3151. [PubMed] [Google Scholar]
  2. Beuzón C. R., Banks G., Deiwick J., Hensel M., Holden D. W. pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium. Mol Microbiol. 1999 Aug;33(4):806–816. doi: 10.1046/j.1365-2958.1999.01527.x. [DOI] [PubMed] [Google Scholar]
  3. Blocker A., Gounon P., Larquet E., Niebuhr K., Cabiaux V., Parsot C., Sansonetti P. The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J Cell Biol. 1999 Nov 1;147(3):683–693. doi: 10.1083/jcb.147.3.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowe F., Lipps C. J., Tsolis R. M., Groisman E., Heffron F., Kusters J. G. At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice. Infect Immun. 1998 Jul;66(7):3372–3377. doi: 10.1128/iai.66.7.3372-3377.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown J. S., Holden D. W. Insertional mutagenesis of pathogenic fungi. Curr Opin Microbiol. 1998 Aug;1(4):390–394. doi: 10.1016/s1369-5274(98)80054-4. [DOI] [PubMed] [Google Scholar]
  6. Chiang S. L., Mekalanos J. J. Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol Microbiol. 1998 Feb;27(4):797–805. doi: 10.1046/j.1365-2958.1998.00726.x. [DOI] [PubMed] [Google Scholar]
  7. Cirillo D. M., Valdivia R. H., Monack D. M., Falkow S. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol. 1998 Oct;30(1):175–188. doi: 10.1046/j.1365-2958.1998.01048.x. [DOI] [PubMed] [Google Scholar]
  8. Cormack B. P., Ghori N., Falkow S. An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science. 1999 Jul 23;285(5427):578–582. doi: 10.1126/science.285.5427.578. [DOI] [PubMed] [Google Scholar]
  9. Coulter S. N., Schwan W. R., Ng E. Y., Langhorne M. H., Ritchie H. D., Westbrock-Wadman S., Hufnagle W. O., Folger K. R., Bayer A. S., Stover C. K. Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol. 1998 Oct;30(2):393–404. doi: 10.1046/j.1365-2958.1998.01075.x. [DOI] [PubMed] [Google Scholar]
  10. Darwin A. J., Miller V. L. Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis. Mol Microbiol. 1999 Apr;32(1):51–62. doi: 10.1046/j.1365-2958.1999.01324.x. [DOI] [PubMed] [Google Scholar]
  11. Deiwick J., Nikolaus T., Erdogan S., Hensel M. Environmental regulation of Salmonella pathogenicity island 2 gene expression. Mol Microbiol. 1999 Mar;31(6):1759–1773. doi: 10.1046/j.1365-2958.1999.01312.x. [DOI] [PubMed] [Google Scholar]
  12. Dussurget O., Smith I. Interdependence of mycobacterial iron regulation, oxidative-stress response and isoniazid resistance. Trends Microbiol. 1998 Sep;6(9):354–358. doi: 10.1016/s0966-842x(98)01307-9. [DOI] [PubMed] [Google Scholar]
  13. Ebel F., Podzadel T., Rohde M., Kresse A. U., Krämer S., Deibel C., Guzmán C. A., Chakraborty T. Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages. Mol Microbiol. 1998 Oct;30(1):147–161. doi: 10.1046/j.1365-2958.1998.01046.x. [DOI] [PubMed] [Google Scholar]
  14. Edelstein P. H., Edelstein M. A., Higa F., Falkow S. 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. 1999 Jul 6;96(14):8190–8195. doi: 10.1073/pnas.96.14.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frankel G., Phillips A. D., Rosenshine I., Dougan G., Kaper J. B., Knutton S. Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol. 1998 Dec;30(5):911–921. doi: 10.1046/j.1365-2958.1998.01144.x. [DOI] [PubMed] [Google Scholar]
  16. Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. García Véscovi E., Soncini F. C., Groisman E. A. Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Cell. 1996 Jan 12;84(1):165–174. doi: 10.1016/s0092-8674(00)81003-x. [DOI] [PubMed] [Google Scholar]
  18. Greene C., McDevitt D., Francois P., Vaudaux P. E., Lew D. P., Foster T. J. Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol Microbiol. 1995 Sep;17(6):1143–1152. doi: 10.1111/j.1365-2958.1995.mmi_17061143.x. [DOI] [PubMed] [Google Scholar]
  19. Groisman E. A., Ochman H. How Salmonella became a pathogen. Trends Microbiol. 1997 Sep;5(9):343–349. doi: 10.1016/S0966-842X(97)01099-8. [DOI] [PubMed] [Google Scholar]
  20. Gulig P. A., Doyle T. J. The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice. Infect Immun. 1993 Feb;61(2):504–511. doi: 10.1128/iai.61.2.504-511.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hensel M., Hinsley A. P., Nikolaus T., Sawers G., Berks B. C. The genetic basis of tetrathionate respiration in Salmonella typhimurium. Mol Microbiol. 1999 Apr;32(2):275–287. doi: 10.1046/j.1365-2958.1999.01345.x. [DOI] [PubMed] [Google Scholar]
  22. Hensel M., Shea J. E., Bäumler A. J., Gleeson C., Blattner F., Holden D. W. Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12. J Bacteriol. 1997 Feb;179(4):1105–1111. doi: 10.1128/jb.179.4.1105-1111.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hensel M., Shea J. E., Gleeson C., Jones M. D., Dalton E., Holden D. W. Simultaneous identification of bacterial virulence genes by negative selection. Science. 1995 Jul 21;269(5222):400–403. doi: 10.1126/science.7618105. [DOI] [PubMed] [Google Scholar]
  24. Hensel M., Shea J. E., Raupach B., Monack D., Falkow S., Gleeson C., Kubo T., Holden D. W. Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2. Mol Microbiol. 1997 Apr;24(1):155–167. doi: 10.1046/j.1365-2958.1997.3271699.x. [DOI] [PubMed] [Google Scholar]
  25. Hensel M., Shea J. E., Waterman S. R., Mundy R., Nikolaus T., Banks G., Vazquez-Torres A., Gleeson C., Fang F. C., Holden D. W. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol Microbiol. 1998 Oct;30(1):163–174. doi: 10.1046/j.1365-2958.1998.01047.x. [DOI] [PubMed] [Google Scholar]
  26. Hueck C. J. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev. 1998 Jun;62(2):379–433. doi: 10.1128/mmbr.62.2.379-433.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kaniga K., Bossio J. C., Galán J. E. The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol Microbiol. 1994 Aug;13(4):555–568. doi: 10.1111/j.1365-2958.1994.tb00450.x. [DOI] [PubMed] [Google Scholar]
  28. Knutton S., Rosenshine I., Pallen M. J., Nisan I., Neves B. C., Bain C., Wolff C., Dougan G., Frankel G. A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 1998 Apr 15;17(8):2166–2176. doi: 10.1093/emboj/17.8.2166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lowe A. M., Beattie D. T., Deresiewicz R. L. Identification of novel staphylococcal virulence genes by in vivo expression technology. Mol Microbiol. 1998 Mar;27(5):967–976. doi: 10.1046/j.1365-2958.1998.00741.x. [DOI] [PubMed] [Google Scholar]
  30. Lowy F. D. Staphylococcus aureus infections. N Engl J Med. 1998 Aug 20;339(8):520–532. doi: 10.1056/NEJM199808203390806. [DOI] [PubMed] [Google Scholar]
  31. Mahan M. J., Slauch J. M., Mekalanos J. J. Selection of bacterial virulence genes that are specifically induced in host tissues. Science. 1993 Jan 29;259(5095):686–688. doi: 10.1126/science.8430319. [DOI] [PubMed] [Google Scholar]
  32. Mei J. M., Nourbakhsh F., Ford C. W., Holden D. W. Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol. 1997 Oct;26(2):399–407. doi: 10.1046/j.1365-2958.1997.5911966.x. [DOI] [PubMed] [Google Scholar]
  33. Miller I., Maskell D., Hormaeche C., Johnson K., Pickard D., Dougan G. Isolation of orally attenuated Salmonella typhimurium following TnphoA mutagenesis. Infect Immun. 1989 Sep;57(9):2758–2763. doi: 10.1128/iai.57.9.2758-2763.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Neyt C., Cornelis G. R. Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG. Mol Microbiol. 1999 Sep;33(5):971–981. doi: 10.1046/j.1365-2958.1999.01537.x. [DOI] [PubMed] [Google Scholar]
  35. Ochman H., Groisman E. A. Distribution of pathogenicity islands in Salmonella spp. Infect Immun. 1996 Dec;64(12):5410–5412. doi: 10.1128/iai.64.12.5410-5412.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ochman H., Soncini F. C., Solomon F., Groisman E. A. Identification of a pathogenicity island required for Salmonella survival in host cells. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7800–7804. doi: 10.1073/pnas.93.15.7800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Polissi A., Pontiggia A., Feger G., Altieri M., Mottl H., Ferrari L., Simon D. Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect Immun. 1998 Dec;66(12):5620–5629. doi: 10.1128/iai.66.12.5620-5629.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rathman M., Barker L. P., Falkow S. The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry. Infect Immun. 1997 Apr;65(4):1475–1485. doi: 10.1128/iai.65.4.1475-1485.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rathman M., Sjaastad M. D., Falkow S. Acidification of phagosomes containing Salmonella typhimurium in murine macrophages. Infect Immun. 1996 Jul;64(7):2765–2773. doi: 10.1128/iai.64.7.2765-2773.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Richter-Dahlfors A., Buchan A. M., Finlay B. B. Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo. J Exp Med. 1997 Aug 18;186(4):569–580. doi: 10.1084/jem.186.4.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schwan W. R., Coulter S. N., Ng E. Y., Langhorne M. H., Ritchie H. D., Brody L. L., Westbrock-Wadman S., Bayer A. S., Folger K. R., Stover C. K. Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models. Infect Immun. 1998 Feb;66(2):567–572. doi: 10.1128/iai.66.2.567-572.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shea J. E., Beuzon C. R., Gleeson C., Mundy R., Holden D. W. Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion system on bacterial growth in the mouse. Infect Immun. 1999 Jan;67(1):213–219. doi: 10.1128/iai.67.1.213-219.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shea J. E., Hensel M., Gleeson C., Holden D. W. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2593–2597. doi: 10.1073/pnas.93.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Smith J. M., Tang C. M., Van Noorden S., Holden D. W. Virulence of Aspergillus fumigatus double mutants lacking restriction and an alkaline protease in a low-dose model of invasive pulmonary aspergillosis. Infect Immun. 1994 Dec;62(12):5247–5254. doi: 10.1128/iai.62.12.5247-5254.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tang C., Holden D. Pathogen virulence genes--implications for vaccines and drug therapy. Br Med Bull. 1999;55(2):387–400. doi: 10.1258/0007142991902448. [DOI] [PubMed] [Google Scholar]
  46. Tran Van Nhieu G., Caron E., Hall A., Sansonetti P. J. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. EMBO J. 1999 Jun 15;18(12):3249–3262. doi: 10.1093/emboj/18.12.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tsolis R. M., Adams L. G., Ficht T. A., Bäumler A. J. Contribution of Salmonella typhimurium virulence factors to diarrheal disease in calves. Infect Immun. 1999 Sep;67(9):4879–4885. doi: 10.1128/iai.67.9.4879-4885.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Uchiya K., Barbieri M. A., Funato K., Shah A. H., Stahl P. D., Groisman E. A. A Salmonella virulence protein that inhibits cellular trafficking. EMBO J. 1999 Jul 15;18(14):3924–3933. doi: 10.1093/emboj/18.14.3924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Valdivia R. H., Falkow S. Fluorescence-based isolation of bacterial genes expressed within host cells. Science. 1997 Sep 26;277(5334):2007–2011. doi: 10.1126/science.277.5334.2007. [DOI] [PubMed] [Google Scholar]
  50. Woestyn S., Allaoui A., Wattiau P., Cornelis G. R. YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol. 1994 Mar;176(6):1561–1569. doi: 10.1128/jb.176.6.1561-1569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhao H., Li X., Johnson D. E., Mobley H. L. Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection. Microbiology. 1999 Jan;145(Pt 1):185–195. doi: 10.1099/13500872-145-1-185. [DOI] [PubMed] [Google Scholar]
  52. de Lorenzo V., Timmis K. N. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol. 1994;235:386–405. doi: 10.1016/0076-6879(94)35157-0. [DOI] [PubMed] [Google Scholar]

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