1887

Abstract

is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type. In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from . Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type . Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry. Analysis of the putative proteins encoded by these loci, designated and , demonstrated similarity to repeats in the structural toxin protein and the secreted Sel-1 protein from , respectively. and mutants display significantly reduced entry into host cells, compared to wild-type bacteria. The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from and other bacterial pathogens.

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2000-06-01
2020-06-01
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References

  1. Aiba S., Tsunekawa H., Imanaka T.. 1982; New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro. Appl Eviron Microbiol43:289–297
    [Google Scholar]
  2. Alexeyev M. F., Shokolenko I. N.. 1995; Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria. Gene160:59–62[CrossRef]
    [Google Scholar]
  3. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J.. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res25:3389–3402[CrossRef]
    [Google Scholar]
  4. Balasubramanian V., Pavelka M. S. Jr, Bardarov S. S., Martin J., Weisbrod T. R., McAdam R. A., Bloom B. R., Jacobs W. R. Jr. 1996; Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol178:273–279
    [Google Scholar]
  5. Bellinger-Kawahara C., Horwitz M. A.. 1990; Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes. J Exp Med172:1201–1210[CrossRef]
    [Google Scholar]
  6. Blanc-Potard A. B., Figueroa-Bossi N., Bossi L.. 1999; Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci. J Bacteriol181:2938–2941
    [Google Scholar]
  7. Blomfield I. C., Vaughn V., Rest R. F., Eisenstein B. I.. 1991; Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol5:1447–1457[CrossRef]
    [Google Scholar]
  8. Boehm D. F., Welch R. A., Snyder I. S.. 1990; Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect Immun58:1959–1964
    [Google Scholar]
  9. Byrne B., Swanson M. S.. 1998; Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect Immun66:3029–3034
    [Google Scholar]
  10. Chandler F. W., Roth I. L., Callaway C. S., Bump J. L., Thomason B. M., Weaver R. E.. 1980; Flagella on Legionnaires’ disease bacteria. Ann Intern Med93:711–714[CrossRef]
    [Google Scholar]
  11. Cheng X., Cirillo J. D., Duhamel G. E.. 1999; Coiling phagocytosis is the predominant mechanism for uptake of Serpulina pilosicoli by human monocytes. Adv Exp Med Biol473:207–214
    [Google Scholar]
  12. Cirillo J. D., Barletta R. G., Bloom B. R., Jacobs W. R. Jr. 1991; A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J Bacteriol173:7772–7780
    [Google Scholar]
  13. Cirillo J. D., Falkow S., Tompkins L. S.. 1994; Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect Immun62:3254–3261
    [Google Scholar]
  14. Cirillo J. D., Cirillo S. L. G., Yan L., Bermudez L. E., Falkow S., Tompkins L. S.. 1999; Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect Immun67:4427–4434
    [Google Scholar]
  15. Davis G. S., Winn W. C. Jr, Gump D. W., Beaty H. N.. 1983; The kinetics of early inflammatory events during experimental pneumonia due to Legionella pneumophila in guinea pigs. J Infect Dis148:823–825[CrossRef]
    [Google Scholar]
  16. Dower W. J., Miller J. F., Ragsdale C. W.. 1988; High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res16:6127–6145[CrossRef]
    [Google Scholar]
  17. Edelstein P. H.. 1981; Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J Clin Microbiol14:298–303
    [Google Scholar]
  18. Engleberg N. C., Drutz D. J., Eisenstein B. I.. 1984; Cloning and expression of Legionella pneumophila antigens in Escherichia coli. Infect Immun44:222–227
    [Google Scholar]
  19. Fang G., Weiser B., Visosky A., Moran T., Burger H.. 1999; PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA. Nat Med5:239–242[CrossRef]
    [Google Scholar]
  20. Fields B. S., Sanden G. N., Barbaree J. M.. 1989; Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr Microbiol18:131–137[CrossRef]
    [Google Scholar]
  21. Gibson F. C. III, Tzianabos A. O., Rodgers F. G.. 1994; Adherence of Legionella pneumophila to U-937 cells, guinea-pig alveolar macrophages, and MRC-5 cells by a novel, complement-independent binding mechanism. Can J Microbiol40:865–872[CrossRef]
    [Google Scholar]
  22. Glaser P., Elmaoglou-Lazaridou A., Krin E., Ladant D., Barzu O., Danchin A.. 1989; Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. EMBO J8:967–972
    [Google Scholar]
  23. Glavin F. L., Winn W. C. Jr, Craighead J. E.. 1979; Ultrastructure of lung in Legionnaires’ disease. Ann Intern Med90:555–559[CrossRef]
    [Google Scholar]
  24. Goebl M., Yanagida M.. 1991; The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci16:173–177[CrossRef]
    [Google Scholar]
  25. Grant B., Greenwald I.. 1996; The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1 encodes a predicted extracellular protein. Genetics143:237–247
    [Google Scholar]
  26. Grant B., Greenwald I.. 1997; Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans. Development124:637–644
    [Google Scholar]
  27. Harb O. S., Venkataraman C., Haack B. J., Gao L.-Y., Abu Kwaik Y.. 1998; Heterogeneity in the attachment and uptake mechanisms of Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol64:126–132
    [Google Scholar]
  28. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R.. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene77:61–68[CrossRef]
    [Google Scholar]
  29. Horwitz M. A.. 1984; Phagocytosis of the Legionnaires’ disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell36:27–33[CrossRef]
    [Google Scholar]
  30. Horwitz M. A., Silverstein S. C.. 1981; Interaction of the Legionnaires’ disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication. J Exp Med153:398–406[CrossRef]
    [Google Scholar]
  31. Husmann L. K., Johnson W.. 1992; Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors. Infect Immun60:5212–5218
    [Google Scholar]
  32. Jacobs W. R., Barrett J. F., Clark-Curtiss J. E., Curtiss R. III. 1986; In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries. Infect Immun52:101–109
    [Google Scholar]
  33. Jones B. D., Falkow S.. 1994; Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization. Infect Immun62:3745–3752
    [Google Scholar]
  34. Kolter R., Inuzuka M., Helinski D.. 1978; Trans-complementation-dependent replication of a low-molecular-weight origin fragment from plasmid R6K. Cell15:1199–1208[CrossRef]
    [Google Scholar]
  35. Krinos C., High A. S., Rodgers F. G.. 1999; Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor in chick embryos. J Appl Microbiol86:237–244[CrossRef]
    [Google Scholar]
  36. Lally E. T., Kieba I. R., Sato A..8 other authors 1997; RTX toxins recognize a β2 integrin on the surface of human target cells. J Biol Chem272:30463–30469[CrossRef]
    [Google Scholar]
  37. Lüthy R., Xenarios I., Bucher P.. 1994; Improving the sensitivity of the sequence profile method. Protein Sci3:139–146
    [Google Scholar]
  38. McAdam R. A., Weisbrod T. R., Martin J., Scuderi J. D., Brown A. M., Cirillo J. D., Bloom B. R., Jacobs W. R. Jr. 1995; In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infect Immun63:1004–1012
    [Google Scholar]
  39. Marra A., Horwitz M. A., Shuman H. A.. 1990; The HL-60 model for the interaction of human macrophages with the Legionnaires’ disease bacterium. J Immunol144:2738–2744
    [Google Scholar]
  40. Miller J. H.. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  41. Miller V. L., Mekalanos J. J.. 1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholera requires toxR. J Bacteriol170:2575–2583
    [Google Scholar]
  42. Mobley H. L., Jarvis K. G., Elwood J. P., Whittle D. I., Lockatell C. V., Russell R. G., Johnson D. E., Donnenberg M. S., Warren J. W.. 1993; Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of alpha Gal(1-4) beta Gal binding in virulence of a wild-type strain. Mol Microbiol10:143–155[CrossRef]
    [Google Scholar]
  43. Mody C. H., Paine R. III, Shahrabadi M. S., Simon R. H., Pearlman E., Eisenstein B. I., Toews G. B.. 1993; Legionella pneumophila replicates within rat alveolar epithelial cells. J Infect Dis167:1138–1145[CrossRef]
    [Google Scholar]
  44. Moffat J. F., Edelstein P. H., Regula D. P. Jr, Cirillo J. D., Tompkins L. S.. 1994; Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig model. Mol Microbiol12:693–705[CrossRef]
    [Google Scholar]
  45. Nash T. W., Libby D. M., Horwitz M. A.. 1984; Interaction between the Legionnaires’ disease bacterium (Legionella pneumophila) and human alveolar macrophages. J Clin Invest74:771–782[CrossRef]
    [Google Scholar]
  46. Nielsen H., Engelbrecht J., Brunak S., von Heijne G.. 1997; Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineer10:1–6[CrossRef]
    [Google Scholar]
  47. O’Sullivan D. J., Walker S. A., West S. G., Klaenhammer T. R.. 1996; Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression. Biotechnology14:82–87[CrossRef]
    [Google Scholar]
  48. Payne N. R., Horwitz M. A.. 1987; Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med166:1377–1389[CrossRef]
    [Google Scholar]
  49. Rechnitzer C., Blom J.. 1989; Engulfment of the Philadelphia strain of Legionella pneumophila within pseudopod coils in human phagocytes. APMIS97:105–114[CrossRef]
    [Google Scholar]
  50. Rittig M., Häupl T., Burmester G. R.. 1994; Coiling phagocytosis – a way for MHC class I presentation of bacterial antigens?. Int Arch Allergy Appl Immunol103:4–10[CrossRef]
    [Google Scholar]
  51. Rittig M. G., Krause A., Hä upl T..7 other authors 1992; Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi. Infect Immun60:4205–4212
    [Google Scholar]
  52. Rittig M. G., Jacoda J. C., Wilske B., Murgia R., Cinco M., Repp R., Burmester G. R., Krause A.. 1998; Coiling phagocytosis discriminates between different spirochetes and is enhanced by phorbol myristate acetate and granulocyte-macrophage colony-stimulating factor. Infect Immun66:627–635
    [Google Scholar]
  53. Rodgers F. G., Greaves P. W., Macrae A. D., Lewis M. J.. 1980; Electron microscopic evidence of flagella and pili on Legionella pneumophila. J Clin Pathol33:1184–1188[CrossRef]
    [Google Scholar]
  54. Rodgers F. G., Gibson F. C. III.. 1993; Opsonin-independent adherence and intracellular development of Legionella pneumophila within U-937 cells. Can J Microbiol39:718–722[CrossRef]
    [Google Scholar]
  55. Rosenberg M., Court D.. 1979; Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet13:319–353[CrossRef]
    [Google Scholar]
  56. Rowbotham T. J.. 1986; Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci22:678–689
    [Google Scholar]
  57. Roy C. R., Berger K. H., Isberg R. R.. 1998; Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol28:663–674[CrossRef]
    [Google Scholar]
  58. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  59. Sandhu G. S., Aleff R. A., Kline B. C.. 1992; Dual asymmetric PCR: one-step construction of synthetic genes. Biotechniques12:14–16
    [Google Scholar]
  60. Sikorski R. S., Boguski M. S., Goebl M., Hieter P.. 1990; A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell60:307–317[CrossRef]
    [Google Scholar]
  61. Silhavy T. J., Berman M. L., Enquist L. W.. 1984; Experiments With Gene Fusions Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  62. Skaliy P., McEachern H. V.. 1979; Survival of Legionnaires’ disease bacterium in water. Ann Intern Med90:662–663[CrossRef]
    [Google Scholar]
  63. Stibitz S., Black W., Falkow S.. 1986; The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene50:133–140[CrossRef]
    [Google Scholar]
  64. Stone B. J., Abu Kwaik Y.. 1998; Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun66:1768–1775
    [Google Scholar]
  65. Szczepanski A., Fleit H. B.. 1988; Interaction between Borrelia burgdorferi and polymorphonuclear leukocytes: phagocytosis and the induction of the respiratory burst. Ann N Y Acad Sci539:425–428[CrossRef]
    [Google Scholar]
  66. Thompson J. D., Higgins D. G., Gibson T.. 1994; Improved sensitivity of profile searches through the use of sequence weights and gap excision. Comput Appl Biosci10:19–29
    [Google Scholar]
  67. Venkataraman C., Haack B. J., Bondada S., Abu Kwaik Y.. 1997; Identification of a Gal/GalNAc lectin in the protozoan Hartmanella vermiformis as a potential receptor for attachment and invasion by the Legionnaires’ disease bacterium, Legionella pneumophila. J Exp Med186:537–547[CrossRef]
    [Google Scholar]
  68. Visick K. L., Ruby E. G.. 1997; New genetic tools for use in the marine bioluminescent bacterium Vibrio fischeri. In Bioluminescence and Chemiluminescence, Proceedings of the 9th International Symposium pp.119–122Edited by Hastings J. W., Kricka L. J., Stanley P. E.. Chichester: Wiley;
    [Google Scholar]
  69. Wadowsky R. M., Butler L. J., Cook M. K., Verma S. M., Paul M. A., Fields B. S., Keleti G., Sykora J. L., Yee R. B.. 1988; Growth-supporting activity of Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors. Appl Environ Microbiol54:2677–2682
    [Google Scholar]
  70. Wei X., Bauer W. D.. 1998; Starvation-induced changes in motility, chemotaxis, and flagellation of Rhizobium meliloti. Appl Environ Microbiol64:1708–1714
    [Google Scholar]
  71. Welch R. A.. 1991; Pore-forming cytolysin of gram-negative bacteria. Mol Microbiol5:521–528[CrossRef]
    [Google Scholar]
  72. Wiater L. A., Dunn K., Maxfield F. R., Shuman H. A.. 1998; Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect Immun66:4450–4460
    [Google Scholar]
  73. Winn W. C. Jr, Myerowitz R. L.. 1981; The pathology of Legionella pneumonias: a review of 74 cases and the literature. Hum Pathol12:401–422[CrossRef]
    [Google Scholar]
  74. Youderian P., Bouvier S., Susskind M. M.. 1982; Sequence determinants of promoter activity. Cell30:843–853
    [Google Scholar]
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