Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Nov;65(11):4778–4783. doi: 10.1128/iai.65.11.4778-4783.1997

Study of the role of the htrB gene in Salmonella typhimurium virulence.

B D Jones 1, W A Nichols 1, B W Gibson 1, M G Sunshine 1, M A Apicella 1
PMCID: PMC175685  PMID: 9353064

Abstract

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.

Full Text

The Full Text of this article is available as a PDF (155.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alpuche Aranda C. M., Swanson J. A., Loomis W. P., Miller S. I. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10079–10083. doi: 10.1073/pnas.89.21.10079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bajaj V., Hwang C., Lee C. A. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol. 1995 Nov;18(4):715–727. doi: 10.1111/j.1365-2958.1995.mmi_18040715.x. [DOI] [PubMed] [Google Scholar]
  3. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bogdan C., Vodovotz Y., Nathan C. Macrophage deactivation by interleukin 10. J Exp Med. 1991 Dec 1;174(6):1549–1555. doi: 10.1084/jem.174.6.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buchmeier N. A., Heffron F. Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium. Infect Immun. 1991 Jul;59(7):2232–2238. doi: 10.1128/iai.59.7.2232-2238.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. D'Andrea A., Aste-Amezaga M., Valiante N. M., Ma X., Kubin M., Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993 Sep 1;178(3):1041–1048. doi: 10.1084/jem.178.3.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dlabac V. The sensitivity of smooth and rough mutants of Salmonella typhimurium to bactericidal and bacteriolytic action of serum, lysozyme and to phagocytosis. Folia Microbiol (Praha) 1968;13(5):439–449. doi: 10.1007/BF02869196. [DOI] [PubMed] [Google Scholar]
  8. Espevik T., Nissen-Meyer J. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods. 1986 Dec 4;95(1):99–105. doi: 10.1016/0022-1759(86)90322-4. [DOI] [PubMed] [Google Scholar]
  9. Fields P. I., Groisman E. A., Heffron F. A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science. 1989 Feb 24;243(4894 Pt 1):1059–1062. doi: 10.1126/science.2646710. [DOI] [PubMed] [Google Scholar]
  10. Fields P. I., Swanson R. V., Haidaris C. G., Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5189–5193. doi: 10.1073/pnas.83.14.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freudenberg M. A., Keppler D., Galanos C. Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin. Infect Immun. 1986 Mar;51(3):891–895. doi: 10.1128/iai.51.3.891-895.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Freudenberg M. A., Ness T., Kumazawa Y., Galanos C. Die Bedeutung von Zytokinen beim Endotoxinschock und bei der Endotoxinüberempfindlichkeit. Immun Infekt. 1993 Apr;21(2):40–44. [PubMed] [Google Scholar]
  13. Galanos C., Lüderitz O., Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem. 1969 Jun;9(2):245–249. doi: 10.1111/j.1432-1033.1969.tb00601.x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Garcia-del Portillo F., Finlay B. B. Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors. J Cell Biol. 1995 Apr;129(1):81–97. doi: 10.1083/jcb.129.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garcia-del Portillo F., Stein M. A., Finlay B. B. Release of lipopolysaccharide from intracellular compartments containing Salmonella typhimurium to vesicles of the host epithelial cell. Infect Immun. 1997 Jan;65(1):24–34. doi: 10.1128/iai.65.1.24-34.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ginocchio C., Pace J., Galán J. E. Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5976–5980. doi: 10.1073/pnas.89.13.5976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldman R. C., Joiner K., Leive L. Serum-resistant mutants of Escherichia coli O111 contain increased lipopolysaccharide, lack an O antigen-containing capsule, and cover more of their lipid A core with O antigen. J Bacteriol. 1984 Sep;159(3):877–882. doi: 10.1128/jb.159.3.877-882.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Groisman E. A., Chiao E., Lipps C. J., Heffron F. Salmonella typhimurium phoP virulence gene is a transcriptional regulator. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7077–7081. doi: 10.1073/pnas.86.18.7077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Groisman E. A., Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J. 1993 Oct;12(10):3779–3787. doi: 10.1002/j.1460-2075.1993.tb06056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grossman N., Schmetz M. A., Foulds J., Klima E. N., Jimenez-Lucho V. E., Leive L. L., Joiner K. A., Jiminez V. Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo. J Bacteriol. 1987 Feb;169(2):856–863. doi: 10.1128/jb.169.2.856-863.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gulig P. A., Caldwell A. L., Chiodo V. A. Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid. Mol Microbiol. 1992 May;6(10):1395–1411. doi: 10.1111/j.1365-2958.1992.tb00860.x. [DOI] [PubMed] [Google Scholar]
  23. Gulig P. A., Curtiss R., 3rd Plasmid-associated virulence of Salmonella typhimurium. Infect Immun. 1987 Dec;55(12):2891–2901. doi: 10.1128/iai.55.12.2891-2901.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gulig P. A., Danbara H., Guiney D. G., Lax A. J., Norel F., Rhen M. Molecular analysis of spv virulence genes of the Salmonella virulence plasmids. Mol Microbiol. 1993 Mar;7(6):825–830. doi: 10.1111/j.1365-2958.1993.tb01172.x. [DOI] [PubMed] [Google Scholar]
  25. Heffernan E. J., Harwood J., Fierer J., Guiney D. The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. J Bacteriol. 1992 Jan;174(1):84–91. doi: 10.1128/jb.174.1.84-91.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heinzel F. P., Hujer A. M., Ahmed F. N., Rerko R. M. In vivo production and function of IL-12 p40 homodimers. J Immunol. 1997 May 1;158(9):4381–4388. [PubMed] [Google Scholar]
  27. 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]
  28. Hueck C. J., Hantman M. J., Bajaj V., Johnston C., Lee C. A., Miller S. I. Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins. Mol Microbiol. 1995 Nov;18(3):479–490. doi: 10.1111/j.1365-2958.1995.mmi_18030479.x. [DOI] [PubMed] [Google Scholar]
  29. Joiner K. A. Complement evasion by bacteria and parasites. Annu Rev Microbiol. 1988;42:201–230. doi: 10.1146/annurev.mi.42.100188.001221. [DOI] [PubMed] [Google Scholar]
  30. Jones B. D., Falkow S. Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization. Infect Immun. 1994 Sep;62(9):3745–3752. doi: 10.1128/iai.62.9.3745-3752.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Karem K. L., Kanangat S., Rouse B. T. Cytokine expression in the gut associated lymphoid tissue after oral administration of attenuated Salmonella vaccine strains. Vaccine. 1996 Nov;14(16):1495–1502. doi: 10.1016/s0264-410x(96)00118-1. [DOI] [PubMed] [Google Scholar]
  33. Karow M., Fayet O., Cegielska A., Ziegelhoffer T., Georgopoulos C. Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media. J Bacteriol. 1991 Jan;173(2):741–750. doi: 10.1128/jb.173.2.741-750.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kawamoto Y., Winger L. A., Hong K., Matsuoka H., Chinzei Y., Kawamoto F., Kamimura K., Arakawa R., Sinden R. E., Miyama A. Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum. Exp Parasitol. 1992 Nov;75(3):361–368. doi: 10.1016/0014-4894(92)90249-a. [DOI] [PubMed] [Google Scholar]
  35. Lee N. G., Sunshine M. G., Engstrom J. J., Gibson B. W., Apicella M. A. Mutation of the htrB locus of Haemophilus influenzae nontypable strain 2019 is associated with modifications of lipid A and phosphorylation of the lipo-oligosaccharide. J Biol Chem. 1995 Nov 10;270(45):27151–27159. [PubMed] [Google Scholar]
  36. Lehmann V., Freudenberg M. A., Galanos C. Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and D-galactosamine-treated mice. J Exp Med. 1987 Mar 1;165(3):657–663. doi: 10.1084/jem.165.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lindberg A. A. Bacterial virulence factors--with particular reference to Salmonella bacteria. Scand J Infect Dis Suppl. 1980;Suppl 24:86–92. [PubMed] [Google Scholar]
  38. Lyman M. B., Steward J. P., Roantree R. J. Characterization of the virulence and antigenic structure of Salmonella typhimurium strains with lipopolysaccharide core defects. Infect Immun. 1976 Jun;13(6):1539–1542. doi: 10.1128/iai.13.6.1539-1542.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lyman M. B., Stocker B. A., Roantree R. J. Comparison of the virulence of O:9,12 and O:4,5,12 Salmonella typhimurium his+ transductants for mice. Infect Immun. 1977 Feb;15(2):491–499. doi: 10.1128/iai.15.2.491-499.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. MacPhee D. G., Krishnapillai V., Roantree R. J., Stocker B. A. Mutations in Salmonella typhimurium conferring resistance to Felix O phage without loss of smooth character. J Gen Microbiol. 1975 Mar;87(1):1–10. doi: 10.1099/00221287-87-1-1. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Mitsui-Yamaguchi T., Abe A., Danbara H., Kawahara K. Induction of TNF-alpha mRNA in murine macrophages by virulent and avirulent strains of Salmonella choleraesuis serovar typhimurium and serovar Choleraesuis. Microb Pathog. 1997 Jan;22(1):59–66. doi: 10.1006/mpat.1996.0091. [DOI] [PubMed] [Google Scholar]
  43. Moore K. W., O'Garra A., de Waal Malefyt R., Vieira P., Mosmann T. R. Interleukin-10. Annu Rev Immunol. 1993;11:165–190. doi: 10.1146/annurev.iy.11.040193.001121. [DOI] [PubMed] [Google Scholar]
  44. Nnalue N. A., Lindberg A. A. Salmonella choleraesuis strains deficient in O antigen remain fully virulent for mice by parenteral inoculation but are avirulent by oral administration. Infect Immun. 1990 Aug;58(8):2493–2501. doi: 10.1128/iai.58.8.2493-2501.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nnalue N. A., Stocker B. A. Some galE mutants of Salmonella choleraesuis retain virulence. Infect Immun. 1986 Dec;54(3):635–640. doi: 10.1128/iai.54.3.635-640.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pamer E. G., Harty J. T., Bevan M. J. Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes. Nature. 1991 Oct 31;353(6347):852–855. doi: 10.1038/353852a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pie S., Matsiota-Bernard P., Truffa-Bachi P., Nauciel C. Gamma interferon and interleukin-10 gene expression in innately susceptible and resistant mice during the early phase of Salmonella typhimurium infection. Infect Immun. 1996 Mar;64(3):849–854. doi: 10.1128/iai.64.3.849-854.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pugliese C., LaSalle M. D., DeBari V. A. Relationships between the structure and function of lipopolysaccharide chemotypes with regard to their effects on the human polymorphonuclear neutrophil. Mol Immunol. 1988 Jul;25(7):631–637. doi: 10.1016/0161-5890(88)90098-3. [DOI] [PubMed] [Google Scholar]
  49. Raetz C. R., Ulevitch R. J., Wright S. D., Sibley C. H., Ding A., Nathan C. F. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 1991 Sep;5(12):2652–2660. doi: 10.1096/fasebj.5.12.1916089. [DOI] [PubMed] [Google Scholar]
  50. Reimann T., Büscher D., Hipskind R. A., Krautwald S., Lohmann-Matthes M. L., Baccarini M. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes. J Immunol. 1994 Dec 15;153(12):5740–5749. [PubMed] [Google Scholar]
  51. Ross S. C., Rosenthal P. J., Berberich H. M., Densen P. Killing of Neisseria meningitidis by human neutrophils: implications for normal and complement-deficient individuals. J Infect Dis. 1987 Jun;155(6):1266–1275. doi: 10.1093/infdis/155.6.1266. [DOI] [PubMed] [Google Scholar]
  52. Schafer R., Eisenstein T. K. Natural killer cells mediate protection induced by a Salmonella aroA mutant. Infect Immun. 1992 Mar;60(3):791–797. doi: 10.1128/iai.60.3.791-797.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shaio M. F., Rowland H. Bactericidal and opsonizing effects of normal serum on mutant strains of Salmonella typhimurium. Infect Immun. 1985 Sep;49(3):647–653. doi: 10.1128/iai.49.3.647-653.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Stinavage P., Martin L. E., Spitznagel J. K. O antigen and lipid A phosphoryl groups in resistance of Salmonella typhimurium LT-2 to nonoxidative killing in human polymorphonuclear neutrophils. Infect Immun. 1989 Dec;57(12):3894–3900. doi: 10.1128/iai.57.12.3894-3900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sunshine M. G., Gibson B. W., Engstrom J. J., Nichols W. A., Jones B. D., Apicella M. A. Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization. J Bacteriol. 1997 Sep;179(17):5521–5533. doi: 10.1128/jb.179.17.5521-5533.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Terakado N., Ushijima T., Samejima T., Ito H., Hamaoka T., Murayama S., Kawahara K., Danbara H. Transposon insertion mutagenesis of a genetic region encoding serum resistance in an 80 kb plasmid of Salmonella dublin. J Gen Microbiol. 1990 Sep;136(9):1833–1838. doi: 10.1099/00221287-136-9-1833. [DOI] [PubMed] [Google Scholar]
  58. Tomás J. M., Ciurana B., Benedí V. J., Juarez A. Role of lipopolysaccharide and complement in susceptibility of Escherichia coli and Salmonella typhimurium to non-immune serum. J Gen Microbiol. 1988 Apr;134(4):1009–1016. doi: 10.1099/00221287-134-4-1009. [DOI] [PubMed] [Google Scholar]
  59. Tripp C. S., Wolf S. F., Unanue E. R. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3725–3729. doi: 10.1073/pnas.90.8.3725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vaara M., Viljanen P., Vaara T., Mäkelä P. H. An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action. J Immunol. 1984 May;132(5):2582–2589. [PubMed] [Google Scholar]
  61. Valtonen M. V., Häyry P. O antigen as virulence factor in mouse typhoid: effect of B-cell suppression. Infect Immun. 1978 Jan;19(1):26–28. doi: 10.1128/iai.19.1.26-28.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Valtonen M. V., Plosila M., Valtonen V. V., Mäkelä P. H. Effect of the quality of the lipopolysaccharide on mouse virulence of Salmonella enteritidis. Infect Immun. 1975 Oct;12(4):828–832. doi: 10.1128/iai.12.4.828-832.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weinstein D. L., O'Neill B. L., Metcalf E. S. Salmonella typhi stimulation of human intestinal epithelial cells induces secretion of epithelial cell-derived interleukin-6. Infect Immun. 1997 Feb;65(2):395–404. doi: 10.1128/iai.65.2.395-404.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Weinstein S. L., Gold M. R., DeFranco A. L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4148–4152. doi: 10.1073/pnas.88.10.4148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Weinstein S. L., Sanghera J. S., Lemke K., DeFranco A. L., Pelech S. L. Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages. J Biol Chem. 1992 Jul 25;267(21):14955–14962. [PubMed] [Google Scholar]
  66. Williamson C. M., Pullinger G. D., Lax A. J. Identification of an essential virulence region on Salmonella plasmids. Microb Pathog. 1988 Dec;5(6):469–473. doi: 10.1016/0882-4010(88)90008-3. [DOI] [PubMed] [Google Scholar]
  67. Wray C., Sojka W. J. Experimental Salmonella typhimurium infection in calves. Res Vet Sci. 1978 Sep;25(2):139–143. [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES