Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Mar;63(3):762–769. doi: 10.1128/iai.63.3.762-769.1995

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

J Lodge 1, G R Douce 1, I I Amin 1, A J Bolton 1, G D Martin 1, S Chatfield 1, G Dougan 1, N L Brown 1, J Stephen 1
PMCID: PMC173068  PMID: 7868245

Abstract

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Full Text

The Full Text of this article is available as a PDF (265.4 KB).

Selected References

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

  1. Allaoui A., Sansonetti P. J., Parsot C. MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins. Mol Microbiol. 1993 Jan;7(1):59–68. doi: 10.1111/j.1365-2958.1993.tb01097.x. [DOI] [PubMed] [Google Scholar]
  2. Altmeyer R. M., McNern J. K., Bossio J. C., Rosenshine I., Finlay B. B., Galán J. E. Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. Mol Microbiol. 1993 Jan;7(1):89–98. doi: 10.1111/j.1365-2958.1993.tb01100.x. [DOI] [PubMed] [Google Scholar]
  3. Amin I. I., Douce G. R., Osborne M. P., Stephen J. Quantitative studies of invasion of rabbit ileal mucosa by Salmonella typhimurium strains which differ in virulence in a model of gastroenteritis. Infect Immun. 1994 Feb;62(2):569–578. doi: 10.1128/iai.62.2.569-578.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Auerswald E. A., Ludwig G., Schaller H. Structural analysis of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):107–113. doi: 10.1101/sqb.1981.045.01.019. [DOI] [PubMed] [Google Scholar]
  5. Baudry B., Maurelli A. T., Clerc P., Sadoff J. C., Sansonetti P. J. Localization of plasmid loci necessary for the entry of Shigella flexneri into HeLa cells, and characterization of one locus encoding four immunogenic polypeptides. J Gen Microbiol. 1987 Dec;133(12):3403–3413. doi: 10.1099/00221287-133-12-3403. [DOI] [PubMed] [Google Scholar]
  6. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  7. Bleasby A. J., Wootton J. C. Construction of validated, non-redundant composite protein sequence databases. Protein Eng. 1990 Jan;3(3):153–159. doi: 10.1093/protein/3.3.153. [DOI] [PubMed] [Google Scholar]
  8. Bliska J. B., Galán J. E., Falkow S. Signal transduction in the mammalian cell during bacterial attachment and entry. Cell. 1993 Jun 4;73(5):903–920. doi: 10.1016/0092-8674(93)90270-z. [DOI] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Douce G. R., Amin I. I., Stephen J. Invasion of HEp-2 cells by strains of Salmonella typhimurium of different virulence in relation to gastroenteritis. J Med Microbiol. 1991 Dec;35(6):349–357. doi: 10.1099/00222615-35-6-349. [DOI] [PubMed] [Google Scholar]
  11. Eichelberg K., Ginocchio C. C., Galán J. E. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol. 1994 Aug;176(15):4501–4510. doi: 10.1128/jb.176.15.4501-4510.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elsinghorst E. A., Baron L. S., Kopecko D. J. Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5173–5177. doi: 10.1073/pnas.86.13.5173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Finlay B. B., Ruschkowski S., Dedhar S. Cytoskeletal rearrangements accompanying salmonella entry into epithelial cells. J Cell Sci. 1991 Jun;99(Pt 2):283–296. doi: 10.1242/jcs.99.2.283. [DOI] [PubMed] [Google Scholar]
  16. Finlay B. B., Starnbach M. N., Francis C. L., Stocker B. A., Chatfield S., Dougan G., Falkow S. Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer. Mol Microbiol. 1988 Nov;2(6):757–766. doi: 10.1111/j.1365-2958.1988.tb00087.x. [DOI] [PubMed] [Google Scholar]
  17. Gahring L. C., Heffron F., Finlay B. B., Falkow S. Invasion and replication of Salmonella typhimurium in animal cells. Infect Immun. 1990 Feb;58(2):443–448. doi: 10.1128/iai.58.2.443-448.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galán J. E., Curtiss R., 3rd Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6383–6387. doi: 10.1073/pnas.86.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Galán J. E., Curtiss R., 3rd Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect Immun. 1990 Jun;58(6):1879–1885. doi: 10.1128/iai.58.6.1879-1885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Giannella R. A., Broitman S. A., Zamcheck N. Salmonella enteritis. I. Role of reduced gastric secretion in pathogenesis. Am J Dig Dis. 1971 Nov;16(11):1000–1006. doi: 10.1007/BF02235012. [DOI] [PubMed] [Google Scholar]
  22. Giannella R. A., Broitman S. A., Zamcheck N. Salmonella enteritis. II. Fulminant diarrhea in and effects on the small intestine. Am J Dig Dis. 1971 Nov;16(11):1007–1013. doi: 10.1007/BF02235013. [DOI] [PubMed] [Google Scholar]
  23. Giannella R. A., Formal S. B., Dammin G. J., Collins H. Pathogenesis of salmonellosis. Studies of fluid secretion, mucosal invasion, and morphologic reaction in the rabbit ileum. J Clin Invest. 1973 Feb;52(2):441–453. doi: 10.1172/JCI107201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Haddix P. L., Straley S. C. Structure and regulation of the Yersinia pestis yscBCDEF operon. J Bacteriol. 1992 Jul;174(14):4820–4828. doi: 10.1128/jb.174.14.4820-4828.1992. [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. Lee C. A., Jones B. D., Falkow S. Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1847–1851. doi: 10.1073/pnas.89.5.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. MacBeth K. J., Lee C. A. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion. Infect Immun. 1993 Apr;61(4):1544–1546. doi: 10.1128/iai.61.4.1544-1546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
  32. Maurelli A. T. Temperature regulation of virulence genes in pathogenic bacteria: a general strategy for human pathogens? Microb Pathog. 1989 Jul;7(1):1–10. doi: 10.1016/0882-4010(89)90106-x. [DOI] [PubMed] [Google Scholar]
  33. Mazodier P., Cossart P., Giraud E., Gasser F. Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes. Nucleic Acids Res. 1985 Jan 11;13(1):195–205. doi: 10.1093/nar/13.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Michiels T., Vanooteghem J. C., Lambert de Rouvroit C., China B., Gustin A., Boudry P., Cornelis G. R. Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol. 1991 Aug;173(16):4994–5009. doi: 10.1128/jb.173.16.4994-5009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miller I. A., Chatfield S., Dougan G., Desilva L., Joysey H. S., Hormaeche C. Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains. Mol Gen Genet. 1989 Jan;215(2):312–316. doi: 10.1007/BF00339734. [DOI] [PubMed] [Google Scholar]
  36. Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miller V. L., Beer K. B., Loomis W. P., Olson J. A., Miller S. I. An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae. Infect Immun. 1992 Sep;60(9):3763–3770. doi: 10.1128/iai.60.9.3763-3770.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ochman H., Gerber A. S., Hartl D. L. Genetic applications of an inverse polymerase chain reaction. Genetics. 1988 Nov;120(3):621–623. doi: 10.1093/genetics/120.3.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pace J., Hayman M. J., Galán J. E. Signal transduction and invasion of epithelial cells by S. typhimurium. Cell. 1993 Feb 26;72(4):505–514. doi: 10.1016/0092-8674(93)90070-7. [DOI] [PubMed] [Google Scholar]
  40. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schmieger H. Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet. 1972;119(1):75–88. doi: 10.1007/BF00270447. [DOI] [PubMed] [Google Scholar]
  43. Shuttleworth H., Taylor J., Minton N. Sequence of the gene for alkaline phosphatase from Escherichia coli JM83. Nucleic Acids Res. 1986 Nov 11;14(21):8689–8689. doi: 10.1093/nar/14.21.8689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stone B. J., Garcia C. M., Badger J. L., Hassett T., Smith R. I., Miller V. L. Identification of novel loci affecting entry of Salmonella enteritidis into eukaryotic cells. J Bacteriol. 1992 Jun;174(12):3945–3952. doi: 10.1128/jb.174.12.3945-3952.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tartera C., Metcalf E. S. Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells. Infect Immun. 1993 Jul;61(7):3084–3089. doi: 10.1128/iai.61.7.3084-3089.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  49. Venkatesan M. M., Buysse J. M., Kopecko D. J. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9317–9321. doi: 10.1073/pnas.85.23.9317. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES