Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 Nov;12(11):4053–4062. doi: 10.1002/j.1460-2075.1993.tb06089.x

Molecular genetic analysis of a locus required for resistance to antimicrobial peptides in Salmonella typhimurium.

C Parra-Lopez 1, M T Baer 1, E A Groisman 1
PMCID: PMC413698  PMID: 8223423

Abstract

The innate immunity of vertebrates and invertebrates to microbial infection is mediated in part by small cationic peptides with antimicrobial activity. Successful pathogens have evolved mechanisms to withstand the antibiotic activity of these molecules. We have isolated a set of genes from Salmonella typhimurium which are required for virulence and resistance to the antimicrobial peptides melittin and protamine. Sequence analysis of a 5.7 kb segment from the wild-type plasmid conferring resistance to protamine contained five open reading frames: sapA, sapB, sapC, sapD and sapF, organized in an operon structure and transcribed as a 5.3 kb mRNA. SapD and SapF exhibited similarity with the 'ATP binding cassette' family of transporters including the bacterial Opp and SpoOK, involved in the uptake of oligopeptides; the yeast STE6, necessary for the export of a peptide pheromone; and the mammalian mdr, which mediates resistance to chemotherapeutic agents in cancer cells. SapA showed identity with other periplasmic solute binding proteins involved in peptide transport. The SapABCDF system constitutes a novel transporter for enteric bacteria and the first one harboring a periplasmic component with a role in virulence.

Full text

PDF
4056

Images in this article

Selected References

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

  1. Abouhamad W. N., Manson M., Gibson M. M., Higgins C. F. Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein. Mol Microbiol. 1991 May;5(5):1035–1047. doi: 10.1111/j.1365-2958.1991.tb01876.x. [DOI] [PubMed] [Google Scholar]
  2. Alloing G., Trombe M. C., Claverys J. P. The ami locus of the gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of gram-negative bacteria. Mol Microbiol. 1990 Apr;4(4):633–644. doi: 10.1111/j.1365-2958.1990.tb00632.x. [DOI] [PubMed] [Google Scholar]
  3. Andrews J. C., Short S. A. opp-lac Operon fusions and transcriptional regulation of the Escherichia coli trp-linked oligopeptide permease. J Bacteriol. 1986 Feb;165(2):434–442. doi: 10.1128/jb.165.2.434-442.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bangalore N., Travis J., Onunka V. C., Pohl J., Shafer W. M. Identification of the primary antimicrobial domains in human neutrophil cathepsin G. J Biol Chem. 1990 Aug 15;265(23):13584–13588. [PubMed] [Google Scholar]
  5. Benjamin W. H., Jr, Hall P., Briles D. E. A hemA mutation renders Salmonella typhimurium avirulent in mice, yet capable of eliciting protection against intravenous infection with S. typhimurium. Microb Pathog. 1991 Oct;11(4):289–295. doi: 10.1016/0882-4010(91)90033-7. [DOI] [PubMed] [Google Scholar]
  6. Boman H. G. Antibacterial peptides: key components needed in immunity. Cell. 1991 Apr 19;65(2):205–207. doi: 10.1016/0092-8674(91)90154-q. [DOI] [PubMed] [Google Scholar]
  7. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen C. J., Chin J. E., Ueda K., Clark D. P., Pastan I., Gottesman M. M., Roninson I. B. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell. 1986 Nov 7;47(3):381–389. doi: 10.1016/0092-8674(86)90595-7. [DOI] [PubMed] [Google Scholar]
  9. Davidson A. L., Shuman H. A., Nikaido H. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2360–2364. doi: 10.1073/pnas.89.6.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deverson E. V., Gow I. R., Coadwell W. J., Monaco J. J., Butcher G. W., Howard J. C. MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters. Nature. 1990 Dec 20;348(6303):738–741. doi: 10.1038/348738a0. [DOI] [PubMed] [Google Scholar]
  11. Diamond G., Zasloff M., Eck H., Brasseur M., Maloy W. L., Bevins C. L. Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3952–3956. doi: 10.1073/pnas.88.9.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dorman C. J., Chatfield S., Higgins C. F., Hayward C., Dougan G. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect Immun. 1989 Jul;57(7):2136–2140. doi: 10.1128/iai.57.7.2136-2140.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Du Pasquier L. Origin and evolution of the vertebrate immune system. APMIS. 1992 May;100(5):383–392. doi: 10.1111/j.1699-0463.1992.tb00888.x. [DOI] [PubMed] [Google Scholar]
  14. Eisenhauer P. B., Harwig S. S., Lehrer R. I. Cryptdins: antimicrobial defensins of the murine small intestine. Infect Immun. 1992 Sep;60(9):3556–3565. doi: 10.1128/iai.60.9.3556-3565.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Ganz T., Selsted M. E., Lehrer R. I. Defensins. Eur J Haematol. 1990 Jan;44(1):1–8. doi: 10.1111/j.1600-0609.1990.tb00339.x. [DOI] [PubMed] [Google Scholar]
  17. Gibson M. M., Price M., Higgins C. F. Genetic characterization and molecular cloning of the tripeptide permease (tpp) genes of Salmonella typhimurium. J Bacteriol. 1984 Oct;160(1):122–130. doi: 10.1128/jb.160.1.122-130.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodell E. W., Higgins C. F. Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli. J Bacteriol. 1987 Aug;169(8):3861–3865. doi: 10.1128/jb.169.8.3861-3865.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Groisman E. A., Casadaban M. J. Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J Bacteriol. 1986 Oct;168(1):357–364. doi: 10.1128/jb.168.1.357-364.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Groisman E. A., Heffron F., Solomon F. Molecular genetic analysis of the Escherichia coli phoP locus. J Bacteriol. 1992 Jan;174(2):486–491. doi: 10.1128/jb.174.2.486-491.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Groisman E. A. In vivo genetic engineering with bacteriophage Mu. Methods Enzymol. 1991;204:180–212. doi: 10.1016/0076-6879(91)04010-l. [DOI] [PubMed] [Google Scholar]
  23. Groisman E. A., Parra-Lopez C., Salcedo M., Lipps C. J., Heffron F. Resistance to host antimicrobial peptides is necessary for Salmonella virulence. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11939–11943. doi: 10.1073/pnas.89.24.11939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Groisman E. A., Sturmoski M. A., Solomon F. R., Lin R., Ochman H. Molecular, functional, and evolutionary analysis of sequences specific to Salmonella. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1033–1037. doi: 10.1073/pnas.90.3.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guilfoile P. G., Hutchinson C. R. A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8553–8557. doi: 10.1073/pnas.88.19.8553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  27. Hiles I. D., Gallagher M. P., Jamieson D. J., Higgins C. F. Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J Mol Biol. 1987 May 5;195(1):125–142. doi: 10.1016/0022-2836(87)90332-9. [DOI] [PubMed] [Google Scholar]
  28. Hiles I. D., Higgins C. F. Peptide uptake by Salmonella typhimurium. The periplasmic oligopeptide-binding protein. Eur J Biochem. 1986 Aug 1;158(3):561–567. doi: 10.1111/j.1432-1033.1986.tb09791.x. [DOI] [PubMed] [Google Scholar]
  29. Jamieson D. J., Higgins C. F. Anaerobic and leucine-dependent expression of a peptide transport gene in Salmonella typhimurium. J Bacteriol. 1984 Oct;160(1):131–136. doi: 10.1128/jb.160.1.131-136.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lambert J., Keppi E., Dimarcq J. L., Wicker C., Reichhart J. M., Dunbar B., Lepage P., Van Dorsselaer A., Hoffmann J., Fothergill J. Insect immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proc Natl Acad Sci U S A. 1989 Jan;86(1):262–266. doi: 10.1073/pnas.86.1.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee J. Y., Boman A., Sun C. X., Andersson M., Jörnvall H., Mutt V., Boman H. G. Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9159–9162. doi: 10.1073/pnas.86.23.9159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lehrer R. I., Barton A., Daher K. A., Harwig S. S., Ganz T., Selsted M. E. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest. 1989 Aug;84(2):553–561. doi: 10.1172/JCI114198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lehrer R. I., Ganz T., Selsted M. E. Defensins: endogenous antibiotic peptides of animal cells. Cell. 1991 Jan 25;64(2):229–230. doi: 10.1016/0092-8674(91)90632-9. [DOI] [PubMed] [Google Scholar]
  34. Macias E. A., Rana F., Blazyk J., Modrzakowski M. C. Bactericidal activity of magainin 2: use of lipopolysaccharide mutants. Can J Microbiol. 1990 Aug;36(8):582–584. doi: 10.1139/m90-102. [DOI] [PubMed] [Google Scholar]
  35. McGrath J. P., Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. doi: 10.1038/340400a0. [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 S. I., Pulkkinen W. S., Selsted M. E., Mekalanos J. J. Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium. Infect Immun. 1990 Nov;58(11):3706–3710. doi: 10.1128/iai.58.11.3706-3710.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Monaco J. J., Cho S., Attaya M. Transport protein genes in the murine MHC: possible implications for antigen processing. Science. 1990 Dec 21;250(4988):1723–1726. doi: 10.1126/science.2270487. [DOI] [PubMed] [Google Scholar]
  39. Moore K. S., Bevins C. L., Brasseur M. M., Tomassini N., Turner K., Eck H., Zasloff M. Antimicrobial peptides in the stomach of Xenopus laevis. J Biol Chem. 1991 Oct 15;266(29):19851–19857. [PubMed] [Google Scholar]
  40. Ouellette A. J., Miller S. I., Henschen A. H., Selsted M. E. Purification and primary structure of murine cryptdin-1, a Paneth cell defensin. FEBS Lett. 1992 Jun 15;304(2-3):146–148. doi: 10.1016/0014-5793(92)80606-h. [DOI] [PubMed] [Google Scholar]
  41. Parham P. Antigen presentation. Flying the first class flag. Nature. 1992 May 21;357(6375):193–194. doi: 10.1038/357193a0. [DOI] [PubMed] [Google Scholar]
  42. Park J. T. Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus. J Bacteriol. 1993 Jan;175(1):7–11. doi: 10.1128/jb.175.1.7-11.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Payne J. W., Gilvarg C. Size restriction on peptide utilization in Escherichia coli. J Biol Chem. 1968 Dec 10;243(23):6291–6299. [PubMed] [Google Scholar]
  44. Perego M., Higgins C. F., Pearce S. R., Gallagher M. P., Hoch J. A. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol Microbiol. 1991 Jan;5(1):173–185. doi: 10.1111/j.1365-2958.1991.tb01838.x. [DOI] [PubMed] [Google Scholar]
  45. Pereira H. A., Erdem I., Pohl J., Spitznagel J. K. Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4733–4737. doi: 10.1073/pnas.90.10.4733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rana F. R., Macias E. A., Sultany C. M., Modzrakowski M. C., Blazyk J. Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure. Biochemistry. 1991 Jun 18;30(24):5858–5866. doi: 10.1021/bi00238a008. [DOI] [PubMed] [Google Scholar]
  47. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  48. Rudner D. Z., LeDeaux J. R., Ireton K., Grossman A. D. The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol. 1991 Feb;173(4):1388–1398. doi: 10.1128/jb.173.4.1388-1398.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Selsted M. E., Miller S. I., Henschen A. H., Ouellette A. J. Enteric defensins: antibiotic peptide components of intestinal host defense. J Cell Biol. 1992 Aug;118(4):929–936. doi: 10.1083/jcb.118.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Selsted M. E., Tang Y. Q., Morris W. L., McGuire P. A., Novotny M. J., Smith W., Henschen A. H., Cullor J. S. Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem. 1993 Mar 25;268(9):6641–6648. [PubMed] [Google Scholar]
  51. Shuman H. A. Active transport of maltose in Escherichia coli K12. Role of the periplasmic maltose-binding protein and evidence for a substrate recognition site in the cytoplasmic membrane. J Biol Chem. 1982 May 25;257(10):5455–5461. [PubMed] [Google Scholar]
  52. Sidén I., Boman H. G. Escherichia coli mutants with an altered sensitivity to cecropin D. J Bacteriol. 1983 Apr;154(1):170–176. doi: 10.1128/jb.154.1.170-176.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Speiser D. M., Ames G. F. Salmonella typhimurium histidine periplasmic permease mutations that allow transport in the absence of histidine-binding proteins. J Bacteriol. 1991 Feb;173(4):1444–1451. doi: 10.1128/jb.173.4.1444-1451.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Spies T., Bresnahan M., Bahram S., Arnold D., Blanck G., Mellins E., Pious D., DeMars R. A gene in the human major histocompatibility complex class II region controlling the class I antigen presentation pathway. Nature. 1990 Dec 20;348(6303):744–747. doi: 10.1038/348744a0. [DOI] [PubMed] [Google Scholar]
  55. Steiner H., Hultmark D., Engström A., Bennich H., Boman H. G. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981 Jul 16;292(5820):246–248. doi: 10.1038/292246a0. [DOI] [PubMed] [Google Scholar]
  56. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tam R., Saier M. H., Jr Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol Rev. 1993 Jun;57(2):320–346. doi: 10.1128/mr.57.2.320-346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Trowsdale J., Hanson I., Mockridge I., Beck S., Townsend A., Kelly A. Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters. Nature. 1990 Dec 20;348(6303):741–744. doi: 10.1038/348741a0. [DOI] [PubMed] [Google Scholar]
  59. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev. 1992 Sep;56(3):395–411. doi: 10.1128/mr.56.3.395-411.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vaara M. Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants. J Bacteriol. 1981 Nov;148(2):426–434. doi: 10.1128/jb.148.2.426-434.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yamauchi K., Tomita M., Giehl T. J., Ellison R. T., 3rd Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect Immun. 1993 Feb;61(2):719–728. doi: 10.1128/iai.61.2.719-728.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zasloff M. Antibiotic peptides as mediators of innate immunity. Curr Opin Immunol. 1992 Feb;4(1):3–7. doi: 10.1016/0952-7915(92)90115-u. [DOI] [PubMed] [Google Scholar]
  64. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5449–5453. doi: 10.1073/pnas.84.15.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES