Abstract
The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways. Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export. Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA. Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP. In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation. These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible candidates for the outer membrane defect are discussed.
Full Text
The Full Text of this article is available as a PDF (1.3 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Attridge S. R., Voss E., Manning P. A. The role of toxin-coregulated pili in the pathogenesis of Vibrio cholerae O1 El Tor. Microb Pathog. 1993 Dec;15(6):421–431. doi: 10.1006/mpat.1993.1091. [DOI] [PubMed] [Google Scholar]
- Bally M., Filloux A., Akrim M., Ball G., Lazdunski A., Tommassen J. Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. 1992 May;6(9):1121–1131. doi: 10.1111/j.1365-2958.1992.tb01550.x. [DOI] [PubMed] [Google Scholar]
- Dupuy B., Taha M. K., Pugsley A. P., Marchal C. Neisseria gonorrhoeae prepilin export studied in Escherichia coli. J Bacteriol. 1991 Dec;173(23):7589–7598. doi: 10.1128/jb.173.23.7589-7598.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Every D. Purification of pili from Bacteroides nodosus and an examination of their chemical, physical and serological properties. J Gen Microbiol. 1979 Dec;115(2):309–316. doi: 10.1099/00221287-115-2-309. [DOI] [PubMed] [Google Scholar]
- Farinha M. A., Conway B. D., Glasier L. M., Ellert N. W., Irvin R. T., Sherburne R., Paranchych W. Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infect Immun. 1994 Oct;62(10):4118–4123. doi: 10.1128/iai.62.10.4118-4123.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Folkhard W., Marvin D. A., Watts T. H., Paranchych W. Structure of polar pili from Pseudomonas aeruginosa strains K and O. J Mol Biol. 1981 Jun 15;149(1):79–93. doi: 10.1016/0022-2836(81)90261-8. [DOI] [PubMed] [Google Scholar]
- Frost L. S., Paranchych W. Composition and molecular weight of pili purified from Pseudomonas aeruginosa K. J Bacteriol. 1977 Jul;131(1):259–269. doi: 10.1128/jb.131.1.259-269.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Genin S., Boucher C. A. A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet. 1994 Apr;243(1):112–118. doi: 10.1007/BF00283883. [DOI] [PubMed] [Google Scholar]
- Geuze H. J., Slot J. W. Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study. Eur J Cell Biol. 1980 Apr;21(1):93–100. [PubMed] [Google Scholar]
- Hardie K. R., Lory S., Pugsley A. P. Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J. 1996 Mar 1;15(5):978–988. [PMC free article] [PubMed] [Google Scholar]
- Herrington D. A., Hall R. H., Losonsky G., Mekalanos J. J., Taylor R. K., Levine M. M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988 Oct 1;168(4):1487–1492. doi: 10.1084/jem.168.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hobbs M., Mattick J. S. Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol Microbiol. 1993 Oct;10(2):233–243. doi: 10.1111/j.1365-2958.1993.tb01949.x. [DOI] [PubMed] [Google Scholar]
- Iredell J. R., Manning P. A. Biotype-specific tcpA genes in Vibrio cholerae. FEMS Microbiol Lett. 1994 Aug 1;121(1):47–54. doi: 10.1111/j.1574-6968.1994.tb07074.x. [DOI] [PubMed] [Google Scholar]
- Jones A. L., Shirasu K., Kado C. I. The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol. 1994 Sep;176(17):5255–5261. doi: 10.1128/jb.176.17.5255-5261.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jonson G., Lebens M., Holmgren J. Cloning and sequencing of Vibrio cholerae mannose-sensitive haemagglutinin pilin gene: localization of mshA within a cluster of type 4 pilin genes. Mol Microbiol. 1994 Jul;13(1):109–118. doi: 10.1111/j.1365-2958.1994.tb00406.x. [DOI] [PubMed] [Google Scholar]
- Kaufman M. R., Seyer J. M., Taylor R. K. Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria. Genes Dev. 1991 Oct;5(10):1834–1846. doi: 10.1101/gad.5.10.1834. [DOI] [PubMed] [Google Scholar]
- Kaufman M. R., Shaw C. E., Jones I. D., Taylor R. K. Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems. Gene. 1993 Apr 15;126(1):43–49. doi: 10.1016/0378-1119(93)90588-t. [DOI] [PubMed] [Google Scholar]
- Kazmierczak B. I., Mielke D. L., Russel M., Model P. pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J Mol Biol. 1994 Apr 29;238(2):187–198. doi: 10.1006/jmbi.1994.1280. [DOI] [PubMed] [Google Scholar]
- Klauser T., Pohlner J., Meyer T. F. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J. 1990 Jun;9(6):1991–1999. doi: 10.1002/j.1460-2075.1990.tb08327.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klauser T., Pohlner J., Meyer T. F. Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Iga beta-mediated outer membrane transport. EMBO J. 1992 Jun;11(6):2327–2335. doi: 10.1002/j.1460-2075.1992.tb05292.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laird M. W., Kloser A. W., Misra R. Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12. J Bacteriol. 1994 Apr;176(8):2259–2264. doi: 10.1128/jb.176.8.2259-2264.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
- Manning P. A., Kaufmann A., Roll U., Pohlner J., Meyer T. F., Haas R. L-pilin variants of Neisseria gonorrhoeae MS11. Mol Microbiol. 1991 Apr;5(4):917–926. doi: 10.1111/j.1365-2958.1991.tb00766.x. [DOI] [PubMed] [Google Scholar]
- Martin P. R., Hobbs M., Free P. D., Jeske Y., Mattick J. S. Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol. 1993 Aug;9(4):857–868. doi: 10.1111/j.1365-2958.1993.tb01744.x. [DOI] [PubMed] [Google Scholar]
- Moore D., Sowa B. A., Ippen-Ihler K. Location of an F-pilin pool in the inner membrane. J Bacteriol. 1981 Apr;146(1):251–259. doi: 10.1128/jb.146.1.251-259.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ogierman M. A., Manning P. A. TCP pilus biosynthesis in Vibrio cholerae O1: gene sequence of tcpC encoding an outer membrane lipoprotein. FEMS Microbiol Lett. 1992 Oct 1;76(1-2):179–184. doi: 10.1016/0378-1097(92)90383-y. [DOI] [PubMed] [Google Scholar]
- Ogierman M. A., Zabihi S., Mourtzios L., Manning P. A. Genetic organization and sequence of the promoter-distal region of the tcp gene cluster of Vibrio cholerae. Gene. 1993 Apr 15;126(1):51–60. doi: 10.1016/0378-1119(93)90589-u. [DOI] [PubMed] [Google Scholar]
- Osek J., Jonson G., Svennerholm A. M., Holmgren J. Role of antibodies against biotype-specific Vibrio cholerae pili in protection against experimental classical and El Tor cholera. Infect Immun. 1994 Jul;62(7):2901–2907. doi: 10.1128/iai.62.7.2901-2907.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Overbye L. J., Sandkvist M., Bagdasarian M. Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae. Gene. 1993 Sep 30;132(1):101–106. doi: 10.1016/0378-1119(93)90520-d. [DOI] [PubMed] [Google Scholar]
- Pannekoek Y., van Putten J. P., Dankert J. Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae. J Bacteriol. 1992 Nov;174(21):6928–6937. doi: 10.1128/jb.174.21.6928-6937.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paranchych W., Frost L. S. The physiology and biochemistry of pili. Adv Microb Physiol. 1988;29:53–114. doi: 10.1016/s0065-2911(08)60346-x. [DOI] [PubMed] [Google Scholar]
- Paranchych W., Sastry P. A., Frost L. S., Carpenter M., Armstrong G. D., Watts T. H. Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO. Can J Microbiol. 1979 Oct;25(10):1175–1181. doi: 10.1139/m79-182. [DOI] [PubMed] [Google Scholar]
- Parge H. E., Bernstein S. L., Deal C. D., McRee D. E., Christensen D., Capozza M. A., Kays B. W., Fieser T. M., Draper D., So M. Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae. J Biol Chem. 1990 Feb 5;265(4):2278–2285. [PubMed] [Google Scholar]
- Parge H. E., Forest K. T., Hickey M. J., Christensen D. A., Getzoff E. D., Tainer J. A. Structure of the fibre-forming protein pilin at 2.6 A resolution. Nature. 1995 Nov 2;378(6552):32–38. doi: 10.1038/378032a0. [DOI] [PubMed] [Google Scholar]
- Parsot C., Taxman E., Mekalanos J. J. ToxR regulates the production of lipoproteins and the expression of serum resistance in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1641–1645. doi: 10.1073/pnas.88.5.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peek J. A., Taylor R. K. Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6210–6214. doi: 10.1073/pnas.89.13.6210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pilipcinec E., Huisman T. T., Willemsen P. T., Appelmelk B. J., de Graaf F. K., Oudega B. Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: effect of LPS core mutations. FEMS Microbiol Lett. 1994 Oct 15;123(1-2):201–206. doi: 10.1016/0378-1097(94)90295-x. [DOI] [PubMed] [Google Scholar]
- Pugsley A. P. Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca. Mol Microbiol. 1993 Jul;9(2):295–308. doi: 10.1111/j.1365-2958.1993.tb01691.x. [DOI] [PubMed] [Google Scholar]
- Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reyss I., Pugsley A. P. Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. Mol Gen Genet. 1990 Jul;222(2-3):176–184. doi: 10.1007/BF00633815. [DOI] [PubMed] [Google Scholar]
- Rudel T., Scheurerpflug I., Meyer T. F. Neisseria PilC protein identified as type-4 pilus tip-located adhesin. Nature. 1995 Jan 26;373(6512):357–359. doi: 10.1038/373357a0. [DOI] [PubMed] [Google Scholar]
- Russel M. Phage assembly: a paradigm for bacterial virulence factor export? Science. 1994 Jul 29;265(5172):612–614. doi: 10.1126/science.8036510. [DOI] [PubMed] [Google Scholar]
- Sandlin R. C., Lampel K. A., Keasler S. P., Goldberg M. B., Stolzer A. L., Maurelli A. T. Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane. Infect Immun. 1995 Jan;63(1):229–237. doi: 10.1128/iai.63.1.229-237.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scherer P. E., Krieg U. C., Hwang S. T., Vestweber D., Schatz G. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J. 1990 Dec;9(13):4315–4322. doi: 10.1002/j.1460-2075.1990.tb07880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sen K., Nikaido H. Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin. J Bacteriol. 1991 Jan;173(2):926–928. doi: 10.1128/jb.173.2.926-928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sengupta D. K., Sengupta T. K., Ghose A. C. Major outer membrane proteins of Vibrio cholerae and their role in induction of protective immunity through inhibition of intestinal colonization. Infect Immun. 1992 Nov;60(11):4848–4855. doi: 10.1128/iai.60.11.4848-4855.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharma D. P., Stroeher U. H., Thomas C. J., Manning P. A., Attridge S. R. The toxin-coregulated pilus (TCP) of Vibrio cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model. Microb Pathog. 1989 Dec;7(6):437–448. doi: 10.1016/0882-4010(89)90024-7. [DOI] [PubMed] [Google Scholar]
- Strom M. S., Lory S. Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions. J Bacteriol. 1987 Jul;169(7):3181–3188. doi: 10.1128/jb.169.7.3181-3188.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strom M. S., Nunn D., Lory S. Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa. J Bacteriol. 1991 Feb;173(3):1175–1180. doi: 10.1128/jb.173.3.1175-1180.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sun D. X., Seyer J. M., Kovari I., Sumrada R. A., Taylor R. K. Localization of protective epitopes within the pilin subunit of the Vibrio cholerae toxin-coregulated pilus. Infect Immun. 1991 Jan;59(1):114–118. doi: 10.1128/iai.59.1.114-118.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Taylor R. K., Miller V. L., Furlong D. B., Mekalanos J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A. 1987 May;84(9):2833–2837. doi: 10.1073/pnas.84.9.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor R., Shaw C., Peterson K., Spears P., Mekalanos J. Safe, live Vibrio cholerae vaccines? Vaccine. 1988 Apr;6(2):151–154. doi: 10.1016/s0264-410x(88)80019-7. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsai W. M., Larsen S. H., Wilde C. E., 3rd Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae. Infect Immun. 1989 Sep;57(9):2653–2659. doi: 10.1128/iai.57.9.2653-2659.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Voss E., Attridge S. R. In vitro production of toxin-coregulated pili by Vibrio cholerae El Tor. Microb Pathog. 1993 Oct;15(4):255–268. doi: 10.1006/mpat.1993.1076. [DOI] [PubMed] [Google Scholar]
- Wandersman C., Létoffé S. Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases. Mol Microbiol. 1993 Jan;7(1):141–150. doi: 10.1111/j.1365-2958.1993.tb01105.x. [DOI] [PubMed] [Google Scholar]
- Ward H. M., Manning P. A. Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680. Mol Gen Genet. 1989 Aug;218(2):367–370. doi: 10.1007/BF00331294. [DOI] [PubMed] [Google Scholar]
- Watts T. H., Worobec E. A., Paranchych W. Identification of pilin pools in the membranes of Pseudomonas aeruginosa. J Bacteriol. 1982 Nov;152(2):687–691. doi: 10.1128/jb.152.2.687-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- d'Enfert C., Reyss I., Wandersman C., Pugsley A. P. Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12. J Biol Chem. 1989 Oct 15;264(29):17462–17468. [PubMed] [Google Scholar]