Epitopes fused to F-pilin are incorporated into functional recombinant pili

Rondot, S.; Anthony, Karen G.; Dübel, Stefan; Ida, Nobuo; Wiemann, Stefan; Beyreuther, Konrad; Frost, Laura S.; Little, Melvyn; Breitling, F.

doi:10.1006/jmbi.1998.1773

Cited by 11 publications

(6 citation statements)

References 58 publications

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“…3B). Residues 64 to 69 of VirB2 correspond to residues 17 to 22 of TraA F , a region also found to be exposed laterally on the pilus by bacteriophage binding and antibody labeling of immunodominant epitopes (30,52,60). The surface display of a stretch of hydrophobicity of VirB2 (marked by Cys77 labeling) is intriguing in view of observations that, in contrast to F pili, which dynamically extend and retract (18), T pili and related pili are sloughed or broken from the cell surface.…”

Section: Discussionmentioning

confidence: 96%

“…If the T pilus has a central lumen analogous to that detected for the F pilus (69,72), these hydrophilic regions might line the pilus lumen. Interestingly, whereas the central domain III of TraA F is also buried in the F pilus, its C terminus is surface displayed and can even accommodate epitope tags that are accessible to antibody (60). We were unable to generate functional forms of VirB2 bearing epitope tags near the N-C junction, preventing conclusions regarding surface display of this region of VirB2 (J. E. Kerr, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

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Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Kerr

Christie

2010

J Bacteriol

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Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Kerr

Christie

2010

J Bacteriol

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“…Immunogold electron microscopy. The procedure for immunogold electron microscopy was as described in Rondot et al (66). The monoclonal antibody (MAb) JEL92 (29) was used at a 1:500 dilution and gold-conjugated anti-mouse goat antibodies (EY Labs) were used at a 1:100 dilution.…”

Section: Methodsmentioning

confidence: 99%

Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation

et al. 1999

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F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.

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“…One of the potential advantages of such a system is that repeated exposure of one or more epitopes along the length of the pilus should increase immunogenicity. The possible use of pili for such purposes is not a new idea (11,39,41,51), but many previous studies failed because insertions prevented polymerization of the pilus filament because of the unpredictable structural constraints. It will be interesting to determine the length and number of the foreign peptides that can be tagged onto PulG, whether there are other permissive sites for epitope insertion, and whether inserted peptides are immunogenic when delivered either on whole bacteria or in purified pili.…”

Section: Pseudopili or Real Pili?mentioning

confidence: 99%

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

Vignon

Köhler

Larquet³

et al. 2003

J Bacteriol

118

148

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The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).The secreton or type II secretion (T2S) system permits the energy-dependent secretion of a limited number of specific proteins from the periplasm in gram-negative bacteria (33). Many of the 12 or more secreton components share extensive sequence similarity with components of the type IV piliation (T4P) system in gram-negative bacteria (24, 33). In the T2S system, these "shared" components include the pilins themselves (called pseudopilins in the secreton system) and prepilin peptidase, the enzyme that removes a short N-terminal peptide and then N-methylates type IV pilin precursors (25,29,35,37) and type IV pilin-like proteins. The N-terminal prepeptide and approximately 20-residue hydrophobic domains of the type IV pilins and pseudopilins are highly conserved (24, 33). In the pullulanase (PulA)-specific secreton from Klebsiella oxytoca, there are five type IV pseudopilins (PulG, PulH, Pull, PulJ, and PulK) (31,36,40).In the absence of any direct evidence for the assembly of pseudopilins into a pilus (34, 38), they were proposed to form a small, intraperiplasmic structure (the ...

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Epitopes fused to F-pilin are incorporated into functional recombinant pili

Cited by 11 publications

References 58 publications

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

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