scholarly journals A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence

2005 ◽  
Vol 73 (3) ◽  
pp. 1411-1422 ◽  
Author(s):  
Rohinee N. Paranjpye ◽  
Mark S. Strom
Keyword(s):  
Vibrio Vulnificus ◽  
Type Ii Secretion ◽  
Secretion Pathway ◽  
Type Iv ◽  
Secreted Factors ◽  
Pilus Biogenesis ◽  
Type Iv Pilin ◽  
Group A ◽  
Prepilin Peptidase ◽  
Reduced Adherence

ABSTRACT Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.

scholarly journals Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG

2007 ◽  
Vol 189 (17) ◽  
pp. 6389-6396 ◽  
Author(s):  
Richard F. Collins ◽  
Muhammad Saleem ◽  
Jeremy P. Derrick
Keyword(s):  
Electron Microscopy ◽  
Neisseria Meningitidis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower “waist” region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

scholarly journals Characterization of Type IV Pilus Genes in Pseudomonas syringae pv. tomato DC3000

1998 ◽  
Vol 11 (11) ◽  
pp. 1048-1056 ◽  
Author(s):  
Elina Roine ◽  
Deanna M. Raineri ◽  
Martin Romantschuk ◽  
Mark Wilson ◽  
David N. Nunn
Keyword(s):  
Pseudomonas Syringae ◽  
Lytic Bacteriophage ◽  
Type Iv Pilus ◽  
Tomato Dc3000 ◽  
Plant Leaf ◽  
Type Iv ◽  
Pilus Biogenesis ◽  
Population Sizes ◽  
Pila Gene ◽  
Prepilin Peptidase

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage φ6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only φ6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage φ6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

scholarly journals Type II Protein Secretion in Pseudomonas aeruginosa: the Pseudopilus Is a Multifibrillar and Adhesive Structure

2003 ◽  
Vol 185 (9) ◽  
pp. 2749-2758 ◽  
Author(s):  
Éric Durand ◽  
Alain Bernadac ◽  
Geneviève Ball ◽  
Andrée Lazdunski ◽  
James N. Sturgis ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Hydrolytic Enzymes ◽  
Macromolecular Complex ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Type Iv Pilin

ABSTRACT The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.

scholarly journals Type IV Pilin Proteins: Versatile Molecular Modules

2012 ◽  
Vol 76 (4) ◽  
pp. 740-772 ◽  
Author(s):  
Carmen L. Giltner ◽  
Ylan Nguyen ◽  
Lori L. Burrows
Keyword(s):  
Posttranslational Modifications ◽  
Electrical Conductance ◽  
Research Area ◽  
Type Ii Secretion ◽  
Dna Uptake ◽  
Multifunctional Protein ◽  
Type Iv ◽  
Type Iv Pilin ◽  
And Function ◽  
Related Proteins

SUMMARYType IV pili (T4P) are multifunctional protein fibers produced on the surfaces of a wide variety of bacteria and archaea. The major subunit of T4P is the type IV pilin, and structurally related proteins are found as components of the type II secretion (T2S) system, where they are called pseudopilins; of DNA uptake/competence systems in both Gram-negative and Gram-positive species; and of flagella, pili, and sugar-binding systems in the archaea. This broad distribution of a single protein family implies both a common evolutionary origin and a highly adaptable functional plan. The type IV pilin is a remarkably versatile architectural module that has been adopted widely for a variety of functions, including motility, attachment to chemically diverse surfaces, electrical conductance, acquisition of DNA, and secretion of a broad range of structurally distinct protein substrates. In this review, we consider recent advances in this research area, from structural revelations to insights into diversity, posttranslational modifications, regulation, and function.

scholarly journals Functional Consequences of Sequence Variation in Bundlin, the Enteropathogenic Escherichia coli Type IV Pilin Protein

2007 ◽  
Vol 75 (10) ◽  
pp. 4687-4696 ◽  
Author(s):  
Paula J. Fernandes ◽  
Qin Guo ◽  
Michael S. Donnenberg
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Sequence Divergence ◽  
Cross Reactivity ◽  
Human Antibody ◽  
Type Iv ◽  
Volunteer Study ◽  
Pilus Biogenesis ◽  
Type Iv Pilin ◽  
Pilin Protein

ABSTRACT The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor. We examined the role of divergent alleles of bfpA encoding bundlin, the BFP pilin protein, in pilus biogenesis, pilus interactions, and immune responses. We found that the BFP biogenesis machine from an EPEC strain that expresses one bundlin type is capable of assembling all other bundlin types. Furthermore, we found that EPEC strains expressing divergent bundlin types are capable of forming mixed autoaggregates, suggesting that different pilin types can intertwine. However, we found that there was a marked difference between alleles in immunogenicity in both rabbits and mice of a peptide derived from a region of bundlin undergoing apparent diversifying selection. In addition, despite a high degree of cross-reactivity between divergent bundlin proteins, in both mice and rabbits responses appeared to be stronger against the homologous pilin protein than against the heterologous protein. This result was verified using sera from a volunteer study, which demonstrated that the human antibody responses after an initial challenge with live EPEC were stronger against the homologous bundlin protein than against a divergent bundlin protein. However, a repeat challenge induced equivalent responses. These results are consistent with the hypothesis that human immune responses against bundlin exert selective pressure on bfpA sequence divergence.

scholarly journals A Type IV Pilin, PilA, Contributes to Adherence of Burkholderia pseudomallei and Virulence In Vivo

2005 ◽  
Vol 73 (2) ◽  
pp. 1260-1264 ◽  
Author(s):  
Angela E. Essex-Lopresti ◽  
Justin A. Boddey ◽  
Richard Thomas ◽  
Martin P. Smith ◽  
M. Gill Hartley ◽  
...  
Keyword(s):  
Murine Model ◽  
Burkholderia Pseudomallei ◽  
Deletion Mutant ◽  
Type Iv Pili ◽  
Structural Protein ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Reduced Adherence ◽  
Multiple Type

ABSTRACT The Burkholderia pseudomallei K96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). A pilA deletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili in B. pseudomallei virulence.

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

2003 ◽  
Vol 185 (11) ◽  
pp. 3416-3428 ◽  
Author(s):  
Guillaume Vignon ◽  
Rolf Köhler ◽  
Eric Larquet ◽  
Stéphanie Giroux ◽  
Marie-Christine Prévost ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Type Ii Secretion ◽  
Type Ii ◽  
High Level Expression ◽  
Polar Localization ◽  
Type Iv ◽  
C Terminus ◽  
Type Iv Pilin ◽  
High Level ◽  
K 12

ABSTRACT 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).

scholarly journals Role of Anaerobiosis in Capsule Production and Biofilm Formation in Vibrio vulnificus

2014 ◽  
Vol 83 (2) ◽  
pp. 551-559 ◽  
Author(s):  
Britney L. Phippen ◽  
James D. Oliver
Keyword(s):  
Biofilm Formation ◽  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Human Infection ◽  
Content Type ◽  
Type Iv ◽  
Virulence Potential ◽  
Capsule Production ◽  
Type Iv Pilin ◽  
Effect Of Oxygen

Vibrio vulnificus, a pervasive human pathogen, can cause potentially fatal septicemia after consumption of undercooked seafood. Biotype 1 strains ofV. vulnificusare most commonly associated with human infection and are separated into two genotypes, clinical (C) and environmental (E), based on the virulence-correlated gene. For ingestion-based vibriosis to occur, this bacterium must be able to withstand multiple conditions as it traverses the gastrointestinal tract and ultimately gains entry into the bloodstream. One such condition, anoxia, has yet to be extensively researched inV. vulnificus. We investigated the effect of oxygen availability on capsular polysaccharide (CPS) production and biofilm formation in this bacterium, both of which are thought to be important for disease progression. We found that lack of oxygen elicits a reduction in both CPS and biofilm formation in both genotypes. This is further supported by the finding thatpilA,pilD, andmshAgenes, all of which encode type IV pilin proteins that aid in attachment to surfaces, were downregulated during anaerobiosis. Surprisingly, E-genotypes exhibited distinct differences in gene expression levels of capsule and attachment genes compared to C-genotypes, both aerobically and anaerobically. The importance of understanding these disparities may give insight into the observed differences in environmental occurrence and virulence potential between these two genotypes ofV. vulnificus.

scholarly journals Role of Type IV Pilins in Persistence of Vibrio vulnificus in Crassostrea virginica Oysters

2007 ◽  
Vol 73 (15) ◽  
pp. 5041-5044 ◽  
Author(s):  
Rohinee N. Paranjpye ◽  
Asta B. Johnson ◽  
Anne E. Baxter ◽  
Mark S. Strom
Keyword(s):  
United States ◽  
Epithelial Cells ◽  
Crassostrea Virginica ◽  
Biofilm Formation ◽  
Vibrio Vulnificus ◽  
The United States ◽  
Type Iv ◽  
Abiotic Surfaces ◽  
Prepilin Peptidase

ABSTRACT Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters.

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