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Hendrickx, Antoni P. A., Claudia M. E. Schapendonk, Miranda van Luit-Asbroek, Marc J. M. Bonten, Willem van Schaik i Rob J. L. Willems. "Differential PilA pilus assembly by a hospital-acquired and a community-derived Enterococcus faecium isolate". Microbiology 156, nr 9 (1.09.2010): 2649–59. http://dx.doi.org/10.1099/mic.0.041392-0.
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Pili are hair-like structures protruding from the cell envelope of bacterial cells. Here, we describe the conditional and differential display of PilA-type pili, and PilE and PilF proteins, encoded from pilin gene cluster 1 at the surface of a hospital-acquired Enterococcus faecium bloodstream isolate (E1165) and a community-derived stool isolate (E1039), at two different temperatures. Both strains have virtually identical pilA gene clusters, as determined by sequencing. Western blotting and transmission immunoelectron microscopy revealed that PilA and PilF assembled into high-molecular-mass pilus-like structures at 37 °C in the E1165 strain, whereas PilE was not produced at either of the temperatures used; at 21 °C, PilA and PilF were cell-wall-anchored proteins. In contrast, in strain E1039, PilA, PilE and PilF pilin proteins were found to be displayed as cell-wall-anchored proteins at 37 °C only, and they were not associated with pilus-like structures. The discrepancy in pilus assembly between E1039 and E1165 cannot be explained by differences in expression of the genes encoding the predicted sortases in the pilA gene cluster, as these had similar expression levels in both strains at 21 and 37 °C. Double-labelling electron microscopy revealed that PilA formed the pilus backbone in E1165, and PilF the minor subunit which was distributed along the PilA pilus shaft and positioned at the tip; however, it was deposited as a cell-wall-anchored protein in a pilA isogenic mutant. The differential deposition of surface proteins from pilin gene cluster 1 and differences in pilus assembly in the two strains suggest a complex post-transcriptional regulatory mechanism of pilus biogenesis in E. faecium.
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Sauvonnet, Nathalie, Pierre Gounon i Anthony P. Pugsley. "PpdD Type IV Pilin of Escherichia coliK-12 Can Be Assembled into Pili in Pseudomonas aeruginosa". Journal of Bacteriology 182, nr 3 (1.02.2000): 848–54. http://dx.doi.org/10.1128/jb.182.3.848-854.2000.
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ABSTRACT Escherichia coli K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However, E. coli K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putativeE. coli piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (ppdD) and the linked assembly genes hofB andhofC (homologues of the Pseudomonas aeruginosatype IV pilus assembly genes pilB and pilC) did not lead to pilus production. However, expression of theppdD gene in P. aeruginosa led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in P. aeruginosawas dependent on the expression of the pilB andpilC genes and independent of expression of the P. aeruginosa pilin structural gene pilA.
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Graupner, Stefan, Verena Frey, Rozita Hashemi, Michael G. Lorenz, Gudrun Brandes i Wilfried Wackernagel. "Type IV Pilus Genes pilA andpilC of Pseudomonas stutzeri Are Required for Natural Genetic Transformation, and pilA Can Be Replaced by Corresponding Genes from Nontransformable Species". Journal of Bacteriology 182, nr 8 (15.04.2000): 2184–90. http://dx.doi.org/10.1128/jb.182.8.2184-2190.2000.
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ABSTRACT Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB andpilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilAabolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of 3H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC andpilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosusand the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.
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Bertrand, Jacob J., Joyce T. West i Joanne N. Engel. "Genetic Analysis of the Regulation of Type IV Pilus Function by the Chp Chemosensory System of Pseudomonas aeruginosa". Journal of Bacteriology 192, nr 4 (11.12.2009): 994–1010. http://dx.doi.org/10.1128/jb.01390-09.
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ABSTRACT The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export.
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Villar, Maria T., Jennifer T. Helber, Becky Hood, Michael R. Schaefer i Rona L. Hirschberg. "Eikenella corrodens Phase Variation Involves a Posttranslational Event in Pilus Formation". Journal of Bacteriology 181, nr 14 (15.07.1999): 4154–60. http://dx.doi.org/10.1128/jb.181.14.4154-4160.1999.
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ABSTRACT The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designatedpilA1, pilA2, pilB, andhagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagAresembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundantpilA1 transcript initiating upstream of pilA1and terminating at a predicted hairpin structure betweenpilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassingpilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.
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Li, Yinuo, Renate Lux, Andrew E. Pelling, James K. Gimzewski i Wenyuan Shi. "Analysis of type IV pilus and its associated motility in Myxococcus xanthus using an antibody reactive with native pilin and pili". Microbiology 151, nr 2 (1.02.2005): 353–60. http://dx.doi.org/10.1099/mic.0.27614-0.
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Myxococcus xanthus possesses a social gliding motility that requires type IV pili (TFP). According to the current model, M. xanthus pili attach to an external substrate and retract, pulling the cell body forward along their long axis. By analogy with the situation in other bacteria employing TFP-dependent motility, M. xanthus pili have been assumed to be composed of pilin (PilA) subunits, but this has not previously been confirmed. The first 28 amino acids of the M. xanthus PilA protein share extensive homology with the N-terminal oligomerization domain of pilins in other bacterial species. To facilitate purification, the authors engineered a truncated form of M. xanthus PilA lacking the first 28 amino acids and purified this protein in soluble form. Polyclonal antibody generated against this protein was reactive with native pilin and pili. Using this antibody, it was confirmed that TFP of M. xanthus are indeed composed of PilA, and that TFP are located unipolarly and required for social gliding motility via retraction. Using tethering as well as motility assays, details of pili function in M. xanthus social motility were further examined.
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Scheuerpflug, Ina, Thomas Rudel, Roland Ryll, Jasmine Pandit i Thomas F. Meyer. "Roles of PilC and PilE Proteins in Pilus-Mediated Adherence of Neisseria gonorrhoeae and Neisseria meningitidis to Human Erythrocytes and Endothelial and Epithelial Cells". Infection and Immunity 67, nr 2 (1.02.1999): 834–43. http://dx.doi.org/10.1128/iai.67.2.834-843.1999.
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ABSTRACT Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coliinteracted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.
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Jelsbak, Lotte, i Dale Kaiser. "Regulating Pilin Expression Reveals a Threshold for S Motility in Myxococcus xanthus". Journal of Bacteriology 187, nr 6 (15.03.2005): 2105–12. http://dx.doi.org/10.1128/jb.187.6.2105-2112.2005.
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ABSTRACT An isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a ΔpilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the ΔpilA strain to wild-type levels. The rate of S-motile swarming was found to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.
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Bakaletz, Lauren O., Beth D. Baker, Joseph A. Jurcisek, Alistair Harrison, Laura A. Novotny, James E. Bookwalter, Rachna Mungur i Robert S. Munson. "Demonstration of Type IV Pilus Expression and a Twitching Phenotype by Haemophilus influenzae". Infection and Immunity 73, nr 3 (marzec 2005): 1635–43. http://dx.doi.org/10.1128/iai.73.3.1635-1643.2005.
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ABSTRACT Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.
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Macdonald, D. L., B. L. Pasloske i W. Paranchych. "Mutations in the fifth-position glutamate in Pseudomonas aeruginosa pilin affect the transmethylation of the N-terminal phenylalanine". Canadian Journal of Microbiology 39, nr 5 (1.05.1993): 500–505. http://dx.doi.org/10.1139/m93-071.
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The pili of Pseudomonas aeruginosa are composed of 15-kDa pilin monomers that are synthesized in the cytoplasm and assembled in the membrane. Processing occurs between the synthesis and assembly steps. The propilin is cleaved by a unique leader peptidase encoded by pilD, which is adjacent to the pilin structural gene pilA. This generates an N-terminal phenylalanine that is subsequently methylated by an as yet uncharacterized transmethylase. The pili of P. aeruginosa belong to the type IV class of pilins, which share a highly conserved N-terminal region 35 amino acids in length, containing a short leader of 6 or 7 amino acids. Two site-specific mutants in the N-terminal region of the mature pilin were constructed. Reestablishing the fifth-position glutamate in a four amino acid deletion mutant (amino acids 4–7) restored the leader peptidase cleavage but not the methylation. A mutation of the fifth-position glutamate to alanine decreased the degree of methylation of the N-terminal phenylalanine. Pili were not assembled by these mutants as assessed by electron microscopy and sensitivity to pilus-specific bacteriophage. Methylation may be required for recognition of the pilin by the assembly machinery and is not residue specific. The fifth-position glutamate appears to play an important role in transmethylase recognition of the pilin subunit.Key words: Pseudomonas aeruginosa, pilin, transmethylase.
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Hansen, Johanna K., Karen P. Demick, John M. Mansfield i Katrina T. Forest. "Conserved Regions from Neisseria gonorrhoeae Pilin Are Immunosilent and Not Immunosuppressive". Infection and Immunity 75, nr 8 (11.06.2007): 4138–47. http://dx.doi.org/10.1128/iai.02015-06.
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ABSTRACT PilE is the primary subunit of type IV pili from Neisseria gonorrhoeae and contains a surface-exposed hypervariable region thought to be one feature of pili that has prevented development of a pilin-based vaccine. We have created a three-dimensional structure-based antigen by replacing the hypervariable region of PilE with an aspartate-glutamine linker chosen from the sequence of Pseudomonas aeruginosa PilA. We then characterized murine immune responses to this novel protein to determine if conserved PilE regions could serve as a vaccine candidate. The control PilE protein elicited strong T-cell-dependent B-cell responses that are specific to epitopes in both the hypervariable deletion and control proteins. In contrast, the hypervariable deletion protein was unable to elicit an immune response in mice, suggesting that in the absence of the hypervariable region, the conserved regions of PilE alone are not sufficient for antibody production. Further analysis of these PilE proteins with suppressor cell assays showed that neither suppresses T- or B-cell responses, and flow cytometry experiments suggested that they do not exert suppressor effects by activating T regulatory cells. Our results show that in the murine model, the hypervariable region of PilE is required to activate immune responses to pilin, whereas the conserved regions are unusually nonimmunogenic. In addition, we show that both hypervariable and conserved regions of pilin are not suppressive, suggesting that PilE does not cause the decrease in T-cell populations observed during gonococcal cervicitis.
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Stone, Barbara J., i Yousef Abu Kwaik. "Natural Competence for DNA Transformation by Legionella pneumophila and Its Association with Expression of Type IV Pili". Journal of Bacteriology 181, nr 5 (1.03.1999): 1395–402. http://dx.doi.org/10.1128/jb.181.5.1395-1402.1999.
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ABSTRACT We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768–1775, 1998). Production of long pili requires a functional pilE L locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeaeare associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophilaafter addition of plasmid DNA, including gspA,ppa, asd, and pilE L. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and apilE L mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilE Lalone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).
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Luke, Nicole R., Amy J. Howlett, Jianqiang Shao i Anthony A. Campagnari. "Expression of Type IV Pili by Moraxella catarrhalis Is Essential for Natural Competence and Is Affected by Iron Limitation". Infection and Immunity 72, nr 11 (listopad 2004): 6262–70. http://dx.doi.org/10.1128/iai.72.11.6262-6270.2004.
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ABSTRACT Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.
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Winther-Larsen, Hanne C., Matthew C. Wolfgang, Jos P. M. van Putten, Norbert Roos, Finn Erik Aas, Wolfgang M. Egge-Jacobsen, Berenike Maier i Michael Koomey. "Pseudomonas aeruginosa Type IV Pilus Expression in Neisseria gonorrhoeae: Effects of Pilin Subunit Composition on Function and Organelle Dynamics". Journal of Bacteriology 189, nr 18 (15.06.2007): 6676–85. http://dx.doi.org/10.1128/jb.00407-07.
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ABSTRACT Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilAPAK pilin subunit in N. gonorrhoeae. We show here that, although PilAPAK pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilAPAK pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilAPAK pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilAPAK pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilAPAK pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.
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Chiang, Poney, Marc Habash i Lori L. Burrows. "Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility". Journal of Bacteriology 187, nr 3 (1.02.2005): 829–39. http://dx.doi.org/10.1128/jb.187.3.829-839.2005.
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ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.
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Mahmoud, Khaled K., i Susan F. Koval. "Characterization of type IV pili in the life cycle of the predator bacterium Bdellovibrio". Microbiology 156, nr 4 (1.04.2010): 1040–51. http://dx.doi.org/10.1099/mic.0.036137-0.
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Bdellovibrio and like organisms (BALOs) are obligate prokaryotic predators of other Gram-negative bacteria. Bdellovibrio bacteriovorus is the most studied organism among BALOs. It has a periplasmic life cycle with two major stages: a motile, non-replicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the Gram-negative prey cell (the growth phase) after forming an osmotically stable body termed the bdelloplast. Within Bdellovibrio, there are also strains exhibiting an epibiotic life cycle. The genome sequence of the type strain B. bacteriovorus HD100T revealed the presence of multiple dispersed pil genes encoding type IV pili. Type IV pili in other bacteria are involved in adherence to and invasion of host cells and therefore can be considered to play a role in invasion of prey cells by Bdellovibrio. In this study, genes involved in producing type IV pili were identified in the periplasmic strain B. bacteriovorus 109J and an epibiotic Bdellovibrio sp. strain JSS. The presence of fibres on attack-phase cells was confirmed by examining negative stains of cells fixed with 10 % buffered formalin. Fibres were at the non-flagellated pole on approximately 25 % of attack-phase cells. To confirm that these fibres were type IV pili, a truncated form of PilA lacking the first 35 amino acids was designed to facilitate purification of the protein. The truncated PilA fused to a His-tag was overexpressed in Escherichia coli BL21(DE3) plysS. The fusion protein, accumulated in the insoluble fraction, was purified under denaturing conditions and used to produce polyclonal antisera. Immunoelectron microscopy showed that polar fibres present on the cell surface of the predator were composed of PilA, the major subunit of type IV pili. Immunofluorescence microscopy showed the presence of pilin on attack-phase cells of B. bacteriovorus 109J during attachment to prey cells and just after penetration, inside the bdelloplast. Antibodies against PilA delayed and inhibited predation in co-cultures of Bdellovibrio. This study confirms that type IV pili play a role in invasion of prey cells by Bdellovibrio.
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Long, Cynthia D., Stanley F. Hayes, Jos P. M. van Putten, Hillery A. Harvey, Michael A. Apicella i H. Steven Seifert. "Modulation of Gonococcal Piliation by Regulatable Transcription of pilE". Journal of Bacteriology 183, nr 5 (1.03.2001): 1600–1609. http://dx.doi.org/10.1128/jb.183.5.1600-1609.2001.
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ABSTRACT The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (ΔpilE1) in which the lacIOPregulatory sequences control pilE transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-β-d-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level ofpilE transcription, without a change in pilEsequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.
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Liu, Xing, Pier-Luc Tremblay, Nikhil S. Malvankar, Kelly P. Nevin, Derek R. Lovley i Madeline Vargas. "A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production". Applied and Environmental Microbiology 80, nr 3 (2.12.2013): 1219–24. http://dx.doi.org/10.1128/aem.02938-13.
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ABSTRACTThe conductive pili ofGeobacterspecies play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili ofGeobacter sulfurreducenshave a metal-like conductivity, independent of the presence ofc-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain ofG. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene ofPseudomonas aeruginosaPAO1. Strain PA expressed and properly assembledP. aeruginosaPilA subunits into pili and exhibited a profile of outer surfacec-type cytochromes similar to that of a control strain expressing theG. sulfurreducensPilA. Surprisingly, the strain PA pili were decorated with thec-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of theG. sulfurreducensPilA that are necessary for conductivity.
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Baynham, Patricia J., Deborah M. Ramsey, Borys V. Gvozdyev, Ellen M. Cordonnier i Daniel J. Wozniak. "The Pseudomonas aeruginosa Ribbon-Helix-Helix DNA-Binding Protein AlgZ (AmrZ) Controls Twitching Motility and Biogenesis of Type IV Pili". Journal of Bacteriology 188, nr 1 (1.01.2006): 132–40. http://dx.doi.org/10.1128/jb.188.1.132-140.2006.
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ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).
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Brissac, Terry, Guillain Mikaty, Guillaume Duménil, Mathieu Coureuil i Xavier Nassif. "The Meningococcal Minor Pilin PilX Is Responsible for Type IV Pilus Conformational Changes Associated with Signaling to Endothelial Cells". Infection and Immunity 80, nr 9 (9.07.2012): 3297–306. http://dx.doi.org/10.1128/iai.00369-12.
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ABSTRACTNeisseria meningitidiscrosses the blood-brain barrier (BBB) following the activation of the β2-adrenergic receptor by the type IV pili (TFP). Two components of the type IV pili recruit the β2-adrenergic receptor, the major pilin PilE and the minor pilin PilV. Here, we report that a strain deleted of PilX, one of the three minor pilins, is defective in endothelial cell signaling. The signaling role of PilX was abolished when pili were not retractable. Purified PilX was unable to recruit the β2-adrenergic receptor, thus suggesting that PilX was playing an indirect role in endothelial cell signaling. Considering the recent finding that type IV pili can transition into a new conformation (N. Biais, D. L. Higashi, J. Brujic, M. So, and M. P. Sheetz, Proc. Natl. Acad. Sci. U. S. A. 107:11358–11363, 2010), we hypothesized that PilX was responsible for a structural modification of the fiber and allowed hidden epitopes to be exposed. To confirm this hypothesis, we showed that a monoclonal antibody which recognizes a linear epitope of PilE bound fibers only when bacteria adhered to endothelial cells. On the other hand, this effect was not observed in PilX-deleted pili. A deletion of a region of PilX exposed on the surface of the fiber had phenotypical consequences identical to those of a PilX deletion. These data support a model in which surface-exposed motifs of PilX use forces generated by pilus retraction to promote conformational changes required for TFP-mediated signaling.
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Liles, Mark R., V. K. Viswanathan i Nicholas P. Cianciotto. "Identification and Temperature Regulation ofLegionella pneumophila Genes Involved in Type IV Pilus Biogenesis and Type II Protein Secretion". Infection and Immunity 66, nr 4 (1.04.1998): 1776–82. http://dx.doi.org/10.1128/iai.66.4.1776-1782.1998.
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ABSTRACT Previously, we had isolated by transposon mutagenesis aLegionella pneumophila mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong similarity to PilB fromPseudomonas aeruginosa. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when L. pneumophila was grown at 30°C as opposed to 37°C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the pilBlocus was conserved within L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.
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Yoshida, Tetsu, Nobuhisa Furuya, Masayuki Ishikura, Toshiaki Isobe, Kazu Haino-Fukushima, Toshio Ogawa i Teruya Komano. "Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili". Journal of Bacteriology 180, nr 11 (1.06.1998): 2842–48. http://dx.doi.org/10.1128/jb.180.11.2842-2848.1998.
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ABSTRACT Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of thepilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of thepilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.
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Daehnel, Katrin, Robin Harris, Lucinda Maddera i Philip Silverman. "Fluorescence assays for F-pili and their application". Microbiology 151, nr 11 (1.11.2005): 3541–48. http://dx.doi.org/10.1099/mic.0.28159-0.
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Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell–cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0·1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.
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Villar, Maria T., Rona L. Hirschberg i Michael R. Schaefer. "Role of the Eikenella corrodens pilALocus in Pilus Function and Phase Variation". Journal of Bacteriology 183, nr 1 (1.01.2001): 55–62. http://dx.doi.org/10.1128/jb.183.1.55-62.2001.
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ABSTRACT The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolateE. corrodens VA1. Earlier work defined the pilAlocus which includes pilA1, pilA2,pilB, and hagA. Both pilA1 andpilA2 predict a type IV pilin, whereas pilBpredicts a putative pilus assembly protein. The role ofhagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilAmutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated forpilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation ofhagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.
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Asikyan, Miranda L., Julianne V. Kus i Lori L. Burrows. "Novel Proteins That Modulate Type IV Pilus Retraction Dynamics in Pseudomonas aeruginosa". Journal of Bacteriology 190, nr 21 (5.09.2008): 7022–34. http://dx.doi.org/10.1128/jb.00938-08.
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ABSTRACTPseudomonas aeruginosauses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles ofpilAinP. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1pilAmutant. Expression of group I or IVpilAgenes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group VpilAgenes but increased significantly when the cognatetfpYortfpZaccessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1pilA-pilTdouble mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed fortfpYsingle andtfpY-pilTdouble knockout mutants of group III strain PA14. A PilAV-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.
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Paranjpye, Rohinee N., i Mark S. Strom. "A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence". Infection and Immunity 73, nr 3 (marzec 2005): 1411–22. http://dx.doi.org/10.1128/iai.73.3.1411-1422.2005.
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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.
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Essex-Lopresti, Angela E., Justin A. Boddey, Richard Thomas, Martin P. Smith, M. Gill Hartley, Timothy Atkins, Nat F. Brown i in. "A Type IV Pilin, PilA, Contributes to Adherence of Burkholderia pseudomallei and Virulence In Vivo". Infection and Immunity 73, nr 2 (luty 2005): 1260–64. http://dx.doi.org/10.1128/iai.73.2.1260-1264.2005.
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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.
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Tan, Felicia Y. Y., Mirka E. Wörmann, Edmund Loh, Christoph M. Tang i Rachel M. Exley. "Characterization of a Novel Antisense RNA in the Major Pilin Locus of Neisseria meningitidis Influencing Antigenic Variation". Journal of Bacteriology 197, nr 10 (9.03.2015): 1757–68. http://dx.doi.org/10.1128/jb.00082-15.
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ABSTRACTExpression of type four pili (Tfp) is essential for virulence inNeisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. InN. meningitidis, the major pilin subunit is encoded by thepilEgene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in thepilElocus, 3′ of thepilEcoding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entirepilEcoding sequence and 5′ untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes inpilEtranscript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenicNeisseria.IMPORTANCEPathogenicNeisseriaspp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains ofN. meningitidisare able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to thepilEgene. In this work, we identified acis-encoded RNA antisense topilEinN. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affectpilEor pilin expression levels but instead appears to modulate pilin variation.
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Harvey, Hanjeong, Marc Habash, Francisca Aidoo i Lori L. Burrows. "Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility". Journal of Bacteriology 191, nr 21 (28.08.2009): 6513–24. http://dx.doi.org/10.1128/jb.00943-09.
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ABSTRACT PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.
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Paranjpye, Rohinee N., J. Cano Lara, Jeffrey C. Pepe, Cynthia M. Pepe i Mark S. Strom. "The Type IV Leader Peptidase/ N -Methyltransferase ofVibrio vulnificus Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice". Infection and Immunity 66, nr 12 (1.12.1998): 5659–68. http://dx.doi.org/10.1128/iai.66.12.5659-5668.1998.
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ABSTRACT Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/ N -methyltransferase (PilD) mutant ofPseudomonas aeruginosa with a V. vulnificusgenomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC fromP. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificusrequires type IV pili and/or extracellular secretion of several exoenzymes.
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van Schaik, Erin J., Carmen L. Giltner, Gerald F. Audette, David W. Keizer, Daisy L. Bautista, Carolyn M. Slupsky, Brian D. Sykes i Randall T. Irvin. "DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili". Journal of Bacteriology 187, nr 4 (15.02.2005): 1455–64. http://dx.doi.org/10.1128/jb.187.4.1455-1464.2005.
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ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.
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Ilver, Dag, Helena Källström, Staffan Normark i Ann-Beth Jonsson. "Transcellular Passage of Neisseria gonorrhoeae Involves Pilus Phase Variation". Infection and Immunity 66, nr 2 (1.02.1998): 469–73. http://dx.doi.org/10.1128/iai.66.2.469-473.1998.
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ABSTRACT Piliated and nonpiliated Neisseria gonorrhoeaeorganisms were added on top of confluent layers of HEC-1-B cells, each maintained on a microporous Transwell-COL membrane. The bacteria released into the lower chamber were characterized with respect to the following virulence determinants: pili, which mediate adherence to target host cells; PilE, the major pilus subunit protein; and PilC, which is involved in pilus biogenesis and adherence. Even if >99% of the added bacteria of N. gonorrhoeae MS11 were piliated, bacteria recovered on the other side of the cell layer were predominantly nonpiliated. The recovered clones still expressed unassembled PilE protein, but 50% had lost PilC production. Nonpiliated gonococci, in which the 5′ end of pilE had been deleted, were released in reduced numbers, and piliatedrecA bacteria added to the cell layer were not released at all, at time points when piliated recA + clones were found at high numbers in the lower chamber. Our data indicate that bacteria producing unassembled PilE protein are selected for during passage through an epithelial cell layer. The finding that thepilE gene sequence was altered in the transmigrants suggests that pilin sequence variation is involved in the transcellular passage of N. gonorrhoeae.
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Takahashi, Hideyuki, Tatsuo Yanagisawa, Kwang Sik Kim, Shigeyuki Yokoyama i Makoto Ohnishi. "Meningococcal PilV Potentiates Neisseria meningitidis Type IV Pilus-Mediated Internalization into Human Endothelial and Epithelial Cells". Infection and Immunity 80, nr 12 (17.09.2012): 4154–66. http://dx.doi.org/10.1128/iai.00423-12.
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ABSTRACTThe type IV pilus ofNeisseria meningitidisis the major factor for meningococcal adhesion to host cells. In this study, we showed that a mutant ofN. meningitidis pilV, a minor pilin protein, internalized less efficiently to human endothelial and epithelial cells than the wild-type strain. Matrix-assisted laser desorption ionization–time of flight mass spectrometry and electrospray ionization tandem mass spectrometry analyses showed that PilE, the major subunit of pili, was less glycosylated at its serine 62 residue (Ser62) in the ΔpilVmutant than in thepilV+strain, whereas phosphoglycerol at PilE Ser93 and phosphocholine at PilE Ser67 were not changed. Introduction of thepglLmutation, which results in complete loss of O-linked glycosylation from Ser62, slightly reducedN. meningitidisinternalization into human brain microvascular endothelial cells, whereas the addition of the ΔpilVmutation greatly reducedN. meningitidisinternalization. The accumulation of ezrin, which is part of the cytoskeleton ERM family, was observed withpilV+, ΔpglL, andpilE(S62A) strains but not with the ΔpilVmutant. These results suggested that whereasN. meningitidispilin originally had an adhesive activity that was less affected by minor pilin proteins, the invasive function evolved with incorporation of the PilV protein into the pili to promote theN. meningitidisinternalization into human cells.
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Meng, Yizhi, Yaxin Li, Cheryl D. Galvani, Guixia Hao, James N. Turner, Thomas J. Burr i H. C. Hoch. "Upstream Migration of Xylella fastidiosa via Pilus-Driven Twitching Motility". Journal of Bacteriology 187, nr 16 (15.08.2005): 5560–67. http://dx.doi.org/10.1128/jb.187.16.5560-5567.2005.
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ABSTRACT Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 μm min−1 against a rapidly flowing medium (20,000 μm min−1). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 μm) and short type I pili (0.4 to 1.0 μm). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.
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Maisey, Heather C., Mary Hensler, Victor Nizet i Kelly S. Doran. "Group B Streptococcal Pilus Proteins Contribute to Adherence to and Invasion of Brain Microvascular Endothelial Cells". Journal of Bacteriology 189, nr 4 (13.10.2006): 1464–67. http://dx.doi.org/10.1128/jb.01153-06.
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ABSTRACT Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.
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Evans, Katy J., Carey Lambert i R. Elizabeth Sockett. "Predation by Bdellovibrio bacteriovorus HD100 Requires Type IV Pili". Journal of Bacteriology 189, nr 13 (6.04.2007): 4850–59. http://dx.doi.org/10.1128/jb.01942-06.
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ABSTRACT Early electron microscopy and more recent studies in our laboratory of Bdellovibrio bacteriovorus cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the B. bacteriovorus HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of B. bacteriovorus HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The pilA gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple Bdellovibrio replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of pilA in replicate isolates abolished Bdellovibrio predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of pil genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory Bdellovibrio throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in Bdellovibrio predation.
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Shimoda, Eriko, Tatsuya Muto, Takayuki Horiuchi, Nobuhisa Furuya i Teruya Komano. "Novel Class of Mutations of pilS Mutants, Encoding Plasmid R64 Type IV Prepilin: Interface of PilS-PilV Interactions". Journal of Bacteriology 190, nr 4 (7.12.2007): 1202–8. http://dx.doi.org/10.1128/jb.01204-07.
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ABSTRACT The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.
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Nunn, D., S. Bergman i S. Lory. "Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili." Journal of Bacteriology 172, nr 6 (1990): 2911–19. http://dx.doi.org/10.1128/jb.172.6.2911-2919.1990.
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Wall, Daniel, Samuel S. Wu i Dale Kaiser. "Contact Stimulation of Tgl and Type IV Pili inMyxococcus xanthus". Journal of Bacteriology 180, nr 3 (1.02.1998): 759–61. http://dx.doi.org/10.1128/jb.180.3.759-761.1998.
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ABSTRACT Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl + cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.
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Kehl-Fie, Thomas E., Sara E. Miller i Joseph W. St. Geme. "Kingella kingae Expresses Type IV Pili That Mediate Adherence to Respiratory Epithelial and Synovial Cells". Journal of Bacteriology 190, nr 21 (29.08.2008): 7157–63. http://dx.doi.org/10.1128/jb.00884-08.
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ABSTRACTKingella kingaeis a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency ofK. kingaedisease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed thatK. kingaeexpresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by apilAhomolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of theK. kingaegenome, we identified two genes in physically separate locations on the chromosome that encode homologs of theNeisseriaPilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of theK. kingaePilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediatingK. kingaeadherence.
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Yang, Zhe, Wei Hu, Kevin Chen, Jing Wang, Renate Lux, Z. Hong Zhou i Wenyuan Shi. "Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus". Microbiology 157, nr 7 (1.07.2011): 1920–28. http://dx.doi.org/10.1099/mic.0.049684-0.
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Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
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Lucas, Claressa E., Ellen Brown i Barry S. Fields. "Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention". Microbiology 152, nr 12 (1.12.2006): 3569–73. http://dx.doi.org/10.1099/mic.0.2006/000497-0.
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Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, which displays reduced intracellular replication, was able to establish itself and persist in the presence of amoebae. Thus, type IV pili and the pre-pilin peptidase facilitate L. pneumophila colonization of biofilms but are not required in the presence of a host for intracellular replication.
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Paranjpye, Rohinee N., Asta B. Johnson, Anne E. Baxter i Mark S. Strom. "Role of Type IV Pilins in Persistence of Vibrio vulnificus in Crassostrea virginica Oysters". Applied and Environmental Microbiology 73, nr 15 (8.06.2007): 5041–44. http://dx.doi.org/10.1128/aem.00641-07.
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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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Smedley, James G., Erica Jewell, Jennifer Roguskie, Joseph Horzempa, Andrew Syboldt, Donna Beer Stolz i Peter Castric. "Influence of Pilin Glycosylation on Pseudomonas aeruginosa 1244 Pilus Function". Infection and Immunity 73, nr 12 (grudzień 2005): 7922–31. http://dx.doi.org/10.1128/iai.73.12.7922-7931.2005.
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ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.
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Mu, Xiang-Qi, Edward H. Egelman i Esther Bullitt. "Structure and Function of Hib Pili from Haemophilus influenzae Type b". Journal of Bacteriology 184, nr 17 (1.09.2002): 4868–74. http://dx.doi.org/10.1128/jb.184.17.4868-4874.2002.
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ABSTRACT Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.
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Orellana, Roberto, Janet J. Leavitt, Luis R. Comolli, Roseann Csencsits, Noemie Janot, Kelly A. Flanagan, Arianna S. Gray i in. "U(VI) Reduction by Diverse Outer Surfacec-Type Cytochromes of Geobacter sulfurreducens". Applied and Environmental Microbiology 79, nr 20 (9.08.2013): 6369–74. http://dx.doi.org/10.1128/aem.02551-13.
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ABSTRACTEarly studies withGeobacter sulfurreducenssuggested that outer-surfacec-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain ofG. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surfacec-type cytochromes ofG. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested thatG. sulfurreducensrequires outer-surfacec-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.
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Chakraborty, Subhra, Michael Monfett, Tamara M. Maier, Jorge L. Benach, Dara W. Frank i David G. Thanassi. "Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence". Infection and Immunity 76, nr 7 (21.04.2008): 2852–61. http://dx.doi.org/10.1128/iai.01726-07.
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ABSTRACT Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.
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Salomonsson, Emelie, Åke Forsberg, Norbert Roos, Claudia Holz, Berenike Maier, Michael Koomey i Hanne C. Winther-Larsen. "Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae". Microbiology 155, nr 8 (1.08.2009): 2546–59. http://dx.doi.org/10.1099/mic.0.028183-0.
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Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure–function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.
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Tytgat, Hanne L. P., Pia Rasinkangas, Jarmo Ritari, Justus Reunanen, Steven Aalvink, Chia-wei Lin, Airi Palva, François P. Douillard i Willem M. de Vos. "Selection and characterization of a SpaCBA pilus-secreting food-grade derivative of Lacticaseibacillus rhamnosus GG". Applied Microbiology and Biotechnology 105, nr 3 (8.01.2021): 1123–31. http://dx.doi.org/10.1007/s00253-020-11051-7.
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AbstractMany studies have established the functional properties of Lacticaseibacillus rhamnosus GG, previously known as Lactobacillus rhamnosus GG, marketed worldwide as a probiotic. The extraordinary capacity of L. rhamnosus GG to bind to human mucus and influence the immune system especially stand out. Earlier, we have shown the key role of its SpaCBA sortase-dependent pili encoded by the spaCBA-srtC1 gene cluster herein. These heterotrimeric pili consist of a shaft pilin SpaA, a basal pilin SpaB, and tip pilin SpaC that contains a mucus-binding domain. Here, we set out to characterize a food-grade non-GMO mutant of L. rhamnosus GG, strain PA11, which secretes its pilins, rather than coupling them to the cell surface, due to a defect in the housekeeping sortase A. The sortase-negative strain PA11 was extensively characterized using functional genomics and biochemical approaches and found to secrete the SpaCBA pili into the supernatant. Given the functional importance and uniqueness of the mucus-binding pili of L. rhamnosus GG, strain PA11 offers novel opportunities towards the characterization and further therapeutic application of SpaCBA pili and their low-cost, large-scale production. Key points •Creation of pilus-secreting mutant (PA11) of the key probiotic LGG. •Strain PA11 is defective in a functional housekeeping sortase SrtA. •Strain PA11 opens novel biotherapeutic application avenues. Graphical abstract
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Stone, Barbara J., i Yousef Abu Kwaik. "Expression of Multiple Pili by Legionella pneumophila: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells". Infection and Immunity 66, nr 4 (1.04.1998): 1768–75. http://dx.doi.org/10.1128/iai.66.4.1768-1775.1998.
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ABSTRACT Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL ). An insertion mutation was constructed in pilEL and introduced into theL. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that thepilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.