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John F Hess - One of the best experts on this subject based on the ideXlab platform.
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prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant moraxella bovis Pilin moraxella bovis cytotoxin iscom matrix adjuvanted vaccine
Veterinary Microbiology, 2007Co-Authors: John A. Angelos, Robert G Bonifacio, Louise M. Ball, John F HessAbstract:Abstract To evaluate the efficacy of a recombinant Moraxella bovis Pilin– M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus + ISCOM matrix (MbxA), or recombinant M. bovis Pilin–cytotoxin carboxy terminus + ISCOM matrix (Pilin–MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the Pilin–MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with Pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and Pilin–MbxA. In addition, vaccination of calves with MbxA and Pilin–MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus Pilin + cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the Pilin–cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.
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prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant moraxella bovis Pilin moraxella bovis cytotoxin iscom matrix adjuvanted vaccine
Veterinary Microbiology, 2007Co-Authors: John A. Angelos, Robert G Bonifacio, Louise M. Ball, John F HessAbstract:Abstract To evaluate the efficacy of a recombinant Moraxella bovis Pilin– M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus + ISCOM matrix (MbxA), or recombinant M. bovis Pilin–cytotoxin carboxy terminus + ISCOM matrix (Pilin–MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the Pilin–MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with Pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and Pilin–MbxA. In addition, vaccination of calves with MbxA and Pilin–MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus Pilin + cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the Pilin–cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.
John A. Angelos - One of the best experts on this subject based on the ideXlab platform.
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relatedness of type iv Pilin pila amongst geographically diverse moraxella bovoculi isolated from cattle with infectious bovine keratoconjunctivitis
Journal of Medical Microbiology, 2021Co-Authors: John A. Angelos, Kristin A Clothier, Regina L Agulto, Boguslav Mandzyuk, Morten TrylandAbstract:Introduction. Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis, is required for corneal attachment. Seven antigenically distinct Pilin serogroups have been described in M. bovis.Hypothesis/Gap Statement. Multiple different serogroups exist amongst type IV Pilin encoded by M. bovis, however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV Pilin that it encodes.Aim. This study was done to characterize a structural Pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences.Methodology. Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.Results. DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1-99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten Pilin sequences identified were only ~74-76 % identical to deduced amino acid sequences of putative Pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.Conclusions. Compared to the diversity reported between structural Pilin proteins amongst different serogroups of M. bovis, M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.
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prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant moraxella bovis Pilin moraxella bovis cytotoxin iscom matrix adjuvanted vaccine
Veterinary Microbiology, 2007Co-Authors: John A. Angelos, Robert G Bonifacio, Louise M. Ball, John F HessAbstract:Abstract To evaluate the efficacy of a recombinant Moraxella bovis Pilin– M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus + ISCOM matrix (MbxA), or recombinant M. bovis Pilin–cytotoxin carboxy terminus + ISCOM matrix (Pilin–MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the Pilin–MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with Pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and Pilin–MbxA. In addition, vaccination of calves with MbxA and Pilin–MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus Pilin + cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the Pilin–cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.
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prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant moraxella bovis Pilin moraxella bovis cytotoxin iscom matrix adjuvanted vaccine
Veterinary Microbiology, 2007Co-Authors: John A. Angelos, Robert G Bonifacio, Louise M. Ball, John F HessAbstract:Abstract To evaluate the efficacy of a recombinant Moraxella bovis Pilin– M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus + ISCOM matrix (MbxA), or recombinant M. bovis Pilin–cytotoxin carboxy terminus + ISCOM matrix (Pilin–MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the Pilin–MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with Pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and Pilin–MbxA. In addition, vaccination of calves with MbxA and Pilin–MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus Pilin + cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the Pilin–cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.
Steven H Seifert - One of the best experts on this subject based on the ideXlab platform.
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analyzing neisseria gonorrhoeae Pilin antigenic variation using 454 sequencing technology
Journal of Bacteriology, 2016Co-Authors: Ella Rotman, David M Webber, Steven H SeifertAbstract:ABSTRACT Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed Pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant Pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of Pilin Av that allows a more robust analysis of Pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect Pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze Pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of Pilin antigenic variation.
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the Pilin n terminal domain maintains neisseria gonorrhoeae transformation competence during pilus phase variation
PLOS Genetics, 2016Co-Authors: Kyle P Obergfell, Steven H SeifertAbstract:The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (Pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of Pilin variants and can produce strains with unproductive Pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of Pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature Pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the Pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-Pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-Pilin cleavage site demonstrated that S-Pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive Pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-Pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.
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neisseria gonorrhoeae muts affects Pilin antigenic variation through mismatch correction and not by pile guanine quartet binding
Journal of Bacteriology, 2015Co-Authors: Ella Rotman, Steven H SeifertAbstract:ABSTRACT Many pathogens use homologous recombination to vary surface antigens to avoid immune surveillance. Neisseria gonorrhoeae achieves this in part by changing the properties of its surface pili in a process called Pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silent pilS sequences into the expressed pilE locus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in Pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis of pilE variants produced in these mutants showed that all three mutS mutants and a mutL mutant had similar increased frequencies of Pilin AV. Moreover, the mutS mutants all showed similar increased levels of Pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on Pilin AV and is not due to pilE G4 binding by MutS. IMPORTANCE Neisseria gonorrhoeae continually changes its outer surface proteins to avoid recognition by the immune system. N. gonorrhoeae alters the antigenicity of the pilus by directed recombination between partially homologous Pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of Pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit Pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.
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neisseria gonorrhoeae recq helicase hrdc domains are essential for efficient binding and unwinding of the pile guanine quartet structure required for Pilin antigenic variation
Journal of Bacteriology, 2013Co-Authors: Laty A Cahoon, Kelly A Manthei, Ella Rotman, James L Keck, Steven H SeifertAbstract:The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent Pilin loci and the expressed Pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For Pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of Pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one “helicase and RNase D C-terminal” (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for Pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of Pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in Pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.
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transcription of a cis acting noncoding small rna is required for Pilin antigenic variation in neisseria gonorrhoeae
PLOS Pathogens, 2013Co-Authors: Laty A Cahoon, Steven H SeifertAbstract:The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent Pilin copies and the expressed Pilin locus (pilE), resulting in the production of variant Pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to Pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks Pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter Pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of Pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted Pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support Pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events.
Willem M De Vos - One of the best experts on this subject based on the ideXlab platform.
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characterization of the spacba pilus fibers in the probiotic lactobacillus rhamnosus gg
Applied and Environmental Microbiology, 2012Co-Authors: Justus Reunane, Ingema Von Ossowski, Antoni P A Hendrick, Airi Palva, Willem M De VosAbstract:Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193–17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC Pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major Pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB Pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.
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mucosal adhesion properties of the probiotic lactobacillus rhamnosus gg spacba and spafed Pilin subunits
Applied and Environmental Microbiology, 2010Co-Authors: Ingemar Von Ossowski, Matti Kankainen, Soile Tynkkynen, Justus Reunanen, Reetta Satokari, Satu Vesterlund, Heikki Huhtinen, Seppo Salminen, Willem M De VosAbstract:Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three Pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the Pilin subunits (SpaC). Here, we report cloning of the remaining Pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF Pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB Pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two Pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single Pilin subunit (SpaF) is potentially responsible for adhesion to mucus.
Lisa Craig - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the minor Pilin cofb the initiator of cfa iii pilus assembly in enterotoxigenic escherichia coli
Journal of Biological Chemistry, 2015Co-Authors: Subramania Kolappa, Guixiang Yang, Lisa CraigAbstract:Type IV pili are extracellular polymers of the major Pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the Pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor Pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major Pilins but are much less abundant. Minor Pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor Pilin. Here we show that the enterotoxigenic E. coli minor Pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor Pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 A crystal structure of N-terminally truncated CofB, revealing a Pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.
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ultrahigh resolution and full length Pilin structures with insights for filament assembly pathogenic functions and vaccine potential
Journal of Biological Chemistry, 2011Co-Authors: Sophia Hartung, Lisa Craig, Andrew S Arvai, Timothy Wood, Subramaniapillai Kolappan, David S Shin, John A TainerAbstract:Pilin proteins assemble into Type IV pili (T4P), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. However, challenges in crystallizing full-length fiber-forming and membrane protein Pilins leave unanswered questions regarding Pilin structures, assembly, functions, and vaccine potential. Here we report Pilin structures of full-length DnFimA from the sheep pathogen Dichelobacter nodosus and FtPilE from the human pathogen Francisella tularensis at 2.3 and 1 A resolution, respectively. The DnFimA structure reveals an extended kinked N-terminal α-helix, an unusual centrally located disulfide, conserved subdomains, and assembled epitopes informing serogroup vaccines. An interaction between the conserved Glu-5 carboxyl oxygen and the N-terminal amine of an adjacent subunit in the crystallographic dimer is consistent with the hypothesis of a salt bridge between these groups driving T4P assembly. The FtPilE structure identifies an authentic Type IV Pilin and provides a framework for understanding the role of T4P in F. tularensis virulence. Combined results define a unified Pilin architecture, specialized subdomain roles in pilus assembly and function, and potential therapeutic targets.
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type iv Pilin structure and assembly x ray and em analyses of vibrio cholerae toxin coregulated pilus and pseudomonas aeruginosa pak Pilin
Molecular Cell, 2003Co-Authors: Lisa Craig, Ronald K Taylo, Michael E Pique, Ia D Adai, Andrew S Arvai, Mona Singh, Sarah J Lloyd, David S Shi, Elizabeth D Getzoff, Mark YeageAbstract:Abstract Pilin assembly into type IV pili is required for virulence by bacterial pathogens that cause diseases such as cholera, pneumonia, gonorrhea, and meningitis. Crystal structures of soluble, N-terminally truncated Pilin from Vibrio cholera toxin-coregulated pilus (TCP) and full-length PAK Pilin from Pseudomonas aeruginosa reveal a novel TCP fold, yet a shared architecture for the type IV Pilins. In each Pilin subunit a conserved, extended, N-terminal α helix wrapped by β strands anchors the structurally variable globular head. Inside the assembled pilus, characterized by cryo-electron microscopy and crystallography, the extended hydrophobic α helices make multisubunit contacts to provide mechanical strength and flexibility. Outside, distinct interactions of adaptable heads contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion, and colony formation.