The Major Pilin Subunit Homolog PilE4 is Essential for the Expression of a Novel Type IV Pilus by Francisella tularensis

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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Faculty Opinions recommendation of Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1013554.192053 ◽

2003 ◽

Author(s):

Virginia L Miller

Keyword(s):

Gene Cluster ◽

Citrobacter Rodentium ◽

Type Iv Pilus ◽

Type Iv ◽

Gastrointestinal Colonization

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Faculty Opinions recommendation of The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019833.226437 ◽

2004 ◽

Author(s):

George Ordal

Keyword(s):

Myxococcus Xanthus ◽

Type Iv Pilus ◽

Type Iv

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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 ◽

10.1128/jb.00943-09 ◽

2009 ◽

Vol 191 (21) ◽

pp. 6513-6524 ◽

Cited By ~ 35

Author(s):

Hanjeong Harvey ◽

Marc Habash ◽

Francisca Aidoo ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Antigenic Variation ◽

Intact Protein ◽

Twitching Motility ◽

Beta Turn ◽

Type Iv ◽

Pilin Subunit ◽

Type Iv Pilin ◽

Intersubunit Interactions ◽

Pilus Assembly

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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Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽

10.1099/mic.0.001080 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Marie Zachary ◽

Susanne Bauer ◽

Maximilian Klepsch ◽

Katharina Wagler ◽

Bruno Hüttel ◽

...

Keyword(s):

Target Genes ◽

Target Prediction ◽

Type Iv Pilus ◽

Content Type ◽

Type Iv ◽

Gene Expression Control ◽

Initial Characterization ◽

Pilus Biogenesis ◽

Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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

Journal of Bacteriology ◽

10.1128/jb.00648-07 ◽

2007 ◽

Vol 189 (17) ◽

pp. 6389-6396 ◽

Cited By ~ 25

Author(s):

Richard F. Collins ◽

Muhammad Saleem ◽

Jeremy P. Derrick

Keyword(s):

Electron Microscopy ◽

Neisseria Meningitidis ◽

Metal Ion ◽

Polyacrylamide Gel Electrophoresis ◽

Three Dimensional ◽

Type Ii Secretion ◽

Type Ii ◽

Type Iv Pilus ◽

Type Iv ◽

Pilus Biogenesis

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

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