Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients

Kus, Julianne V.; Tullis, Elizabeth; Cvitkovitch, Dennis G.; Burrows, Lori L.

doi:10.1099/mic.0.26822-0

Cited by 131 publications

(213 citation statements)

References 68 publications

(55 reference statements)

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“…The N-terminal amino acid sequence of PilA is highly conserved, even among distantly related species (35,45,46), whereas the C-terminal domains of pilins, both major and minor, can be extremely divergent (24). Heterologous P. aeruginosa pilins, plus the naturally truncated pilin from G. sulfurreducens, interacted with PilS ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…However, the first ∼24 amino acids of mature pilins are highly conserved (Fig. S1), whereas the C-terminal domains are diverse (24). In contrast, PilS in P. aeruginosa strains is invariant (28), regardless of the strain's pilin type.…”

Section: Overexpression Of Heterologous Pilins Reduces Pila Levels Bymentioning

confidence: 99%

“…Twitching motility assays were performed as described previously (24). Briefly, 1% LB agar plates were stab inoculated to the plate-agar interface with a single P. aeruginosa colony and incubated at 37°C for 24 h. The agar was then removed, and plates were stained with 1% (wt/vol) crystal violet for 20 min and washed with water to remove excess dye.…”

Section: Methodsmentioning

confidence: 99%

“…P. aeruginosa strains carry one of five pilA alleles (groups I-V) (24), encoding pilins that vary in size and sequence of the C-terminal domain (9), plus the presence or absence of posttranslational modifications (25,26) and accessory proteins (25,27). However, the first ∼24 amino acids of mature pilins are highly conserved (Fig.…”

Section: Overexpression Of Heterologous Pilins Reduces Pila Levels Bymentioning

confidence: 99%

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Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

Kilmury

Burrows

2016

Proc. Natl. Acad. Sci. U.S.A.

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Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa. Transcription of the major pilin gene—pilA—is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS’s phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA–PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression.

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Section: Discussionmentioning

confidence: 99%

Section: Overexpression Of Heterologous Pilins Reduces Pila Levels Bymentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Overexpression Of Heterologous Pilins Reduces Pila Levels Bymentioning

confidence: 99%

See 2 more Smart Citations

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

Kilmury

Burrows

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Twitching Motility Assays-Twitching assays were performed as described previously (5,47,48,58). Briefly, single colonies were stab inoculated to the bottom of a 1% LB agar plate.…”

Section: Methodsmentioning

confidence: 99%

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

Leighton

Yong

Howell

et al. 2016

Journal of Biological Chemistry

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Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo. Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo-or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo-and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.Many bacteria, including Pseudomonas aeruginosa, use type IV pili (T4P) 3 for surface attachment/adhesion, biofilm formation, and twitching motility (1-6). T4P are divided into T4aP and T4bP subgroups, based on differences in pilin size and organization of the assembly machinery (6, 7). Here we focus on T4aP, henceforth referred to as T4P. T4P are long, thin, fibrous appendages, which extend from the bacteria, attach to a surface, and are retracted back into the cell, winching the cell forward. This flagellum-independent form of locomotion is called twitching motility. Four subcomplexes make up a T4P machine in P. aeruginosa as follows: the outer membrane (OM) secretin (PilQ) and its pilotin (PilF) (8 -12); the inner membrane (IM) platform protein (PilC) and cytoplasmic motor proteins (ATPases PilBTU) (13,14); the helical pilus fiber, composed mainly of the major pilin subunit (PilA) plus minor pilins (PilVWXE and FimU) and adhesin PilY1 (5, 15, 16); and the alignment subcomplex proteins (PilMNOP) that span from the cytoplasm to the OM secretin (17-20). PilN and PilO are bitopic IM proteins with similar predicted secondary structures. These proteins are essential for T4P function and connect the cytoplasmic alignment subcomplex component, PilM, with the IM-associated lipoprotein PilP that interacts with the N0 domain of the secretin monomer PilQ (19,21). Proposed functions of the PilMNOP subcomplex include ali...

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Flagella and Pili ofPseudomonas aeruginosa

Jyot

Ramphal

2008

Pseudomonas

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IntroductionMotility is one of the most impressive features of microbial life and requires a large percent of cellular energy. Its roles include increased efficiency of nutrient acquisition, evasion of toxic substances, ability to translocate to preferred hosts and access to optimal colonization sites within them, as well as dispersal in the environment. Movement in aqueous environments by swimming or along surfaces by using different modes of translocation has been classified into several distinct types [1]. Pseudomonas aeruginosa exhibits three types of motility -flagellum-mediated swimming, flagellum and type IVpilus-mediated swarming [2,3], and type IVpilus-mediated twitching [4][5][6]. Swarming motility can be distinguished from swimming motility in that swarming is required to move across a hydrated, viscous semisolid surface, while swimming allows movement through a relatively low-viscosity liquid environment. The direction of movement is biased by chemotactic responses to chemical stimuli [7][8][9]. This chapter focuses on the two motility organelles of P. aeruginosa, i.e. the flagellum and the pilus, their structure, regulation and role in pathogenesis. 4.2 Flagellum of P. aeruginosa P. aeruginosa has a single, unsheathed polar flagellum similar to that of Caulobacter crescentus and Vibrio parahaemolyticus, but unlike the polar, sheathed flagellum observed in Helicobacter and the peritrichous flagella seen in Escherichia coli and Salmonella enterica serovar Typhimurium. However, swarmer cells of P. aeruginosa appear to possess on average two polar flagella and no lateral flagella [2,10]. At the genetic level, the expression of flagellar genes in P. aeruginosa is tightly regulated, involving transcriptional activators and multiple s factors as in other enteric bacteria. Flagellar structural genes are usually conserved across the genomes Pseudomonas. Model Organism, Pathogen, Cell Factory. Edited by Bernd H.A. Rehm

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Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients

Cited by 131 publications

References 68 publications

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

Flagella and Pili ofPseudomonas aeruginosa

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