Dataset Information

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

ABSTRACT: Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.

SUBMITTER: Marko VA

PROVIDER: S-EPMC5979040 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Publications

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

Marko Victoria A VA Kilmury Sara L N SLN MacNeil Lesley T LT Burrows Lori L LL

PLoS pathogens 20180518 5

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated b ...[more]

PMID: 29775484

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Project description:During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second-messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact are still poorly understood, although mechanosensing is often invoked, with few data supporting this claim. Using a combination of molecular genetics and single-cell analysis, with biophysical, biochemical, and genomics techniques, we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that TFP/PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain of PilY1 results in modestly lower surface adhesion forces, reduced sustained conformational changes, and increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate a role for TFP in twitching motility from surface-sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains and its absence in the ∼720 sequenced strains of P. aeruginosa PAO1 may contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. IMPORTANCE Most bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second-messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade is unclear. Here, we provide key insight into this question; we show that the eukaryote-like vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives the production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that may account for differing surface colonization strategies.

Project description:The intricate biological process of cutaneous wound healing is achieved through precise and highly programmed events. Dermal fibroblasts and keratinocytes play a significant role in the process of reepithelialization during wound healing. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) may delay the proliferative phase of wound repair by secreting their proteins leading to delayed or impaired wound healing. We have analyzed three virulent strains of P. aeruginosa isolated from the wound environment which also differed in their ability to produce biofilms. Mass spectrometric analysis of differentially expressed secreted proteins by three virulent strains of P. aeruginosa revealed peptides from pseudolysin and protease IV expressed from lasB and prpL genes. Pseudolysin and protease IV recombinant proteins were tested for their ability to modulate wound healing in several cell types of wound microenvironment in in vitro and in vivo models. Both pseudolysin and protease IV inhibited migration and survival of fibroblasts, keratinocytes, and endothelial cells. In three dimensional spheroid endothelial models and matrigel assays these proteins impeded sprouting and tube formation. In a mouse model of excision wound, pseudolysin and protease IV treatment showed reduced collagen content, inhibited neovascularization and epithelialization, and delayed wound contraction. Furthermore, pseudolysin and protease IV treatment resulted in a significant increase in plasma IL-6 levels when compared to vehicle control and control, suggesting the induction of a state of prolonged inflammation. Taken together, our data indicate pseudolysin and protease IV secreted from biofilm producing and antibiotic resistant P. aeruginosa in wound microenvironment produce both local and systemic effects that is detrimental to the maintenance of tissue homeostasis. Hence, these proteins may serve as potential therapeutic targets toward better clinical management of wounds.

Dataset Information

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

Publications

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

Similar Datasets

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