Biology subjects

Kilmury, S. L. N.

Publications and source records attributed to Kilmury, S. L. N..

3 recordsLinked to original sources

bioRxiv2018

The Pseudomonas aeruginosa PilSR two-component system regulates both twitching and swimming motilities

Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. Motility structures - pili and flagella - are also involved in sensing surface contact, which modulates pathogenicity. In Pseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies in Geobacter sulfurreducens and Dichelobacter nodosus implicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used RNAseq analysis to identify genes in addition to pilA with changes in expression in the absence of pilR. Among these were 10 genes inversely dysregulated by loss of pilA versus pilR, even though both pilA and pilR mutants lack T4P and pilus-related phenotypes. The products of those genes - many of which were hypothetical - may be important for virulence and surface-associated behaviours, as mutants had altered swarming motility, biofilm formation, type VI secretion, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription of fleSR, and thus many genes in the FleSR regulon. As a result, pilSR deletion mutants had defects in swimming motility that were independent of the loss of PilA. Together these data suggest that in addition to controlling T4P expression, PilSR have a broader role in the regulation of P. aeruginosa motility and surface sensing behaviours.

microbiology↗

bioRxiv2017

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

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 is 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 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 minor pilin operon and other members of the AlgR regulon. Reporter assays confirmed that FimS-AlgR were required for the increased expression from the minor pilin operon promoter upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via point mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on 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 and reduced expression of acute-phase virulence factors. 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.\n\nAuthor summaryPseudomonas aeruginosa causes dangerous infections, including chronic lung infections in cystic fibrosis patients. It uses many strategies to infect its hosts, including deployment of grappling hook-like fibres called type IV pili. Among the components involved in assembly and function of the pilus are five proteins called minor pilins that - along with a larger protein called PilY1 - may help the pilus attach to surfaces. In a roundworm infection model, loss of PilY1 and specific minor pilins delayed killing, while loss of other pilus proteins did not. We traced this effect to increased activation of the FimS-AlgR regulatory system that inhibits expression of virulence factors used to initiate infections, while positively regulating chronic infection traits such as alginate production, a phenotype called mucoidy. A disruption in the appropriate timing of FimS-AlgR-dependent virulence factor expression when select minor pilins or PilY1 are missing may explain why those pilus-deficient mutants have reduced virulence compared with others whose products are not under FimS-AlgR control. Increased FimS-AlgR activity upon loss of PilY1 and specific minor pilins could help to explain the frequent co-occurrence of the non-piliated and mucoid phenotypes that are hallmarks of chronic P. aeruginosa lung infections.

microbiology↗

bioRxiv2017

cAMP-Independent Control Of Twitching Motility In Pseudomonas aeruginosa

FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through activation of the adenylate cyclase, CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimVs cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, required both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds - lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH, or expressing constitutively-active CyaB - pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV-dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimVs role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.\n\nIMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility, and are involved in surface sensing which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements - including the PilSR two-component system - to cell poles for optimal function.

microbiology↗