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

PLoS Pathog.. 2018;

MarkoVictoria A，KilmurySara L N，MacNeilLesley T，BurrowsLo

Products/Services Used	Details	Operation
Gene Synthesis	Deletion constructs were designed to contain 500–1000 bp homology upstream and downstream the gene to be deleted. Deletion constructs for PA14 fimU, pilV, pilW, pilX, pilY1, and pilE were synthesized by Genscript in the pUC57Kan vector. pEX18Gm-sadC was created by amplifying the sadC deletion region from PA14 sadC roeA [42], followed by digestion and ligation into pEX18Gm. pEX18Gm-fimS, pEX18Gm-algRD54A, and pEX18Gm-algRD54E were made by overlap extension PCR [97]. Restriction digestion followed by ligation of theupstream and downstream fragments was used to create the deletion constructs pEX18GmalgR, pEX18Gm-algU, and pEX18Gm-pilD. pMS402-PfimU and pMS402-PcdrA were created by amplifying and digesting the promoter regions of the PA14 MP operon and cdrA gene, respectively. Digested pBADGr was treated with alkaline phosphatase prior to ligation to avoid re-circularization of the vector. Constructs were verified by Sanger sequencing (MOBIX lab, McMaster, Hamilton, ON).	Get A Quote

摘要

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.