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.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, recently listed as one of the highest priority antimicrobial-resistant threats by the World Health Organization, due to its intrinsic antibiotic resistance and recalcitrance to therapy [1]
We found that slow killing (SK) of C. elegans by pilW, pilX, and pilY1 mutants was significantly delayed compared to WT or a pilA mutant, and this delay was dependent on FimS-AlgR, because
An E. coli OP50 plate was included as a negative control for pathogenicity; worms began to senesce on these plates around day 7–8, consistent with published data regarding temperature-dependent effects on lifespan [46]
Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, recently listed as one of the highest priority antimicrobial-resistant threats by the World Health Organization, due to its intrinsic antibiotic resistance and recalcitrance to therapy [1]. T4P are composed of hundreds to thousands of copies of small proteins called major pilins (PilA in P. aeruginosa) along with the low abundance minor pilins (MPs) FimU-PilVWXE [12,13,14,15,16]. The MPs are encoded in a polycistronic operon with the pilY1 gene that codes for a large ~125 kDa non-pilin protein. The operon is positively regulated by the virulence factor regulator Vfr, and the two-component system (TCS) FimS (AlgZ)-AlgR. Mature pilins are polymerized into a T4P fibre via an envelope-spanning assembly machinery, where individual PilA subunits are added or removed at the platform protein, PilC, via action of the ATPases PilB and PilT, respectively [2, 26]
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