Pseudomonas aeruginosa minor pilins regulate virulence via modulation of FimS-AlgR activity

Abstract

The type IV pilus is a motility organelle found in a range of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The principle structural protein of the pilus is the major pilin, PilA, while a set of low abundance “minor pilins” are proposed to constitute the pilus tip. The minor pilins, FimU and PilVWXE, along with the non-pilin protein PilY1, prime assembly of surface-exposed pili. The fimU-pilVWXY1E operon is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is an adhesin and mechanosensor that, along with PilW and PilX, triggers virulence upon surface attachment. Here, we aimed to uncover the mechanism for PilWXY1-mediated virulence. We hypothesized that loss of PilWXY1 would relieve feedback inhibition on FimS-AlgR, resulting in increased transcription of the minor pilin operon and dysregulation of virulence factors in the AlgR regulon. Caenorhabditis elegans slow killing assays revealed that pilW, pilX, and pilY1 mutants had reduced virulence relative to a pilA mutant, implying a role in virulence independent of pilus assembly. FimS-AlgR were required for the increased promoter activity of the minor pilin operon upon loss of pilV, pilW, pilX, or pilY1. Overexpression or hyperactivation of AlgR by point mutation led to reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression. We propose that PilWXY1 inhibit their own expression at the level of FimS-AlgR, such that loss of pilW, pilX, or pilY1 leads to FimS-mediated activation of AlgR, and reduced expression of acute-phase virulence factors. Accumulation of mutations in the minor pilin operon may represent an evolutionary strategy for P. aeruginosa populations in chronic lung infections, as loss of PilWXY1 would upregulate the expression of AlgR-dependent virulence factors – such as alginate – characteristic of such infections.