The Regulation and Dynamics of Type IV Pili

Abstract

Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo.