Investigating mutants of the PilA helix in Pseudomonas aeruginosa

Abstract

Type IV pili are a vital component to bacterial surface sensing. It is a polymer that is composed of the major pilin PilA – it extends from the surface of the cell, attaches to a surface and pulls the cell forward upon retraction. This motion is called twitching motility. The retraction of the Type IV pilus forms the core of a major surface-sensing mechanism. The PilA protein has an alpha-helical domain that sits at the core of the pilus and serves a number of functions – it is theorised to contribute to the “stretchability” of the pilus and its ability to withstand tensile force. It has also been implicated in signalling events that help the cell switch to a superficial lifestyle, and the upregulation of virulence factors. The domain contains two helix-breaking residues that flank a short stretch of amino acids that unfold from an alpha-helical conformation in an assembled pilus. In this study, random mutagenesis was used to mutate the two helix-breaking residues of the alpha-helical domain of PilA in Pseudomonas aeruginosa. The effect of these mutations were tested for pilus formation, motility and signalling. Large and/or charged residues disrupt the formation or functioning of the pilus, but single mutations to small non-polar residues retain quite a lot of the original function. The P22 residue especially appears to absorb mutations well. Two double mutations were also attempted, but they exhibited no functionality at all. Signalling phenotypes matched the twitching phenotypes closely for a small sample of the mutants, suggesting that they follow from one another. It is clear that neither of the two residues are independently essential to pilus assembly or signalling, but further study will be required to understand the true structural impact of these mutations.