Type IV pili (T4P) are important virulence factors that allow bacteria to adhere to and rapidly colonize their hosts. T4P are primarily composed of major pilins that undergo cycles of extension and retraction and minor pilins that initiate pilus assembly. Bacteriophages use T4P as receptors and exploit pilus dynamics to infect their hosts. Some bacteria encode pilin accessory proteins that post-translationally glycosylate major pilins to evade phage binding. TfpY is an accessory protein of unknown function that is widespread and structurally conserved among T4P-expressing bacteria. Here, we use Pseudomonas aeruginosa as a model to characterize the functional role of TfpY and its homologues in pilus assembly. TfpY expression is required for optimal pilus assembly and function; however, it does not provide phage defence, unlike previously characterized accessory proteins. TfpY can cross-complement twitching in strains expressing heterologous P. aeruginosa pilins, suggesting TfpY and its homologues play a common role in pilus assembly. We showed that TfpY likely interacts with the major pilin and specific minor pilins but is not incorporated into the pilus itself. We propose that TfpY, along with the minor pilins at the pilus tip, primes pilus assembly. We identified two unique gain-of-function mutations in T4P regulatory genes that non-specifically restore twitching in tfpY mutants by increasing levels of cAMP and expression of T4P components. This study enhances our understanding of the complex functional and regulatory relationships between pilin and accessory proteins.
Importance: Type IV pili are surface filaments that enable versatile pathogens, like Pseudomonas aeruginosa, to adhere to and colonize surfaces. Pili are composed of diverse proteins called pilins, which serve as host receptors for phages. P. aeruginosa uses specific accessory proteins to glycosylate pilins to evade phage infection. Here, we show that TfpY is a conserved accessory protein that does not mediate phage defence. Instead, we propose a mechanism where TfpY facilitates efficient pilus assembly and function. A better understanding of TfpY function will provide insight into how its associated pilins have evolved to resist phage infection in the absence of post-translational modification, how some phages overcome this barrier to infection, and how this can guide the design of phage-based therapeutics.
Keywords: Pseudomonas aeruginosa; accessory proteins; bacteriophage; type IV pili.