UCSF

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili, which are required for colonization of host tissues and for a type of flagella-independent surface translocation termed twitching motility. Twitching motility is controlled by a number of factors including a putative chemosensory system referred to as the Chp system. We conducted comprehensive genetic analysis of the function of Chp system genes by making in-frame deletions of all of the genes in the Chp cluster and assaying them for type IV pilus functions, including twitching motility, phage sensitivity, and pilin assembly. The majority of work in the current study focused on the core signaling components of the Chp system, which include a putative histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. It remains unclear how components of the Chp system interface with the type IV pili motor proteins PilB, PilT, and PilU. Here we present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and that of PilH and PilT in regulating pilus retraction. In addition, we present evidence that the diguanylate phosphodiesterase, FimX, is involved in regulating pilus extension, consistent with recent reports showing interactions with PilB. We demonstrate that ChpA, PilG, and PilB function upstream of PilH, PilT and PilU, that PilH functions upstream of PilT, and that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. We present evidence to suggest that despite its homology to PilT, PilU does not appear to play a role in the regulation of pilus retraction, and we show that it continues to localize to the cell pole in Chp system mutant backgrounds. We show that the histidine kinase domain of ChpA is required for function as are the phospho-acceptor sites of both PilG and PilH. These findings suggest that ChpA-mediated control through PilG and PilH likely occurs through phosphorylation, and that this is important in the regulation of pilus extension and retraction. We present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export. Finally we demonstrate that both ChpA and PilH play a role in the regulation of virulence factor production. ChpA regulates production of the type III secreted virulence factor, ExoT, and PilH regulates the production of a number of quorum sensing-regulated secreted virulence factors including pyocyanin and rhamnolipid.