Genetic Analysis of the Regulation of Type IV Pilus Function by the Chp Chemosensory System of Pseudomonas aeruginosa

Abstract

The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, 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.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

“…Inhibiting TFP extension in the pilB mutant nearly abolished mechanosensation, because a PilB mutant lacks surface TFP (17). PilA negatively regulates itself, so that pilTU and pilB mutants have similar levels of intracellular PilA, and likely similar perisplamic levels because pilTU maintains the ability to extend fulllength TFP (17). The difference of signals measured in these two mutants is therefore an effect of TFP extension and retraction rather than an artifact generated by depletion of PilA.…”

“…Thus, PilJ could interact with PilA at the stretched TFP base despite the lack of retraction, so pilTU mutants remain partially sensitive to surface attachment, but with reduced sensitivity. Inhibiting TFP extension in the pilB mutant nearly abolished mechanosensation, because a PilB mutant lacks surface TFP (17). PilA negatively regulates itself, so that pilTU and pilB mutants have similar levels of intracellular PilA, and likely similar perisplamic levels because pilTU maintains the ability to extend fulllength TFP (17).…”

“…Because the Chp system is homologous to a chemotaxis system, this raises the possibility that Chp senses a signal specifically induced upon surface contact to regulate cAMP production. the Chp system do not twitch (17) and mutants in TFP components affect cAMP production (9). However, whether TFP could also activate Chp upon surface contact remained unknown.…”

“…Our results suggest that the amplitude of the transcriptional response scales with the frequency of signaling events (i.e., how often PilJ senses TFP under tension). Simultaneously, the Chp system increases PilB and PilT activity (17), stimulating TFP extension and retraction, prompting a positive feedback loop that increases surface sensitivity and twitching motility.…”

Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis

“…Specifically, the expression profile in different in vitro models of growth (planktonic and biofilm mode of growth) were characterised to identify whether there are differences between the PSE305 (which can undergo twitching motility and biofilm formation) and PSE306 (which cannot). The expression profiles of the genes pilT and flgD both of which are involved in type IV fimbrial biogenesis and motility (Bertrand et al, 2010) was characterised. In addition, pslD, pslA, pelB and algD expression profiles, which are involved in biofilm architecture and formation (Campisano et al, 2006;Ghafoor et al, 2011) and lasR which is involved in the regulation of QS network (Winstanley and Fothergill, 2009) were determined.…”