The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity

Abstract

SummaryMultiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3Ј, 5Ј-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

“…Classic chemotaxis systems use CheY as the response regulator downstream of CheA (18). The Chp system has two apparent CheY homologs, PilG and PilH, which have opposite effects on cAMP levels (9). The pilH mutant exhibits elevated cAMP levels and, as predicted, demonstrated constitutive induction of PaQa expression in liquid (Fig.…”

High Specificity in CheR Methyltransferase Function

“…Here we show that the mutation of the WspC homologue in P. putida reduces biofilm formation, which is consistent with the above findings. The chP chemosensory pathway of P. aeruginosa was found to mediate type IV pilibased motility (32)(33)(34). To assess whether CheR3 may potentially be involved in mediating this type of motility, we have thus studied the twitching motility of P. putida KT2440.…”

“…The third pathway (wsp) (31) regulates cyclic diguanylate concentrations (formed by the cluster III gene products), which in turn was found to modulate biofilm formation. The fourth pathway, chP (cluster IV genes), modulates the cAMP level (32) and consequently several other features including type IV pili synthesis and twitching motility (33,34). Because the signaling proteins of these pathways are paralogous, there thus exists the possibility of cross-talk between pathways.…”

Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System

“…Single substitutions at predicted phosphorylation sites gave a variety of twitching phenotypes in P. aeruginosa PAO1 (27), and the ChpA catalytic domain and phosphorylation sites of PilG and PilH are required for T4P function (28). pilG deletion mutants phenocopy chpA deletions in multiple assays (21,28), suggesting that PilG functions as an output response regulator. In contrast, pilG and pilH deletion mutants give opposite phenotypes for surface pili abundance (21,28) and intracellular cAMP levels (21).…”

“…Twitching motility in P. aeruginosa is regulated by the Pil-Chp chemosensory system (9) and involves repeated cycles of extrusion, surface adhesion, and retraction of T4P (10,19,20). In addition to regulating twitching motility, the P. aeruginosa Pil-Chp chemosensory system also regulates intracellular levels of cAMP (21), a second messenger that activates Vfr, a master transcriptional regulator of genes involved in P. aeruginosa pathogenesis (22). The relationship between Pil-Chp regulation of cAMP levels and twitching motility has yet to be fully elucidated (21,23); however, it has been recently proposed that Pil-Chp regulation of twitching motility and cAMP synthesis is linked to mechanical tension resulting from surface attachment of T4P (24).…”