Characterization of the Chp system of Pseudomonas aeruginosa
2017-03-02T23:19:51Z (GMT) by
P. aeruginosa is a pervasive opportunistic pathogen and its success can be attributed to its agility in responding to environmental stimuli using its five chemosensory systems. The chemosensory system known as the Chp system is responsible for the regulation of several cell processes including surface attachment, biofilm formation, twitching motility, swarming motility and cAMP regulation. Prior to this study only preliminary bioinformatic analysis of the Chp gene cluster had been performed and little was known regarding the transcription of the cluster. To this end RT-PCR was used to demonstrate that the Chp gene cluster is present in a single operon consisting of 10 genes including pilG, pilH, pilI, pilJ, pilK, chpA, chpB, chpC, chpD and chpE. Several putative promoter-binding sites for a number of sigma factors were also identified upstream of the Chp operon suggesting complex regulation of the operon. Bioinformatic analysis showed that although the structure of the Chp operon is well conserved within P. aeruginosa it is not conserved in other Pseudomonas spp. or bacteria where genes pilK, chpB, chpD and chpE are absent. These structural changes in the operon coincide with a large truncation in chpA that removes its Hpt6 domain suggesting an unknown function for this domain that is specific to P. aeruginosa. Previous work had shown the adaptor protein ChpC, unlike other elements of the Chp system, has little effect on twitching motility suggesting that it may be linking an unknown sensor to the Chp system to mediate a response to unknown environmental signals. Mutational analysis performed in this study to identify these environmental signals, identified that the Chp system through ChpC is responsible for mediating a twitching motility response to tryptone, mucin and BSA. Further work was performed using an ordered, non redundant library of PA14 transposon insertion mutants and identified that two methyl-accepting chemotaxis proteins, PctA and CtpH, and two solute binding proteins, DppA1 and DppA2, from a known peptide transport system were also mediating a specific response to tryptone. To gain a deeper understanding of the twitching motility response to tryptone live cell time-lapse video microscopy of the twitching motility zone was performed. Several novel behaviours were observed including affects on raft behaviour and rate of motility that were affected by cell number, cell-cell contact, cell shedding and a novel “internal raft” that appears to recruit cells to the outer edge of the colony. Further to this the variety of raft speed and raft size at the leading edge appears to be required for efficient colony expansion. ChpC mediates the response to tryptone by controlling the speed of individual cells rather than by coordinated cell movement enabling the development of a dynamic twitching motility zone required for fast and efficient colony expansion. This study has also shown that affect on virulence in response to tryptone is inverse to the twitching response with pyocyanin and elastase production being repressed. This repression was not mediated by ChpC or ChpA. Further to this and contrary to previous studies, ChpA mutants made using different antibiotic cartridges had vastly different virulence profiles suggesting acquisition of compensatory mutation. One mutant had severe defects in quorum sensing controlled virulence and both mutants had elevated HSL levels suggesting that ChpA is intersecting with the quorum sensing system to mediate virulence. Overall this work has broadened the understanding of the Chp system and has shown that the Chp operon is complex in its regulation with an overall structure that is unique to P. aeruginosa. The Chp system is also potentially intersecting with two other cell systems to mediate peptide taxis and virulence suggesting intersection with multiple networks in P. aeruginosa.