Regulation of the virulence factor PlcH in Pseudomonas aeruginosa

University Of Vermont & St Agric College, Burlington VT

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Abstract

Pseudomonas aeruginosa (Pa) is an important opportunistic human pathogen that deploys a variety of virulence factors, each playing roles in different infection sites or with different cell types. One important virulence factor of Pa is the hemolytic phospholipase C/sphingomyelinase, PlcH. plcH mutants are defective in a wide range of infection models and PlcH expression by strains has been correlated to human disease severity and differential treatment efficacy. There are three currently known inputs to PlcH expression: induction by phosphate starvation, induction by glycine betaine, and repression by low oxygen environments. While each of these regulatory paths have been described, there remain important gaps in our understanding of PlcH regulation as well as alterations in these regulatory pathways in specific disease contexts. In this proposal, we will determine the identity and mechanisms of previously unidentified transcriptional and posttranslational regulation of PlcH and determine the role of altered PlcH regulation during Pa adaptation in the lungs of people with cystic fibrosis. Despite the contribution of PlcH to Pa virulence, study of its regulation lags behind that of other virulence factors. Here we present preliminary data for novel transcriptional regulation of plcH, identification of a new post- translational PlcH regulatory step, and present changes to metabolic pathways directly upstream of plcH induction that occur within the cystic fibrosis lung. Our hypothesis is that PlcH production and export is regulated at multiple levels and that these regulatory steps work to titrate the flux of enzymatic products and to avoid damage to producing cells. We have longstanding experience with PlcH and a large experimental toolkit with which to approach its regulation. We are well positioned to understand the mechanisms behind uncharacterized PlcH regulatory pathways and to identify new regulatory inputs. We will address PlcH regulation by (i) determining the mechanisms of uncharacterized plcH transcriptional regulation and the importance of each mechanism of transcriptional induction, (ii) deciphering post-translational control mechanisms for PlcH, and (iii) examining alteration in PlcH expression by pathoadaptive mutations occurring during long term cystic fibrosis lung infections. The work proposed here will allow us to have a more complete understanding of PlcH regulation, providing biological insight and exposing potential targets for inhibition of PlcH production.