Investigation of the ion networks in pseudomonas aeruginosa : roles in physiology and pathogenesis

Abstract

Growth defects range in intensity, and phenotypic changes vary by mutant, however the results of this characterization point to a greater role for these ion transporters in the regulation of pathogenesis in P. aeruginosa. This study reveals NQR as the major NADH dehydrogenase employed by P. aeruginosa and an important player in infection-relevant virulence traits. Our findings suggest that regulation of cellular NADH may convey virulence ques in P. aeruginosa. We define the four Na+/H+ antiporters present in P. aeruginosa and characterize the physiological impacts of the deletion of all for antiporters from the P. aeruginosa genome. The profiles revealed in this study begin to outline the roles played by each of these enzymes in P. aeruginosa and inform our understanding of ion regulation in this microbe.

Pseudomonas aeruginosa is a remarkably ubiquitous microorganism featuring an extensive environmental presence, broad infectious profile, and multi-drug resistance. This pervasiveness is a product of P. aeruginosa’s ability to adapt in the face of environmental stress. At the cellular level, environmental stress comes in the form of variable H+ and Na+ concentrations. Thus, survival across microbial habitats necessitates response to and regulation of these ions. The primary proteins responsible for H+ and Na+ regulation are the NADH dehydrogenases NDH-2, NUO and NQR and Na+/H+ antiporters NhaP, NhaP2, NhaB and Mrp. Together, these enzymes are responsible for the generation of energy efficient electrochemical ion gradients and the maintenance of cellular pH and Na+ homeostasis. In addition, these transporters have been previously implicated in the viability and infectious success of P.aeruginosa, indicating that they may play a greater role in cell physiology at large. To define the cellular utility of possessing such complementary transporters and determine their contributions to the adaptive physiology of the cell, this work investigates the kinetic parameters and survival phenotypes in a series of gene deletion mutants. NADH dehydrogenase activity is measured spectrophotometrically via the monitoring of NADH consumption. Antiport activity is measured as a function of substrate-stimulated H+ flux. Cell survival is assessed via growth under a variety of conditions of pH, salinity, and nutrient availability, and the impacts of transporter absence on cell physiology are probed through transcriptome analysis by RNA sequencing. Observed deviations from the activity and growth profiles of wild-type P. aeruginosa are attributed to the absent transporter, outlining the contributions made by each enzyme.