Aaron Hinz

Molecular & Cellular Biology Ph.D. Program
Manoil Lab
ahinz [ a t ] u.washington.edu

Research:
Efflux pump overexpression in Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa infects the airways of cystic fibrosis patients, causing persistent inflammation. During this chronic infection, P. aeruginosa evolves resistance to antibiotics used to restrain the infection. Resistance often results from regulator mutations that increase expression of efflux pumps, leading to enhanced export of drugs out of the cell. P. aeruginosa encodes ~12 such pumps on its chromosome, each with distinct substrate profiles. They are capable of exporting most anti-pseudomonal drugs, including b-lactams, aminoglycosides, and macrolides. The aminoglycoside-specific MexXY pump is frequently found to be upregulated in clinical isolates of P. aeruginosa.

Efflux pump overexpression has been found to compromise virulence, interfere with cell-to-cell signaling, and alter expression of other efflux pumps and porins in P. aeruginosa. However, the effects of MexXY overexpression are unknown. I am investigating the physiological effects of MexXY overexpression using Affymetrix transcriptional arrays. I have compared global RNA levels of a strain overexpressing the efflux pump to a strain not expressing the pump. Genes differentially regulated might indicate the effects of overexpression on cell physiology and point to the natural function or substrates of this pump. In three microarray experiments, two genetic loci are consistently found to be downregulated in MexXY overexpressing strains. One four-gene locus encodes a transcriptional regulator and putative efflux pump of unknown function. The second five-gene locus encodes enzymes of diverse hypothetical functions. Homology searches suggest that they could provide protective roles by degrading toxic metabolites. I am in the process of validating the microarray results by constructing transcriptional lacZ fusions to the promoters of these differentially expressed genes. Once the expression results are verified, I will investigate the functions of these hypothetical genes via phenotypic analysis of transposon mutants and explore hypotheses concerning what these expression results indicate about MexXY function and the consequences of overexpression.