This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Most cystic fibrosis (CF) patients will ultimately succumb to a chronic Pseudomonas infection of the lung. The factors responsible for susceptibility to infection and disease pathogenesis are not entirely clear. However, bacteria can modulate gene expression in response to environmental conditions, and the osmotic stress encountered during growth in the CF airway may induce virulence factor expression in P. aeruginosa. DNA microarrays were used to determine gene expression in log phase P. aeruginosa PA14 cells grown under conditions of osmotic stress in vitro. Sixty-five genes were up-regulated three- to eight-fold in cells grown in the presence of either 0.3M NaCl or 0.7M sucrose. Among the up-regulated genes were two open reading frames (ORFs)?PA3459 and PA3460?encoding putative enzymes involved in the synthesis of the osmoprotectant N-acetylglutaminylglutamine amide (NAGGN). Mutants with a transposon insertion in either of these ORFs were impaired for growth under osmotic stress. Other up-regulated genes include those encoding a type III secretion system and the effector proteins (type III cytotoxins) ExoT and ExoY. Additionally, cells grown in a high-salt medium containing the osmoprotectant betaine (0.5mM) showed a three-fold induction of phoP and phoQ?encoding the PhoPQ two-component signal transduction system?and the PhoP-regulated gene oprH. Expression of the outer membrane protein OprH has been shown by others to confer resistance to antimicrobial peptides and aminoglycoside antibiotics phenotypes that may contribute to evasion of host defense and treatment of infection, respectively. These data suggest that the physical conditions in the CF airway may promote disease pathogenesis by inducing virulence factor expression in P. aeruginosa. Future experiments will focus on identifying determinants involved in regulating expression of the osmoresponsive genes, and characterization of the functional role of the proteins encoded in ORFs PA3459 and PA3460.
Showing the most recent 10 out of 165 publications
