Characterization of antioxidant enzymes among pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa (PA) is subjected to endogenous oxidative stress from aerobic metabolism and exogenous oxidative stress as a result of exposure to oxidantproducing neutrophils. Like other bacteria, laboratory strains of PA contain anrtoxidant enzymes: catalase and two forms of Superoxide dismutase (MnSOD and FeSOO). The SOD form expressed varies with growth conditions. However, detailed analysis of these enzymes among PA clinical isolates Is lacking. Therefore, SOD and catalase from PA clinical Isolates was studied with emphasis on the site of PA isolation and varying in vitro growth conditions. Fifty-one PA Isolates previously obtained from predominantly bloodstream, urinary tract, and respiratory tract sites were studied. Cell extracts containing SOD and catalase were prepared from PA grown to mid-logarithmic phase in tryptic soy broth (TSB) or an iron-limited succinate media (SM). These extracts subjected polyacrylamide native protein gel electrophoresis following which the gel was stained for SOD or catalase activity. Band intensity was quantttated by gel spectrometry. SOD activity was attributed to FeSOD by its ability to be inhibited by H2O2. MnSOD activity was defined by its resistance to NaCN and H2O2 inactivation. Isolates grown in TSB exhibited FeSOD as the predominant SOD form in similar quantities. However, when they were grown in SM, MnSOD predominated. Two different migration patterns of MnSOD among the isolates were observed which has not been previously described. When the SodA gene which codes for MnSOD was cloned by PCR from strains expressing the lower migrating form of MnSOD, the DNA sequence was identical to that of the strains expressing the more common higher migrating form. TSB grown isolates had qualitatively and quantitatively similar catalase activities. The same Isolates grown in SM showed a 36-56% decrease in catalase activity. Sixteen paired mucosal and bloodstream isolates from 8 patients revealed no significant differences based on site of PA isolation. PA clinical isolates demonstrate different SOD forms and varying catalase activities in response to iron-limited growth conditions. These findings further define the oxidative stress defense strategy of PA in a clinical context.

Duke Scholars

Author Rachel Ann Miller Medicine, Infectious Diseases