Properties of antibiotic resistance in H. pylori and enzymatic studies of P. aeruginosa lipoxygenase
Physical and Biological Sciences
Department of Microbiology and Environmental Toxicology
Helicobacter pylori is a pathogenic, microaerophilic microorganism that colonizes the stomach, and upon infection can lead to gastric cancer and ulcers. To navigate throughout the stomach into its preferred niche, it utilizes chemotaxis to drive directed movement. Chemotaxis is an important factor during H. pylori infection because it allows for navigation within the gastric region. Previous studies in the lab have shown that in a mouse model infection, non-chemotactic mutants do not colonize the antrum as well as wild-type parent strain. This shift in distribution may cause H. pylori to become more susceptible to antibiotic agents when their chemotactic system has been disrupted. Currently, there are many antibiotic therapies that are used to treat H. pylori infection; however treatment failures are becoming increasingly frequent. In this study, we examined whether there was a link between the ability of H. pylori to carry out chemotaxis and its antibiotic susceptibility. H. pylori strains SS1 wild-type (WT) and a non-chemotactic isogenic mutant SS1 ΔcheW::kan (cheW) were used. To correlate chemotaxis to antibiotic susceptibility, we compared the susceptibility of the cheW mutant to that of WT during stomach colonization; preliminary results showed that the cheW strain was more susceptible to the antibiotic amoxicillin. We examined whether the cheW strain was more susceptible to antibiotics in vitro as well as in vivo. We determined the minimum inhibitory concentration (MIC) of commonly used antibiotics toward both strains in vitro and found them to be generally the same for wild-type and non-chemotactic strains. Our determined MICs are as follows: clarithromycin (0.030 ug/ml), tetracycline (9 ug/ml), amoxicillin (0.128 ug/ml), metronidazole (9 ug/ml), and omeprazole (64 ug/ml). These concentrations represent the minimal antibiotic concentration needed to inhibit growth on blood agar plates and in Brucella broth culture media. These experiments support that WT and cheW H. pylori are similarly susceptible to antibiotics in vitro. In extension, this allows us to attribute a difference in antibiotic susceptibility in vivo to the gastric environment, or other external factor rather than the antibiotic profile observed in the in vitro assays. We examined whether the antibiotics might act in part by altering bacterial swimming ability. In vitro cultures were grown in liquid media and exposed to 10, 2, 1, and 0.5 times the determined MIC of each antibiotic for 5 hours and observed for any changes in motility. Amoxicillin and clarithromycin treated cells showed a modest loss of motility, while metronidazole and tetracycline gave a more severe effect. This finding suggests that some antibiotics compromise motility in addition to killing bacteria, and may partially explain why antibiotics are effective. In conclusion, we propose that a loss of chemotactic ability in vivo causes H. pylori to become more susceptible to antibiotics specifically in the stomach.
Recent studies have shown that Pseudomonas aeruginosa expresses a lipoxygenase that is normally expressed by mammalian cells. Genomic sequence comparison of p15-LOX and h15-LOX-2 shows 43.6% similarity and 25.7% identity (Vance, 2004). It is unclear how this microorganism utilizes this enzyme or how it differs from the lipoxygenases found in other organisms. To address this, we probed several biochemical properties of this enzyme using chemical and biochemical techniques in order to compare it with its mammalian homolog. Initial experimental approaches necessitated optimization of the expression of P. aeruginosa lipoxygenase (p15-LOX) in BL21 E. coli cells at different induction temperature conditions. It was found that induction at 22 °C exhibited optimum activity compared to 18 °C and 26 °C. Kinetic studies to analyze the catalytic properties of p15-LOX at different pH’s were performed and found that the catalytic efficiency was optimum at pH 7.0 (3.04 [±0.841]). Further studies under different conditions suggested that increased acidity correlates to increased catalytic efficiency. Comparative analysis of product formation between this novel lipoxygenase and the heavily studied soybean lipoxygenase for a number of biologically relevant substrates was performed. The products formed from the two enzymes are similar in identity but differ slightly in their abundance. Biochemical analysis of the bacterial lipoxygenase derived from Pseudomonas aeruginosa supports that product formation has a high degree of similarity between this enzyme and its eukaryotic homologues.
