Dottorato in BIOCHIMICA

Titolo della tesi: Investigating the effect of gasotransmitters and oxidative stress on bacterial aerobic respiration: the role of bd-type oxidases

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are gaseous signalling molecules that exert pivotal roles in pathophysiological processes of both eukaryotes and prokaryotes. They are better known for the toxic effects exerted at high concentrations, which can impair cell viability and aerobic respiration. Bacteria, on the other hand, have a high degree of respiratory flexibility and a branched respiratory chain with different terminal oxidases that are differentially expressed depending on growth conditions. Notably, the bd-type oxidases, terminal oxidases found only in the microbial world, have recently been recognised as enzymes responsible for bacterial adaptation to different stress conditions and thus identified as potential drug targets due to their unique functional and structural features. Although knowledge on the impact of NO, CO and H2S on bacterial physiology is still limited, emerging evidence highlights a significant role for these “gasotransmitters” in host-pathogen interactions as well as in infection and antibiotic resistance. The aim of the present PhD project was to investigate the effect of these gases and oxidative/nitrosative stress on bacterial aerobic respiration in order to gain insights into the role of cytochromes bd in bacterial pathophysiology. For this purpose, the study investigated the effect of NO, CO, H2S and hydrogen peroxide (H2O2) on terminal oxidases of two opportunistic microbes, Escherichia coli and Pseudomonas aeruginosa, which is listed among the “critical” group of pathogens that urgently need new antibiotic therapies. To achieve this goal, the project was divided into three parts: Part I: The role of Pseudomonas aeruginosa Cyanide Insensitive Oxidase (CIO) in sulfide-resistance bacterial respiration and nitrosative stress response In this first part we investigated the effect of H2S and NO on P. aeruginosa aerobic terminal oxidases, with particular attention on the bd-type oxidase cyanide-insensitive oxidase (CIO). Working on membrane fractions of P. aeruginosa PAO1 and isogenic mutants, depleted of CIO only or all other terminal oxidases except CIO, we show that in the presence of even high levels of H2S O2 consumption by CIO is unaltered, under such stressful conditions CIO expression is enhanced and supports bacterial growth. We also report that CIO is reversibly inhibited by NO but recovery of activity after NO exhaustion is complete and rapid, suggesting a protective role of CIO under NO stress conditions. As P. aeruginosa is exposed to H2S and NO during infection, tolerance of CIO towards these stressors agrees with a role of this terminal oxidase in P. aeruginosa virulence. Part II: The effect of carbon monoxide on Escherichia coli growth and respiration In this second part we investigated the effect of CO on the growth and respiration of the three different Escherichia coli mutant strains each expressing a single terminal oxidase: cytochrome bd-I or bd-II or bo3. Our data show that only cytochrome bd-I promotes growth and respiration in the presence of toxic concentrations of CO, conferring resistance to this gas. Surprisingly, we found a different susceptibility to CO inhibition between cytochromes bd-I and bd-II that can be assigned to structural differences between the two cytochromes bd. These results support the hypothesis that a diversification of the common architecture occurred within the cytochromes bd superfamily to accomplish different functions in different ecological niches. Part III: Escherichia coli cytochrome bd-II oxidases and its hydrogen peroxide scavenging activity In this third part, we investigated whether preparations of detergent-solubilized untagged cytochrome bd-II of E. coli can decompose H₂O₂ to O2. Our findings indicate that cytochrome bd-II, like cytochrome bd-I, possesses a significant H2O2 scavenging activity and suggests that this enzyme play a role in bacterial physiology by conferring resistance to peroxide-mediated stress. These findings provide further evidence that bd-type oxidases are key enzyme for bacterial adaptation to environmental stressors.