Until recently ammonia (NH3) has been known merely as a metabolic waste product, it is now emerging as key regulatory molecule, contributing to different patho-physiological processes. NH3 is produced at high concentration in the human gastrointestinal tract, in the same niche occupied by E.coli. Along with NO, CO and H2S, NH3 is both a gasotransmitter and a known inhibitor of mitochondrial haem-copper oxidase, the effect of NH3 on bacterial terminal oxidases being yet unknown.
The E. coli respiratory chain is branched and relies on three terminal oxidases, a haem-copper, cytochrome bo3, and the two bd-type oxidases, bd-I and bd-II. This prompted us to address the following question: how can E. coli survive in the human intestine, if NH3 inhibits the oxidases? Our working hypothesis is that a bd-type oxidase, could be more resistant to NH3 inhibition than a haem-copper oxidase (cytochrome bo3).
Relevant to human pathophysiology, cytochrome bd oxidases are key respiratory enzymes in many bacteria living inside humans and, in some pathogens, they promote bacterial virulence, making these enzymes of interest also as potential drug targets. In collaboration with Vitaly Borisov (Moscow State University), we contributed to show that cytochrome bd confers resistance to oxidative and nitrosative stress. Interestingly, this oxidase promotes also bacterial respiration and growth in the sulphide rich environments like the human gut.
This project aims at expanding these studies to the reaction mechanisms between terminal oxidases and gaseous molecules, particularly NH3, and their patho-physiological relevance.
Research will focus on E.coli oxidases, used as models, as well as on the bd oxidase from a gut pathogen, S. flexneri, either purified or expressed in cells. Particular emphasis will be given to bd oxidases, to better understand the role played by this enzyme within the framework of host-microbiota relationships.
Microbes are an integral part of the human gastrointestinal system. They control many aspects of human physiology and play vital functions such as facilitating digestion, supplying vitamins and providing resistance to invading pathogens. An imbalance in the number or composition of the gut microbial communities can therefore trigger pathological consequences. To gain insight into the mechanisms by which microbes can maintain health and trigger disease a better knowledge of the inter-relationship between the microbiota and the host is required [2].
It is becoming quite apparent that metabolism is critically important to the growth and colonization potential of intestinal microorganisms [17, 18]. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O2 yields the most energy while diversifying the type of substrates that bacteria can use. In E.coli the expression of the respiratory complexes finely tuned in response to NO and O2 concentration [14], but the function of the two respiratory chain branches in response to the high NH3 concentration has not been evaluated yet. Is the oxygen reductase activity of the E.coli respiratory complexes resistant to high NH3 levels? Is their expression modulated by NH3? Can their expression favour bacterial growth in the presence of ammonia? By answering these questions we will contribute to the understanding of the molecular mechanisms allowing E.coli and possibly other enterobacteria to sustain bacterial energetics also in the presence of this toxic compound, thereby helping microorganisms to live and grow in the gut.
Presently there is strong interest in exploiting the link between oxidative phosphorylation and pathogenesis since there is evidence suggesting that energy metabolism might be an important signal used by bacterial pathogens to identify specific host environments [18]. Importantly, cytochrome bd oxidase has been identified in a number of pathogens, such as Shigella, Salmonella, Enterococcus and in entero-pathogenic E.coli. Like for other pathogens in Shigella (S.) flexneri, a gram-negative bacterium, which causes one of the most communicable of bacterial dysenteries by invading the intestinal epithelium, a positive correlation between the enzyme level and virulence was observed [19]. Thus bd-type oxidases expression may represent an advantage for pathogenic bacteria and this makes cytochromes bd of interest also as drug targets since they are not expressed in humans and selective inhibitors of this oxidase should not have negative effects on the host energy metabolism [20]. Relevant to the pathophysiology of the human intestine, E.coli cytochrome bd was recently reported to enable bacterial respiration and growth in environments not only rich in NO but also in sulphide, both gases present at high levels in the gut and inhibitors of mitochondrial respiratory oxidase [14]. This evidence strongly suggests that the bd-type oxidases are required for adaptation to the adverse conditions present in the host, though the molecular mechanisms through which the enzyme operates to enhance bacterial survival have been only partly clarified. In this context the results of the biochemical assays quantifying the respiratory activity and growth in ammonia rich medium of Shigella cytochrome bd expressing cells will allow us to understand if this enzyme plays a role in protecting the respiratory chain also against NH3 toxicity while fuelling bioenergetics, thereby potentiating virulence of a clinically relevant pathogen.
In conclusion the data that will be obtained will allow us to understand the molecular mechanism by which bd-type terminal respiratory oxidases with their unique properties enhance bacterial resistance to different stresses, and will provide clues to the role played by cytochrome bd within the framework of host-pathogen relationships.
REFERENCES in "external partners section" (below)