Hydrogen sulphide (H2S), as the other well-known gasotransmitter nitric oxide (NO), is a highly toxic gas that interferes with cellular respiration; however, at low physiological amounts, it plays an important role in cell signalling and in fuelling bioenergetics in mammals. The role of H2S in bacterial energy metabolism remains poorly investigated, notwithstanding the emerging link between energy production and pathogenesis. Remarkably, endogenously produced H2S has been recently recognized as a general protective molecule, which renders multiple bacterial species highly resistant to oxidative stress, various classes of antibiotics and host immune responses. Therefore, understanding the mechanisms underlying H2S-mediated resistance would help in developing new therapeutic strategies against human pathogens.
Relevant to pathophysiology, of the different enzymes synthesizing H2S in E.coli, 3-mercaptopyruvate sulfurtransferase (MST) appears crucial in the defence against hydrogen peroxide toxicity and in antimicrobials resistance. Hence, defects in its expression and function result in lower virulence. Moreover, MST has been recently related to the production of sulfane sulphur, reactive species currently thought to mediate the signalling and protective properties previously associated to free H2S. Our working hypothesis is that MST endows E.coli with the ability to successfully resist NO and derived species produced by the host to counteract infections.
This project aims at studying the involvement of bacterial H2S in the defence mechanisms against nitrosative stress and in energy metabolism using E.coli wild type and MST mutant strains as model system. Research will focus on the quantification of expression level and activity of enzymes as well as on the determination of viability, redox state and bioenergetic parameters of cells in response to NO/ H2S to better understand the role played by sulphide within the framework of host-microbe relationships.
The emergence and spread of drug-resistant bacteria and the absence of definitive pharmacological agents or vaccines against many pathogenic agents has prompted research and characterization of new targets for the development of radically novel antimicrobials. In this context, we aim at investigating the role of an E.coli enzyme involved in H2S metabolism, MST, in response to NO, which is produced as part of the immune response to control microbial proliferation.
It is becoming quite apparent, in fact, that H2S metabolism is critically important to the growth and colonization potential of microorganisms, including pathogens in which H2S has been implicated in bacterial defence against ROS and antibiotics-induced damage as well as in increased virulence. Therefore targeting the H2S metabolic enzymes is now receiving a strong interest as an innovative strategy for therapeutic intervention in infectious diseases [2,3,6]. Bacterial cells use a variety of proteins to synthesize H2S in different metabolic processes [4]. Of all these enzymatic activities available to bacteria, that of MST yields the most efficient response to oxidative stress in E.coli [1,6]. Indeed, MST antioxidant activity has been recently related to the production of sulfane sulphur [21], reactive species, among other sulfur-containing molecules, now recognized to mediate a great part of the signalling effects and antioxidants properties originally attributed to H2S. These reactive sulfur species together with ROS and RNS occupy a central place in redox biology.
Since bacteria may experience NO exposure during infection in addition to H2S, potential complementary interactions between these two gases may affect cellular biochemistry and metabolism as well as constitute new ways to regulate protein function and cellular responses to environmental conditions changes. The role of these interactions in bacterial pathogenesis is unclear and represents a gap in the field.
Our working hypothesis is that MST can have a crucial role in bacterial response to NO. Are MST deficient cells more susceptible to nitrosative stress? Can NO induce MST expression? Can H2S modulate NO bioavailability in vivo? By answering these questions we will contribute to the understanding of the molecular mechanisms allowing E.coli, and possibly other pathogens, to survive under stress conditions, grow and colonize the host. The characterization of new biochemical pathways required for the success of infections is important to shed some new light on bacterial pathogenesis.
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 [10]. Preserving energy metabolism and ATP production is indeed crucial for growth and survival during infections [14]. Bacteria can use several 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. Relevant to human pathophysiology, E.coli requires modulation of terminal oxidase expression for adaptation to the adverse conditions present in the host, though the molecular mechanisms through which the enzymes operate to enhance bacterial survival have been only partly clarified. Whereas the expression of the respiratory complexes is finely tuned in response to NO and O2 concentration [23], the function of the two E.coli respiratory chain branches in response to low physiological levels of H2S concentration or in combination with NO has not been evaluated yet.
The hypothesis that bacterial sulphide may support cellular proliferation and energy metabolism, similarly to mammalian cells [24], has never been addressed. Preliminary data indicate a correlation between respiratory activity and sulphide. The biochemical assays proposed in this project will allow us to explore the impact of the sulfane sulfur species and the role of H2S metabolism in the regulation of the electron transfer chain complexes expression and in cell bioenergetics.
The understanding of how bacterial-generated sulphide species regulate energy metabolism as well as the effect of H2S-NO cross-interaction on bacterial survival, is relevant within the framework of host-pathogen relationships, as it may help to identify key therapeutic targets and it contributes to a broader comprehension of how gasotransmitters can be engineered as an approach to therapy.
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