Mycobacterium tuberculosis antibiotic resistance represents an increasing threat to global public health as prevents the effective treatment of tuberculosis, one of the top 10 causes of death worldwide. Therefore, understanding how this bacterial pathogen resists the immune system and identifying novel drug targets for therapeutic intervention is crucial for the successful cure of this infectious disease.
Recently targeting bacterial respiration and ATP synthesis has received strong interest as a new strategy for combating the obligate aerobe M. tuberculosis. Our working hypothesis is that the respiratory chain endows this pathogen with the ability to successfully resist NO and related nitrosative stress produced by the host immune response during infection. Mycobacterial respiratory chain is branched and relies on two terminal oxidases, the aa3- cytochrome c oxidase, forming a supercomplex with cytochrome bcc, and the bd quinol oxidase.
Relevant to human pathophysiology, bd type oxidases are key respiratory enzymes that confer bacterial resistance to nitro-oxidative stress and promote virulence in some pathogens, including mycobacteria. Since these enzymes are found only in prokaryotes, they are of great interest as potential drug targets, particularly for combined therapeutic approaches to strengthen the bactericidal effects of antimicrobials targeting other systems.
The present project aims at studying the M. tuberculosis respiratory chain complexes to understand the role played by these enzymes within the framework of host-pathogen relationships.
Research will focus on the identification of selective inhibitors of cytochrome bd activity, on the purification of an active mycobacterial cytochrome bd, as well as on the interaction of NO with the mycobacterial bcc/ aa3 supercomplex to shed light on the reaction mechanisms and their patho-physiological relevance.
The innovative approaches proposed in this project to fight the increasingly serious threat of antibiotic resistance are the characterization of new drug targets and the identification of novel antimicrobials. In particular we aim at investigating the role of the two mycobacterial respiratory chain branches in response to nitrosative stress produced as part of the immune response to control microbial proliferation and at finding and optimising new inhibitors of cytochrome bd oxidase activity.
During evolution infectious microorganisms have developed systems to survive and /or detoxify NO produced as part of the immune response to control microbial proliferation. Our published data on cytochrome bd from E.coli as a model system, showing an unusually high dissociation rate of NO from the active site and an unexpected hydrogen peroxide-degrading activity (3), suggest that this oxidase allows bacterial energetics also in the presence of toxic compounds released from macrophages, thereby helping a bacterial pathogen to evade the host immune defence. In the obligate aerobic pathogen M. tuberculosis the expression of the respiratory complexes, including cytochrome bd, is finely tuned during infection when NO is produced, but the function of the two mycobacterial respiratory chain branches in response to nitrosative stress has not been evaluated yet. Is the oxygen reductase activity of the mycobacterial respiratory complexes resistant to NO? Do they metabolize NO in turnover under aerobic conditions? Do they possess NO reductase activity? By answering these questions we will contribute to the understanding of the molecular mechanisms allowing M. tuberculosis to sustain energy metabolism also in the presence of toxic NO and survive nitrosative stress.
Presently there is strong interest in exploiting oxidative phosphorylation as a target for new antimycobacterial drugs and drug combinations (11). The development of these novel antimicrobials by the world pharmaceutical industry is only in its infancy, yet few compounds are under clinical development. The expression of cytochrome bd oxidase has been shown to lower the bactericidal efficacy mediated by inhibitors of other energy metabolism components, particularly the inhibitor of ATP synthase bedaquiline (7), therefore the choice of cytochrome bd as a prospective target for drug discovery and development may particularly relevant for combined therapeutic approaches, directed against more targets and appears to have the potential for a successful treatment of the mycobacterial infections. Moreover, cytochrome bd is found only in bacteria and selective inhibitors of this oxidase should not have negative effects on the host energy metabolism. In this context the results of the biochemical assays quantifying the activity of cytochrome bd after addition of selected and optimized haem and quinol inhibitors will allow to identify potential antimicrobials expected to strengthen the bacteriostatic/bactericidal effects of drugs targeting other systems in microbial metabolism.
Since M. tuberculosis cytochrome bd oxidase has not been purified yet, our goal is to develop well-designed protocols to express and purify this enzyme in order to obtain an active form of the protein. This will allow further functional investigations for a better understanding on the role of cytochrome bd in the mycobacterial metabolism as well as future crystallographic studies useful for drug design.