Deciphering the role of metals in mycobacterial virulence & host defence against tuberculosis – TB-MET

A pan-genome siRNA and small compounds libraries were screened in macrophages for their ability to alter zinc accumulation in vacuoles. Several hits were obtained, which are under characterization currently.
The M. tuberculosis P-ATPases CtpC is under cloning for fine biochemical characterization and inhibitors identification.

Several hits were obtained, which are under characterization currently; they will provide a better understanding of the molecular and cellular mechanisms involved in microbial zinc poisoning in phagocytes.

We will now characterize the mechanisms behind zinc poisoning in macrophages, and continue the molecular and biochemical characterization of the M. tuberculosis P-ATPase CtpC and other mycobacterial P-ATPases.

None for now.

A better understanding of the cellular and molecular bases of host-pathogen interactions is required for the development of better treatments for infectious diseases. Innate immune defenses against pathogens involve various phagocytic cell types, including macrophages, and subcellular microbial killing mechanisms. These mechanisms include the acidification of the phagocytosis vacuole, or phagosome, the generation of reactive oxygen and nitrogen species, the synthesis of antimicrobial peptides and the exclusion of important nutrients, such as the transition metals iron and manganese, from phagosomes.
The role of transition metals in host-pathogen interactions has mostly been viewed in the context of the NRAMP model of metal starvation, according to which host organisms use soluble chelators (e.g. lactoferrin) or membrane transporters (e.g. NRAMP1) to deprive microbes of vital metal nutrients (e.g. iron), thereby limiting their development and pathogenic potential. In this context, recent results from our laboratory and others have opened up an entirely new field of research into the metallobiology of host-pathogen interactions and, more generally, of the so-called emerging concepts of “nutritional immunology” and “nutritional virulence”. Indeed, others and we have recently reported that phagocytic cells also use the opposite mechanism — i.e. the poisoning microbial cells through the accumulation of transition metals (zinc and copper) within microbial vacuoles — to restrict pathogen growth. Consistent with these findings, microbes, such as Escherichia coli and the tuberculosis (TB) bacillus Mycobacterium tuberculosis, have been shown to require various members of the P-ATPase family of metal efflux pumps to resist metal poisoning within vacuoles and to survive in phagocytes.
The TB-MET project aims to decipher, for the first time, the cellular and molecular mechanisms involved in zinc accumulation within phagosomes in human macrophages, and to determine the role of the microbial P-ATPase in M. tuberculosis physiology and virulence, an area of research that has been neglected to date. For this purpose, we have brought together three highly complementary partners: the laboratory of Dr. O. Neyrolles (IPBS, CNRS-University of Toulouse), which recently made pioneering discoveries concerning the role of zinc in microbial intoxication within macrophages and the role of the P- ATPase CtpC in M. tuberculosis virulence; the laboratory of Drs. Elisabeth Mintz and Patrice Catty (CEA, Grenoble), which has expertise and international recognition in the field of biology and biochemistry of eukaryotic and prokaryotic P-ATPases; and the laboratory of Dr. Priscille Brodin (Institut Pasteur, Lille), which is internationally recognized for its expertise in the use of high-throughput screening of chemicals, mutants and siRNA in the field of mycobacterial chemogenomics. Through a combination of microbial genetics, protein biochemistry, cell biology, functional genomics and high-throughput screening approaches, this project specifically aims to: i) elucidate the mechanism of action of several M. tuberculosis P-ATPases and their function in mycobacterial physiology and virulence in vitro and in vivo, ii) identify potential inhibitors of M. tuberculosis P-ATPases and evaluate their potential as novel anti-TB drugs, iii) identify the cellular and molecular partners involved in microbial poisoning with zinc in human macrophages.
The TB-MET project will provide new and profound insight into the cellular and microbial mechanisms underlying these newly described concepts of metal intoxication and detoxification in host defenses against pathogens and microbial virulence. It may also promote the design of better control strategies for TB and other infections.