Molecular mechanism of Mtb nuoG/NDH-1 complex mediated apoptosis inhibition

The discovery that a bacterial gene, nuoG, which is a cytosolic component of the NADH-dehydrogenase (NDH-1) is involved in host cell apoptosis inhibition was completely unexpected and was only possible due to the unbiased nature our genetic screen. The initial analysis of the molecular mechanism of host cell apoptosis inhibition by the nuoG protein was performed by characterizing the pro-apoptotic phenotype of the nuoG mutant (Mtb∆nuoG) using the multitude of reagents available to study apoptosis signaling. In summary, our results suggest that NuoG of Mtb, via its implication in the NDH-1 dehydrogenase activity, may play a role in the neutralization of host cell generated superoxides. This neutralization may contribute to the capacity of Mtb to inhibit host cell apoptosis, since the mutant bacteria are functionally complemented in mice deficient (NOX2 ^-/-) in the generation of phagosomal superoxides. Our recent results point to a unique and underappreciated function of the NOX2 activity in macrophage which is to help the host cell in sensing persisting intracellular pathogens that are not susceptible to reactive oxygen species (ROS) and to subsequently induce host cell apoptosis as a second defense line of the innate immune response (manuscript in preparation).

The current working hypothesis is that in the lumen of the phagosome the NDH-1 generated protons specifically interact with NOX2 generated superoxides and with the help of bacterial superoxide dismutase and catalase detoxify the superoxide to water and oxygen. How this controlled reaction is achieved in the microenvironment of the phagosome is currently under investigation. Another important question is how the cytosolic components of the apoptosis signaling pathways sense the increase in ROS production in the phagosome? Is there a leakage of ROS in the cytosol or is it a change in the oxidation of phagosome proteins and lipids that is being sensed? Finally, other important human pathogens are known to inhibit NOX2 activity (Salmonella, Leishmania, Helicobacter and Francisella) but does this capacity also influence their propensity to induce host cell apoptosis?

Determine the Host-Pathogen interactome for M. tuberculosis mediated host cell apoptosis manipulation by identifying and characterizing additional anti-apoptosis genes in M. tuberculosis

            A “gain-of-function” genetic screen for anti-apoptosis genes in Mtb identified three genomic regions (named J21, M24 and K20) in Mtb that have anti-apoptotic capacity when transferred into an apoptosis-inducing, nonpathogenic strain of mycobacteria (Velmurugan et al., PLOS Pathogens, 2007). These regions span about 30kbp of the Mtb genome and contain about 30 genes each. A major focus of the lab at the moment is to identify the anti-apoptosis genes within these regions via several genetic approaches and sub-screens that have proven successful in the identification of the first described anti-apoptosis gene, nuoG, of Mtb. The effect of the deletion of the mutant genes on the virulence of Mtb will be analyzed using the mouse model of tuberculosis and if necessary, in collaboration with Johns Hopkins University, the guinea pig model.

The major focus will subsequently be on answering the question of how the identified gene products mediate apoptosis inhibition of the host cell. It is impossible to predict how each of the identified genes will do this job but we will use state-of the art technology to address the basic molecular mechanism of the interaction of each gene product with the host cell. These techniques will include bioinformatics, proteomics, lipidomics, Y2H technology and others.