Identification of Host-Dependent Survival Factors for Intracellular Mycobacterium tuberculosis through an siRNA Screen

Abstract

The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb) involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a “proof-of-concept” demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including multiple drug-resistance. Thus the host factors identified here may potentially be exploited for the development of anti-tuberculosis drugs. The adaptation of Mycobacterium tuberculosis (Mtb) involves dynamic interactions with the molecular components of the host cellular machinery. Therefore, targeting relevant host factors may provide an alternate approach for the chemotherapy of tuberculosis (TB). To test this, we first performed an siRNA screen targeting all known kinases and phosphatases in murine macrophages infected with a virulent strain of Mtb. A subsequent validation of this screen then identified several host molecules whose depletion severely affected the intracellular survival of mycobacteria. We also then screened against the identified host targets in cells infected with independent isolates of MDR-Mtb. This exercise identified those host molecules that were indispensable for supporting infection, independent of the phenotypic variations exhibited by the pathogen. Then, by using a pharmacological inhibitor that simultaneously targeted two of these molecules, we were able to demonstrate clearance of both drug-sensitive and drug-resistant strains of Mtb from infected cells. Importantly, this inhibitor was also effective in mice infected with the virulent strain of Mtb. Thus, in addition to demonstrating the feasibility of targeting host molecules involved in supporting intracellular persistence of pathogen for TB therapy, our studies also identify several such molecules that may be exploited for the purposes of drug development.