Role of Rv3167c in necrosis and autophagy induction by and in virulence of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb), an intracellular pathogen targeting lung phagocytes has been reported to manipulate apoptotic and necrotic host cell death. Mtb induces necrosis allowing for bacteria to escape from the host cell and infect naive cell populations. Mtb genes that influence host cell death responses as well as host cell pathways that are implicated in survival outcomes during mycobacterial infection represent interesting areas of investigation. In this study we identified the Mtb gene Rv3167c to be required for the inhibition of Mtb induced, caspase-independent necrotic cell death. The Rv3167c mutant strain induced higher levels of necrosis in macrophages compared to Mtb or the complement strain. Using a combination of chemical inhibitors and bone marrow derived macrophages from different knock-out mice, we screened for host factors required for the Rv3167c mutant mediated cell death and were able to eliminate involvement of known regulated necrosis pathways. Necrosis induction by the mutant strain was however found to be dependent on reactive oxygen species (ROS) generated by mitochondria and on inhibition of Akt activation. Apart from necrosis, Rv3167c was also found to be required for inhibition of host cell autophagy. Mitochondrial ROS, inhibition of Akt activation and upregulation of JNK activation were required for Rv3167c mutant -induced autophagy. Recent studies on Mtb induced autophagy have shown that Mtb co-localizes with autophagosomes, leading to lysosomal mediated bacterial destruction. Surprisingly, we did not observe co-localization of Mtb or the mutant strain with autophagosomes indicating Mtb has the ability to inhibit selective autophagy mediated killing. Deletion of Rv3167c results in an increase in Mtb virulence as demonstrated by the higher bacterial burden in the lungs and extra-pulmonary organs and the lower median survival time observed in the Rv3167c mutant infected mice compared to Mtb and complement strain infected control animals. In conclusion, this work indicates that Mtb utilizes Rv3167c to regulate necrosis and autophagy induction and to temper its own virulence.