RV3165C AND RV3167C ARE TWO NOVEL ANTI-APOPTOTIC GENES OF MYCOBACTERIUM TUBERCULOSIS

Abstract

Tuberculosis continues to kill close to two million people every year. This is due to the ease with which the bacteria spreads, the ineffectiveness of the vaccine currently in use, as well as the long drug regimen that is required for treatment of the disease. In order to develop new vaccines or drugs which require shorter treatment regimens, it is important to understand the interaction of the immune system with Mycobacterium tuberculosis (Mtb). One of the mechanisms by which Mtb is thought to survive in the human host is the inhibition of the apoptosis of infected cells. I was able to identify Rv3165c and Rv3167c as two novel anti-apoptotic genes of Mtb. Mtb knock-outs lacking Rv3165c or Rv3167c (ΔRv3165c, ΔRv3167c) failed to inhibit apoptosis of infected macrophages and the apoptosis of infected macrophages depended on caspase-8 and 3. Inhibiting reactive oxygen species (ROS) production or scavenging ROS inhibited the apoptosis of ΔRv3165c or ΔRv3167c infected macrophages. ΔRv3165c and ΔRv3167c failed to upregulate the expression of the only catalase of Mtb, katG. Therefore, it seems that Mtb needs to upregulate katG expression in order to neutralize host cell produced ROS to inhibit apoptosis. Rv3165c was found to reside mainly in the cell envelope while Rv3167c, which is predicted to be a probable transcriptional regulator, was found exclusively in the cytosol of mycobacteria. Rv3165c was required for the upregulation of Rv3167c expression. These data led to a working model in which Rv3165c sits in the plasma membrane of Mtb and detects a signal while Mtb is in the phagosome. This signal detection results in upregulation of Rv3167c expression. Increased Rv3167c expression enables Rv3167c to change the expression of Mtb genes like katG. Rv3167c upregulates expression of anti-apoptotic genes and downregulates the expression of pro-apoptotic genes. RNA sequencing and antibody microarray experiments identified the host cell components that are differentially regulated in ΔRv3167c infected cells compared to Mtb infected cells. Validation of these targets will help find the host cell pathways Mtb manipulates to inhibit host cell apoptosis.