NlaA: A protein of Mycobacterium tuberculosis mediates evasion of host cell apoptosis

Abstract

Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming nearly two million lives every year. This pathogen exhibits extraordinary capacities to evade host responses in order to survive within the host. One of the hallmarks of M. tuberculosis is its ability to inhibit apoptosis to promote its intracellular survival. In this context, surface associated or exported and secreted proteins are of particular importance, since they are ideally positioned to interact with the host. In the present study, a Tn552'phoA containing M. tuberculosis cosmid libraries were integrated into the M. smegmatis genome in single copy and colonies were screened for active phoA fusions which represent secreted or exported proteins. Based on this screen, 15 new secreted and membrane proteins were identified, that had not been reported earlier. A clone that we isolated encoded an exported protein, which we name nlaA shares significant homology to the Nurim protein of humans and to Isoprenyl Cysteine Carboxyl Methyltransferase (ICMT) of bacteria. Inhibition of ICMT in human vascular endothelial cells is known to induce apoptosis by interfering with the cell-signaling pathway. We hypothesized that NlaA is a bacterial effector molecule that enhances the survival of M. tuberculosis in a mammalian host by inhibiting macrophage apoptosis. We show by TUNEL labeling followed by flow cytometry that THP-1 cells infected with the DeltanlaA mutant induced significantly more apoptosis than M. tuberculosis H37Rv or the DeltanlaA mutant complemented with the wild-type copy of the nlaA gene. Analysis of TUNEL stained tissue sections from the DeltanlaA mutant and wild-type infected mice further confirmed our in vitro results. Our studies also show that the DeltanlaA mutant is attenuated, grows significantly less than the M. tuberculosis H37Rv or the complemented strain and inflicts less pathological disease in the organs of infected mice. We conclude that the nlaA gene product is a mycobacterial effector molecule that is involved in interactions with the phagocytic cells and inhibits the death signals in monocytes. Understanding the mechanism of inhibition of infection-induced apoptosis and the signaling pathway involved may help reveal salient components of host defense.