IDENTIFICATION OF A NOVEL ANTI-APOPTOTIC GENE OF MYCOBACTERIUM TUBERCULOSIS

Abstract

Over the next 20 years, more than 36 million people are expected to die of <italic>Mycobacterium tuberculosis</italic> (Mtb) related illness. We may prevent this only by learning as much as possible about the Mtb-mediated exploitation of the human immune system and successfully implementing that knowledge to combat the pathogen. One Mtb virulence mechanism involves inhibition of host apoptosis. An Mtb laden macrophage will attempt cell suicide, subsequently destroying any intracellular bacteria. In prior work, a large gain-of-function screen identified Mtb genomic regions involved in host apoptotic suppression; one such region is "K20." A loss-of-"gain-of-function" (LoGoF) screen involving <italic>in vitro</italic> transposon (Tn) mutagenesis of a K20 expressing vector identified individual gene(s) of K20 potentially responsible the virulence phenotype. This LoGoF screen found two unique K20 Tn-clones that consistently induced significant host apoptosis. These Tn's disrupted expression of K20 genes <italic>Rv2666</italic> ( probable truncated transposase) and <italic>Rv2667</italic> ( possible Clp ATPase).

A Himar1 Tn mutant of <italic>Rv2666</italic> was obtained through the TARGET mutant library project. Upon infection of human macrophages, Tn<italic>Rv2666</italic> induced significantly more host apoptosis than wild type (WT) and complement, confirming <italic>Rv2666</italic> as an anti-apoptotic gene of Mtb. There are no current publications suggesting a specific role for transposase-based virulence in Mtb; Rv2666 may affect an anti-apoptotic phenotype by modulating transcription of factors important for the suppression of host apoptosis.

The second LoGoF identified gene, <italic>Rv2667/clpC2/clpX'</italic>, was not available through TARGET; a recombinant &#916;<italic>clpC2</italic> mutant was generated. Comparing induced host apoptosis of &#916;<italic>clpC2</italic> infected macrophages to WT revealed <italic>Rv2667/clpC2</italic> has no essential role as an anti-apoptotic gene of Mtb. &#916;<italic>clpC2</italic> was further characterized to explain the discrepancy between the initial LoGoF data and the &#916;<italic>clpC2</italic> Mtb results. Study of &#916;<italic>clpC2</italic> determined that it bears no significant differences with WT in terms of <italic>in vitro</italic> growth, host necrosis-induction, <italic>in vivo</italic> survival and induced host TNF&#945; secretion levels. However, &#916;<italic>clpC2</italic> induces significantly more host IL-1&#946; release than WT Mtb. The reason for this effect is unknown; ClpC2 may aid Mtb pathogenicity by limiting host inflammation, thus permitting infecting Mtb a "head start" against a host adaptive immune response.