Evaluation of Mycobacterium tuberculosis Genes Involved in Resistance to Killing by Human Macrophages

Abstract

A coinfection assay was developed to examine Mycobacterium tuberculosis genes suspected to be involved in resistance to killing by human macrophages. THP-1 macrophages were infected with a mixture of equal numbers of recombinant Mycobacterium smegmatis LR222 bacteria expressing an M. tuberculosis gene and wild-type M. smegmatis LR222 bacteria expressing the xylE gene. At various times after infection, the infected macrophages were lysed and the bacteria were plated. The resulting colonies were sprayed with catechol to determine the number of recombinant colonies and the number ofxylE-expressing colonies. M. smegmatis bacteria expressing the M. tuberculosis glutamine synthetase A (glnA) gene or open reading frame Rv2962c orRv2958c demonstrated significantly increased survival rates in THP-1 macrophages relative to those of xylE-expressing bacteria. M. smegmatis bacteria expressing M. tuberculosis genes for phospholipase C (plcA andplcB) or for high temperature requirement A (htrA) did not.