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

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 of xylE-expressing colonies. M. smegmatis bacteria expressing the M. tuberculosis glutamine synthetase A (glnA) gene or open reading frame Rv2962c or Rv2958c 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 and plcB) or for high temperature requirement A (htrA) did not.