Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

In Mycobacterium tuberculosis , the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel Mtb ) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel Mtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel Mtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel Mtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel Mtb point mutants that were either synthetase dead (Rel Mtb H344Y ) or hydrolase dead (Rel Mtb H80A ). M. tuberculosis strains expressing the synthetase-dead Rel Mtb H344Y mutant did not persist in mice, demonstrating that the Rel Mtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel Mtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel Mtb mutant, Rel Mtb H80A , that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel Mtb H80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel Mtb -mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.