Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence of Mycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in the sodC gene. In this report, we show that the M. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parental M. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-␥)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-␥ or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages.

Mycobacterium tuberculosis and Salmonella enterica cause very different diseases and are only distantly related. However, growth within macrophages is crucial for virulence in both of these intracellular pathogens. Here, we demonstrate that in spite of the phylogenetic distance, M. tuberculosis and Salmonella employ a parallel survival strategy for growth within macrophage phagosomes. Previous studies established that the Salmonella mgtC gene is required for growth within macrophages and for virulence in vivo. M. tuberculosis contains an open reading frame exhibiting 38% amino acid identity with the Salmonella MgtC protein. Upon inactivation of mgtC, the resulting M. tuberculosis mutant was attenuated for virulence in cultured human macrophages and impaired for growth in the lungs and spleens of mice. Replication of the mgtC mutant was inhibited in vitro by a combination of low magnesium and mildly acidic pH suggesting that the M. tuberculosis-containing phagosome has these characteristics. The similar phenotypes displayed by the mgtC mutants of M. tuberculosis and Salmonella suggest that the ability to acquire magnesium is essential for virulence in intracellular pathogens that proliferate within macrophage phagosomes.

Mycobacterium tuberculosis grows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth of M. tuberculosis and seven other mycobacterial species. In a defined medium, the growth of M. tuberculosis was very restricted by acidic pH. Higher levels of Mg 2؉ were required for growth of M. tuberculosis in mildly acidic media (pH 6.0 to 6.5) compared to pH 7.0 medium.

In this report we examine the phosphorylation state of cytosolic phospholipase A2 (cPLA2) in C3HA fibroblasts that have been treated with TNF, cycloheximide (CHI), or a combination of both compounds. Our experiments show that TNF and CHI, when used independently, caused the rapid phosphorylation of cPLA2 (within 10 min). In both cases, cPLA2 was subsequently dephosphorylated to pretreatment levels by 40 min. In addition, under these conditions [3H]arachidonic acid was not released, and we could not detect a change in the activity of cPLA2 in vitro. In contrast, in cells treated with a combination of TNF and CHI, we found that the dephosphorylation of cPLA2 was inhibited, and cPLA2 remained phosphorylated for up to 2 h. In vitro we found that sustained phosphorylation of cPLA2 was accompanied by a 60 to 80% increase in the activity of cPLA2. The sustained phosphorylation of cPLA2 also occurred in cells infected with the adenovirus mutant dl309, suggesting that sustained phosphorylation may be a general requirement for the activation of cPLA2 in apoptotic cells. We also found that sustained phosphorylation of phosphoproteins is not a general consequence of apoptotic death, since the phosphorylation of p42 mitogen-activated protein kinase was not sustained. Finally, we show that the phosphatase inhibitor orthovanadate acts as does CHI to render cells susceptible to TNF, suggesting that resistance to TNF may depend on TNF’s ability to induce the expression of tyrosine or dual specificity phosphatase(s).