Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst.

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-gamma)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-gamma 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.

Growth of Mycobacterium tuberculosis in a defined medium is very restricted by acid pH and Mg(2+) levels.

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. The divalent cations Ca(2+), Zn(2+), or Mn(2+) could not replace Mg(2+) during growth at pH 6.25, but Ca(2+) could at least partially substitute for Mg(2+) during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg(2+) levels. M. tuberculosis was the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg(2+). M. kansasii and M. smegmatis also grew very poorly in acidic media with limiting Mg(2+). M. fortuitum, M. marinum, M. scrofulaceum, M. avium, and M. chelonae grew at pH 6.0 in an unrestricted manner. These results demonstrate that M. tuberculosis is unique among the mycobacteria in its extreme sensitivity to acid and indicate that M. tuberculosis must acquire sufficient Mg(2+) in order to grow in a mildly acidic environment such as within the phagosome of macrophages.

Sustained phosphorylation of cytosolic phospholipase A2 accompanies cycloheximide- and adenovirus-induced susceptibility to TNF.

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).