| During the course of infection Mycobacterium tuberculosis (Mtb) predominantly resides within macrophages, where it encounters and is often able to resist the anti-bacterial mechanisms of the host. In this work we examined the role of two macrophage processes in defense against Mtb: phagosome acidification and free fatty acid production by phospholipases. Mtb blocks phagosome acidification, but interferon-gamma (IFNgamma) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear if acid kills Mtb, if the intrabacterial pH of any pathogen falls in the phagosome and if acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and therefore appears resistant to phagolysosomal concentrations of acid. We used transposon mutagenesis to identify genes responsible for Mtb's acid resistance. Twenty-one of 10,100 mutants were acid-sensitive. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated serine protease, was sensitive to acid under all conditions tested. As monitored with an intrabacterial fluorescent protein, wild type Mtb maintained its pH when suspended in acid or ingested by activated macrophages. In contrast, the mutant was unable to maintain its pH and was severely attenuated in mice. Thus, phagosomal acid alone does not kill Mtb. This is due in large part to Rv3671c. Disruption of Rv3671 c renders Mtb vulnerable to the host environment, commending a microbial protease as a drug target.;Mouse bone marrow derived macrophages (BMDMs) expressed the following phospholipases: cPLA2-IVA, cPLA2-IVB, iPLA2-VI, sPLA2-IIE and sPLA2-XIIA. The expression of cPLA 2-IVA was increased in response to Mtb, IFNYgamma or their combination, and cPLA2-IVA mediated the release of arachidonic acid that was stimulated by Mtb in activated, but not unactivated macrophages. We confirmed that arachidonic acid is highly mycobactericidal in a concentration and pH dependent manner in vitro. However, when Mtb infected macrophages were treated with PLA2 inhibitors, intracellular survival of Mtb was not affected, even in iNOS-deficient macrophages in which a major bactericidal mechanism is removed. Moreover, intracellular survival of Mtb was similar in cPLA2-IVA deficient and wild type macrophages. These results demonstrate that the cytosolic PLA2s are not required by murine BMDMs to kill Mtb. |
