Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199–3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250–258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

Tuberculosis (TB) is a chronic disease mostly affecting the lungs and, despite the availability of a vaccine and antibiotic therapy, remains the leading cause of death due to a single bacterium. An estimated 2 billion people are thought to be infected with Mycobacterium tuberculosis, the causative agent of TB, with 10 million new infections each year and 1 million deaths (1). M. tuberculosis success as a pathogen can be attributed to its ability to adapt and persist within the host. This intracellular pathogen replicates within macrophages and must be able to withstand host-imposed stresses as well as gain access to nutrients to survive and proliferate. M. tuberculosis has been observed inside lipid-rich lesions during infection in humans and in animal models of TB (2, 3). Its genome contains an extensive number of redundant genes dedicated to β-oxidation necessary for lipid breakdown, indicating the importance of lipid catabolism (4). In addition to β-oxidation, lipid utilization depends on two key enzymes, isocitrate lyase (ICL) required for the glyoxylate shunt and phosphoenolpyruvate carboxykinase (PEPCK) catalyzing the first committed step of gluconeogenesis. Mutants lacking either ICL or PEPCK cannot grow with lipids as a sole carbon source and are severely attenuated in the TB mouse model (5–7). While M. tuberculosis can simultaneously use several different carbon sources to grow in vitro (8), lipids seem to be the primary source of carbon during infection (5, 7, 9–11). Whether this is because glycolytic carbon sources are scarce or inaccessible or because M. tuberculosis requires lipids to grow during infection is unknown.Despite its ability to block phagosome–lysosome fusion, a notable fraction of phagocytosed M. tuberculosis is rapidly trafficked toward acidified compartments, and this proportion increases upon macrophage activation by T cell produced interferon-γ (IFN-γ) (12–14). Moreover, lung tissues from TB patients were found to have a median pH of pH 5.5 (15) supporting that M. tuberculosis faces acid stress during its infectious cycle in humans. M. tuberculosis can survive at a pH as low as pH 4.5 by maintaining its intracellular pH close to neutral at least in part through sustained peptidoglycan hydrolysis (16, 17). While the identification of mechanisms that enable M. tuberculosis to survive at acidic pH has taken much attention, how the pathogen adapts to an acidified environment to grow and promote disease remains ill defined. At acidity lower than pH 5.8, M. tuberculosis enters a nonreplicating state in vitro (18). This growth arrest at mildly acidic pH is surprising considering that the bacterium likely replicates in more acidic environments during infection.The media used to culture mycobacteria contain glycerol and glucose as main carbon sources. Previous work demonstrated that growth of M. tuberculosis at acidic pH is improved by changing the carbon source (18, 19). Pyruvate promoted growth of M. tuberculosis at pH 5.7 (19) indicating that growth at acidic pH is regulated by available carbon sources. Because lipids appear to be the primary carbon source M. tuberculosis utilizes to grow in vivo (5, 7, 9–11), we hypothesized that providing host-relevant carbon sources to M. tuberculosis, such as lipids, would serve as a more physiologically relevant model to examine how M. tuberculosis responds to acidic pH. Here, we demonstrate that providing oleic acid (OA) (and other host-relevant lipids) as a carbon source to M. tuberculosis, resulted in sustained growth in acidic cultures at pH 5.5 and below. We applied metabolomics, genetic, and biochemical approaches to investigate this pH-driven use of lipids to support growth. Our work helps explain the dependence of M. tuberculosis on lipids as a primary carbon source during infection and demonstrates that M. tuberculosis is well adapted to the acidic environments it encounters during infection; in fact, it is not only able to maintain its neutral intrabacterial pH, as previously reported, but can replicate in acidic conditions similar to those within phagolysosomes.