Omar Vandal

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification, but interferon-γ (IFN-γ) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-γ–activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtbs acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtbs acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.

Acid Resistance in Mycobacterium tuberculosis

Responses to acid have been studied extensively in enteric pathogens, such as Escherichia coli, Vibrio cholerae, and Helicobacter pylori that encounter the extremely low pH (pH 2 to 3) of the stomach during ingestion. In contrast, much less is known about how obligate or facultative intracellular bacterial pathogens like Mycobacterium tuberculosis respond, resist, and persist in the moderately acid environment of the phagosome or phagolysosome. The pH of the macrophage compartment, in which M. tuberculosis resides, ranges from pH 6.2 to 4.5, depending on the activation state of the macrophage (55, 81, 99). Phagosomal acidity may provide a critical cue for adaptation of M. tuberculosis to the host niche. At the same time, to ensure survival for what can be decades, the bacterium must prevent excessive entry of protons into its cytosol and expel them when their concentrations threaten the pH homeostasis that most organisms maintain. Here we review current understanding of the ability of M. tuberculosis to adapt to phagosomal levels of acid. It has been challenging to dissect the role of phagosome acidification in the pathogenesis of tuberculosis, because it is accompanied by and synergizes with other host defenses. Similarly, M. tuberculosis acid resistance mechanisms appear to be cross-protective against other forms of stress, making it difficult to directly relate a defect in acid resistance to impaired virulence. Notwithstanding, the phenomenon is central to the pathogenesis of tuberculosis and thus might offer points of vulnerability that could be exploited by new chemotherapeutics.

Selective Killing of Nonreplicating Mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.

Acid-Susceptible Mutants of Mycobacterium tuberculosis Share Hypersusceptibility to Cell Wall and Oxidative Stress and to the Host Environment

Mycobacterium tuberculosis can persist in macrophage phagosomes that acidify to a pH of approximately 4.5 after activation of the macrophage with gamma interferon. How the bacterium resists the low pH of the acidified phagosome is incompletely understood. A screen of 10,100 M. tuberculosis transposon mutants for mutants hypersensitive to pH 4.5 led to the discovery of 21 genes whose disruption attenuated survival of M. tuberculosis at a low pH (41). Here, we show that acid-sensitive M. tuberculosis mutants with transposon insertions in Rv2136c, Rv2224c, ponA2, and lysX were hypersensitive to antibiotics, sodium dodecyl sulfate, heat shock, and reactive oxygen and nitrogen intermediates, indicating that acid resistance can be associated with protection against other forms of stress. The Rv2136c mutant was impaired in intrabacterial pH homeostasis and unable to maintain a neutral intrabacterial pH in activated macrophages. The Rv2136c, Rv2224c, and ponA2 mutants were attenuated in mice, with the Rv2136c mutant displaying the most severe level of attenuation. Pathways utilized by M. tuberculosis for acid resistance and intrabacterial pH maintenance are potential targets for chemotherapy.

A philosophy of anti-infectives as a guide in the search for new drugs for tuberculosis

How we develop antibiotics is shaped by how we view infectious disease. Given the urgent need for new chemotherapeutics for tuberculosis and other infectious diseases, it is timely to reconsider a view of infectious disease that is strongly supported by contemporary evidence but that has rarely been applied in antibiotic development. This view recognizes the importance of nonreplicating bacteria in persistent infections, acknowledges the heterogeneity and stringency of chemical environments encountered by the pathogen in the host, and emphasizes metabolic adaptation of the host and the pathogen during their competition. For example, efforts in our lab are guided by the perspective that Mycobacterium tuberculosis (Mtb) has co-evolved with the human immune response, with the result that Mtb turns host-imposed metabolic adversity to its own advantage. We seek chemotherapeutics that turn Mtbs adversity to the hosts advantage.

Host-Directed Therapeutics for Tuberculosis: Can We Harness the Host?

SUMMARY Treatment of tuberculosis (TB) remains challenging, with lengthy treatment durations and complex drug regimens that are toxic and difficult to administer. Similar to the vast majority of antibiotics, drugs for Mycobacterium tuberculosis are directed against microbial targets. Although more effective drugs that target the bacterium may lead to faster cure of patients, it is possible that a biological limit will be reached that can be overcome only by adopting a fundamentally new treatment approach. TB regimens might be improved by including agents that target host pathways. Recent work on host-pathogen interactions, host immunity, and host-directed interventions suggests that supplementing anti-TB therapy with host modulators may lead to shorter treatment times, a reduction in lung damage caused by the disease, and a lower risk of relapse or reinfection. We undertook this review to identify molecular pathways of the host that may be amenable to modulation by small molecules for the treatment of TB. Although several approaches to augmenting standard TB treatment have been proposed, only a few have been explored in detail or advanced to preclinical and clinical studies. Our review focuses on molecular targets and inhibitory small molecules that function within the macrophage or other myeloid cells, on host inflammatory pathways, or at the level of TB-induced lung pathology.

Detection and treatment of subclinical tuberculosis.

Reduction of active disease by preventive therapy has the potential to make an important contribution towards the goal of tuberculosis (TB) elimination. This report summarises discussions amongst a Working Group convened to consider areas of research that will be important in optimising the design and delivery of preventative therapies. The Working Group met in Cape Town on 26th February 2012, following presentation of results from the GC11 Grand Challenges in Global Health project to discover drugs for latent TB.

Killing the hypnozoite – drug discovery approaches to prevent relapse in Plasmodium vivax

Abstract The eradication of malaria will only be possible if effective, well-tolerated medicines kill hypnozoites in vivax and ovale malaria, and thus prevent relapses in patients. Despite progress in the 8-aminoquinoline series, with tafenoquine in Phase III showing clear benefits over primaquine, the drug discovery challenge to identify hypnozoiticidal or hypnozoite-activating compounds has been hampered by the dearth of biological tools and assays, which in turn has been limited by the immense scientific and logistical challenges associated with accessing relevant human tissue and sporozoites. This review summarises the existing drug discovery series and approaches concerning the goal to block relapse.

Substrate specificity of MarP, a periplasmic protease required for resistance to acid and oxidative stress in Mycobacterium tuberculosis

Background: MarP is a serine protease critical for pH homeostasis in Mycobacterium tuberculosis. Results: MarP is localized in the periplasm, and its substrate specificity was uncovered using synthetic peptides. Conclusion: The periplasmic location of MarP is essential for function and the substrate profile shows high selectivity at multiple subsites. Significance: Understanding the MarP proteolytic pathway may lead to the development of novel anti-tuberculosis chemotherapeutics. The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). Previous structural studies revealed that MarP contains a chymotrypsin fold and a disulfide bond that stabilizes the protease active site in the substrate-bound conformation. Here, we determined that MarP is located in the Mtb periplasm and showed that this localization is essential for function. Using the recombinant protease domain of MarP, we identified its substrate specificity using two independent assays: positional-scanning synthetic combinatorial library profiling and multiplex substrate profiling by mass spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine at P2, arginine or hydrophobic residues at P1, and alanine or asparagine at P1′. Guided by these data, we designed fluorogenic peptide substrates and characterized the kinetic properties of MarP. Finally, we tested the impact of mutating MarP cysteine residues on the peptidolytic activity of recombinant MarP and its ability to complement phenotypes of Mtb ΔMarP. Taken together, our studies provide insight into the enzymatic properties of MarP, its substrate preference, and the importance of its transmembrane helices and disulfide bond.

Cytosolic Phospholipase A2 Enzymes Are Not Required by Mouse Bone Marrow-Derived Macrophages for the Control of Mycobacterium tuberculosis In Vitro

ABSTRACT During the course of infection Mycobacterium tuberculosis predominantly resides within macrophages, where it encounters and is often able to resist the antibacterial mechanisms of the host. In this study, we assessed the role of macrophage phospholipases A2 (PLA2s) in defense against M. tuberculosis. Mouse bone marrow-derived macrophages (BMDMs) expressed cPLA2-IVA, cPLA2-IVB, iPLA2-VI, sPLA2-IIE, and sPLA2-XIIA. The expression of cPLA2-IVA was increased in response to M. tuberculosis, gamma interferon, or their combination, and cPLA2-IVA mediated the release of arachidonic acid, which was stimulated by M. tuberculosis 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 M. tuberculosis-infected macrophages were treated with PLA2 inhibitors, intracellular survival of M. tuberculosis was not affected, even in inducible nitric oxide synthase-deficient macrophages, in which a major bactericidal mechanism is removed. Moreover, intracellular survival of M. tuberculosis was similar in cPLA2-IVA-deficient and wild-type macrophages. Our results demonstrate that the cytosolic PLA2s are not required by murine BMDMs to kill M. tuberculosis.