Mycobacterium tuberculosis Strains Lacking Surface Lipid Phthiocerol Dimycocerosate Are Susceptible to Killing by an Early Innate Host Response

Abstract

The innate immune response plays an important but unknown role in host defense against Mycobacterium tuberculosis. To define the function of innate immunity during tuberculosis, we evaluated M. tuberculosis replication dynamics during murine infection. Our data show that the early pulmonary innate immune response limits M. tuberculosis replication in a MyD88-dependent manner. Strikingly, we found that little M. tuberculosis cell death occurs during the first 2 weeks of infection. In contrast, M. tuberculosis cells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficient M. tuberculosis was not alleviated by the absence of interferon gamma (IFN-␥), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protects M. tuberculosis from an early innate host response that is independent of IFN-␥, reactive nitrogen intermediates, and reactive oxygen species. By employing a pathogen replication tracking tool to evaluate M. tuberculosis replication and death during infection, we identify both host and pathogen factors affecting the outcome of infection. Phagocyte innate antimicrobial mechanisms are crucial in host defense against intracellular pathogens. For Mycobacterium tuberculosis and many other pathogens, interferon gamma (IFN-␥)-mediated activation of infected macrophages is central in controlling bacterial growth. Indeed, IFN-␥ is required to induce the majority of known macrophage effector functions, including the generation of reactive nitrogen and oxygen intermediates, phagosome-lysosome fusion, and autophagy (1). In the case of M. tuberculosis, antigen-specific CD4 ϩ T cells are the primary in vivo source of IFN-␥, but this adaptive immune response does not begin to develop until after 2 weeks postinfection (p.i.) (2). Whether innate immunity contributes to M. tuberculosis control prior to the arrival of antigen-specific T cells in the lung remains an outstanding question in the field.Recent work bearing on this question revealed that innate responses dependent on the central signaling molecule MyD88 are critical in defense against M. tuberculosis, as MyD88 Ϫ/Ϫ mice were highly susceptible despite normal adaptive immune responses (3). Moreover, human studies indicate that genetic variation in the innate immune response influences human susceptibility to tuberculosis (4). These observations suggest that innate immunity may play an important role in host defense against mycobacteria; however, the precise mechanisms remain unknown.To define the function of innate immunity in controlling M. tuberculosis growth, we monitored M. tuberculosis replication dynamics during murine infection. This previously validated technique (5) exploits an unstable plasmid as a replication clock, allowing bacterial replication and death rates to be determined from a simple differential plating technique. In this study, we apply this app...

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

“…For analysis of PDIM lipids, 6 l of each M. marinum lipid sample was resolved on a 12-cm by 9-cm TLC silica gel 60 plate (Merck KGaA, Darmstadt, Germany) using low-boiling-point petroleum ether-ethyl acetate (50:1; Fisher Scientific) for ϳ28 min (82). The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis strain H37Rv purified phthiocerol dimycocerosate (PDIM; catalog number NR-20328).…”

F 420 H 2 Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria

“…One of these reductases (Ddn, encoded by the Rv3547 ORF) acts as a nitroreductase to activate a prodrug called PA-824, a promising anti-TB drug that acts in concert with F 420 H 2 (45,60,63,64). The hydroxymycolic acid dehydrogenase (fHMAD), which generates ketomycolic acids (K-MA) from hydroxymycolic acids (H-MA), and fPKR studied here exemplify the critical role of F 420 in the functionalization of the formidable cell wall lipids that protect M. tuberculosis from the host immune attack and from anti-TB drugs (12,39,61,(65)(66)(67)(68)(69)(70). Also, fHMAD is one of the targets of PA-824 (39).…”

“…The pathways for the biosynthesis of DPs are of great interest because these compounds are virulence factors for Mycobacterium tuberculosis and Mycobacterium leprae, which cause tuberculosis (TB) and leprosy, respectively (3)(4)(5)(6)(7)(8)(9)(10)(11). It was recently shown that these polyketide derivatives protect M. tuberculosis from the early innate immune response of an infected host (12). The DPs are generated by esterifying ␤-glycol-containing long-chain polyketides (PK) with long-chain multimethyl-branched fatty acids ( Fig.…”

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication

“…Moreover, the PDIM lipids have been recently implicated in host immune evasion by masking pathogen–associated molecule patterns (PAMPs), and the absence of PDIMs promoted the recruitment of macrophages producing reactive nitrogen species (Cambier et al, 2014). However, another study showed that the attenuation of PDIM-deficient M. tuberculosis strain is RNIs independent (Day et al, 2014), indicating some controversy in the role of PDIM in the protection of mycobacteria from host-mediated killing.…”