Phthiocerol Dimycocerosate Transport Is Required for Resisting Interferon‐γ–Independent Immunity

Murry, Jeffrey P.; Pandey, Amit; Sassetti, Christopher M.; Rubín, Eric J.

doi:10.1086/605128

Cited by 55 publications

(52 citation statements)

References 48 publications

(70 reference statements)

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“…In contrast, following infection with the PDIM-negative H37Rv clone, NOS2 Ϫ/Ϫ mice survived significantly longer than IFN-␥ Ϫ/Ϫ mice (P Ͻ 0.001). Our results are also consistent with the results of a recent screen for mutants that specifically alter growth of M. tuberculosis in NOS2 Ϫ/Ϫ mice, which identified two independent mutations in the drrA gene encoding a putative PDIM transporter (22). The immune mechanism responsible for this IFN-␥-dependent, NOS2-independent attenuation and the countermechanism by which PDIM confers protection are currently unknown.…”

Section: Discussionsupporting

confidence: 90%

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

et al. 2011

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Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-␥)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2 ؊/؊ mice but were growth-proficient and virulent in IFN-␥ ؊/؊ mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-␥-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2 ؊/؊ and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-␥-mediated immune response that is independent of NOS2.Pathogenic mycobacteria possess a unique array of complex cell wall-associated lipids. The most abundant of these lipids, the phthiocerol dimycocerosates (PDIMs) (Fig. 1), are among the best characterized (23). PDIMs contain long-chain diols esterified by methyl-branched fatty acid chains. As early as 1974, it was recognized that a spontaneously arising PDIMdeficient variant of the laboratory strain H37Rv was attenuated in a guinea pig model of infection (11). Shortly thereafter, it was shown that the in vivo survival of an avirulent Mycobacterium tuberculosis strain was enhanced by coating the bacteria with cholesterol oleate and purified PDIM (16). A genetic link between PDIM production and virulence was not established until a quarter century later, when a large chromosomal locus was identified as playing an essential role in the biosynthesis and export of PDIM (3,4,6). Transposon insertions within the fadD26, fadD28, mmpL7, and drrC genes, and in the putative transcriptional promoter region upstream of the fadD26 gene, were identified in strains deficient in surface-localized PDIM. The fadD26 and fadD28 mutants apparently fail to synthesize PDIM, whereas the mmpL7 and drrC mutants produce PDIM but accumulate it intracellularly, thus implicating these ...

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Section: Discussionsupporting

confidence: 90%

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

et al. 2011

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“…Others have demonstrated upregulation of genes in this pathway in response to acid exposure (29,62), hypoxia (58,62), and treatment with antibiotics, such as clofazamine and rifapentine (7). Furthermore, M. tuberculosis strains with de- fects in this pathway have been shown to have increased cell envelope permeability (10) and are more susceptible to IFN-␥-mediated and IFN-␥-independent immunity (46,56). Importantly, PDIM deficiency appears to be particularly important to M. tuberculosis growth in the host environment, as isolates with deficiencies in this pathway have pronounced growth defects in the spleens and lungs of infected mice (11,19,47) and are more susceptible to the nitric oxide-dependent killing of macrophages (61).…”

Section: Discussionmentioning

confidence: 99%

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Bisson

Mehaffy²,

Broeckling

et al. 2012

J Bacteriol

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h Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominant Mycobacterium tuberculosis mutations in the rpoB gene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains of M. tuberculosis (wild type) to those of their respective rifampin-resistant, rpoB mutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon-and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. Both rpoB mutants showed significant upregulation of the polyketide synthase genes ppsA-ppsE and drrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids in M. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins. ppsA (Rv2931), ppsB (Rv2932), and ppsC (Rv2933) were also found to be upregulated more than 10-fold in the Beijing rpoB mutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in both rpoB mutant isolates. These data suggest that rpoB mutation in M. tuberculosis may trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistant M. tuberculosis infections.A pproximately 9 million people develop active tuberculosis (TB) each year, resulting in nearly 2 million deaths annually (75). Recent progress controlling drug-susceptible TB has been made in many regions (50); however, drug resistance in Mycobacterium tuberculosis, including strains resistant to both first-line drugs, isoniazid and rifampin (multidrug-resistant [MDR] TB), has emerged as a threat to TB control worldwide (26, 65). Although international surveillance systems are inadequate, data from over 80 countries indicate that approximately 1 in 10 new cases of active TB have primary drug resistance (77), and MDR TB has been reported in essentially every country in which drug resistance has been studied (76). Treatment of MDR TB requires the use of costly and toxic second-line drugs for nearly 2 years and is associated with high rates of morbidity and mortality (65), which makes the spread of drug-resistant TB a major public health concern.Drug resistance in M. tuberculosis is due primarily to singlenucleotide polymorphisms in genes encoding key mycobacterial enzymes (6). The rpoB gene encodes the ␤-subunit of bacterial RNA polymerase, which is the target of rifampin (12...

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“…We therefore hypothesized that the increased death of H37Rv:att was due to loss of PDIM from the bacterial cell surface. To test this hypothesis, we obtained a mutant H37Rv strain in which drrA, a gene involved in PDIM transport, was disrupted by transposon insertion (9). As previously described, this strain synthesizes PDIM but is unable to transport the lipid to the cell surface.…”

Section: Innate Immunity Limits the Replication Rate Of M Tuberculosismentioning

confidence: 99%

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

et al. 2014

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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...

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Phthiocerol Dimycocerosate Transport Is Required for Resisting Interferon‐γ–Independent Immunity

Cited by 55 publications

References 48 publications

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

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

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