PE/PPE proteins mediate nutrient transport across the outer membrane of Mycobacterium tuberculosis

Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate–dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.

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

ABSTRACTThe innate immune response plays an important but unknown role in host defense againstMycobacterium tuberculosis. To define the function of innate immunity during tuberculosis, we evaluatedM. tuberculosisreplication dynamics during murine infection. Our data show that the early pulmonary innate immune response limitsM. tuberculosisreplication in a MyD88-dependent manner. Strikingly, we found that littleM. tuberculosiscell death occurs during the first 2 weeks of infection. In contrast,M. tuberculosiscells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficientM. tuberculosiswas not alleviated by the absence of interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protectsM. tuberculosisfrom an early innate host response that is independent of IFN-γ, reactive nitrogen intermediates, and reactive oxygen species. By employing a pathogen replication tracking tool to evaluateM. tuberculosisreplication and death during infection, we identify both host and pathogen factors affecting the outcome of infection.

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

ABSTRACTMultidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominantMycobacterium tuberculosismutations in therpoBgene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains ofM. tuberculosis(wild type) to those of their respective rifampin-resistant,rpoBmutant 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. BothrpoBmutants showed significant upregulation of the polyketide synthase genesppsA-ppsEanddrrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids inM. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins.ppsA(Rv2931),ppsB(Rv2932), andppsC(Rv2933) were also found to be upregulated more than 10-fold in the BeijingrpoBmutant 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 bothrpoBmutant isolates. These data suggest thatrpoBmutation inM. tuberculosismay 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-resistantM. tuberculosisinfections.

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

ABSTRACTOnset of the adaptive immune response in mice infected withMycobacterium tuberculosisis 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-γ)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identifyM. tuberculosis“counterimmune” mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in therv0072,rv0405, andrv2958cgenes. These mutants were impaired for replication and virulence in NOS2−/−mice but were growth-proficient and virulent in IFN-γ−/−mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-γ-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-typeppsDgene and reversion of theppsDgene to the wild-type sequence that theppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2−/−and wild-type C57BL/6 mice, and a growth advantagein vitroin liquid culture. We conclude that PDIM biosynthesis is required forM. tuberculosisresistance to an IFN-γ-mediated immune response that is independent of NOS2.