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

Abstract

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.