Replication dynamics of Mycobacterium tuberculosis in chronically infected mice

The dynamics of host-pathogen interactions have important implications for the design of new antimicrobial agents to treat chronic infections such as tuberculosis (TB), which is notoriously refractory to conventional drug therapy. In the mouse model of TB, an acute phase of exponential bacterial growth in the lungs is followed by a chronic phase characterized by relatively stable numbers of bacteria. This equilibrium could be static, with little ongoing replication, or dynamic, with continuous bacterial multiplication balanced by bacterial killing. A static model predicts a close correspondence between "viable counts" (live bacteria) and "total counts" (live plus dead bacteria) in the lungs over time. A dynamic model predicts the divergence of total counts and viable counts over time due to the accumulation of dead bacteria. Here, viable counts are defined as bacterial CFU enumerated by plating lung homogenates; total counts are defined as bacterial chromosome equivalents (CEQ) enumerated by using quantitative real-time PCR. We show that the viable and total bacterial counts in the lungs of chronically infected mice do not diverge over time. Rapid degradation of dead bacteria is unlikely to account for the stability of bacterial CEQ numbers in the lungs over time, because treatment of mice with isoniazid for 8 weeks led to a marked reduction in the number of CFU without reducing the number of CEQ. These observations support the hypothesis that the stable number of bacterial CFU in the lungs during chronic infection represents a static equilibrium between host and pathogen.

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

Neeraj Dhar, John McKinney

Tuberculosis (TB) is notoriously difficult to cure, requiring administration of multiple antibiotics for 6 mo or longer. Conventional anti-TB drugs inhibit biosynthetic processes involved in cell growth and division, such as DNA replication, RNA transcription, protein translation, and cell wall biogenesis. Although highly effective against bacteria cultured in vitro under optimal growth conditions, these antibiotics are less effective against bacteria grown in vivo in the tissues of a mammalian host. The factors that contribute to the antibiotic tolerance of bacteria grown in vivo are unknown, although altered metabolism and sluggish growth are hypothesized to play a role. To address this question, we identified mutations in Mycobacterium tuberculosis that impaired or enhanced persistence in mice treated with isoniazid (INH), a front-line anti-TB drug. Disruption of cydC, encoding a putative ATP-binding cassette transporter subunit, accelerated bacterial clearance in INH-treated mice without affecting growth or survival in untreated mice. Conversely, transposon insertions within the rv0096-rv0101 gene cluster attenuated bacterial growth and survival in untreated mice but paradoxically prevented INH-mediated killing of bacteria in treated mice. These contrasting phenotypes were dependent on the interaction of the bacteria with the tissue environment because both mutants responded normally to INH when grown in macrophages ex vivo or in axenic cultures in vitro. Our findings have important implications because persistence-impairing mutations would be missed by conventional genetic screens to identify candidate drug targets. Conversely, persistence-enhancing mutations would be missed by standard diagnostic methods, which are performed on bacteria grown in vitro, to detect drug resistance.

2010

Spontaneous phthiocerol dimycocerosate-deficient variants of Mycobacterium tuberculosis are susceptible to gamma interferon-mediated immunity

John McKinney, Anna De Graff Tischler, Swapna Uplekar

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-γ)-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-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-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-γ-mediated immune response that is independent of NOS2.

2011

Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients

John McKinney

Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.

2003