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

Abstract

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.

Fig. 1.

Comparative screening of M. tuberculosis mutants in untreated vs. INH-treated mice. (A) Screening strategy to identify M. tuberculosis mutants with impaired or enhanced persistence in INH-treated mice. (1) Individual signature-tagged mutants are arrayed in 48-well microplates and pooled. (2) C57BL/6 mice are inoculated with pools of mutants and left untreated or treated with INH from 4 wk postinfection onward. (3) Bacteria are recovered by plate culture of lung homogenates. (4) Signature tags from untreated and INH-treated mice are radiolabeled and measured by membrane hybridization and PhosphorImager analysis. (B) Correlation plot of 576 mutants screened in untreated vs. INH-treated mice. Each symbol represents one tag, expressed as a percentage of the total hybridization signal for all 48 tags in the same pool. The x axis plots the ratio of each tag's representation in untreated mice at 6 vs. 0 wk. Mutants to the left of the vertical dashed line are attenuated for growth/survival in untreated mice. The y axis plots the ratio of each tag's representation in INH-treated mice vs. untreated mice at 6 wk. Mutants below the horizontal dashed line are persistence-impaired, and mutants above the horizontal solid line are persistence-enhanced. Arbitrary values of 0.33 and 3.0 were chosen as cutoffs for selection of mutants for further analysis, indicated by red circles and letters. Tag hybridization signals are shown for mutants with the following phenotypes: normal (e.g., WT) in untreated and INH-treated mice (C); persistence-impaired in INH-treated mice (D); persistence-enhanced in INH-treated mice but growth/survival-attenuated in untreated mice (E); and growth/survival-attenuated in untreated mice (F). Each symbol represents the tag from one mouse. H, INH.

Fig. 2.

Persistence of cydC::Tn and 5′Tn::rv0097 bacteria in INH-treated mice. C57BL/6 mice were infected i.v. with ∼1 × 10⁶ cfus of WT (A and B), cydC::Tn (C and D), or 5′Tn::rv0097 (E and F) bacteria. From 4 wk postinfection onward, groups of mice were left untreated (filled symbols) or were treated with INH (empty symbols). Bacterial cfus were measured in the lungs (A, C, and E) and spleens (B, D, and F) at the indicated time points. Symbols represent mean ± SEM (n = 4). Results are representative of three experiments. Tukey's honestly significant difference test was used to compare the effectiveness of drug therapy (untreated vs. treated mice) against WT vs. mutant bacteria. P values are indicated for each time point where P < 0.05.

Fig. 3.

INH-mediated killing of cydC::Tn bacteria grown in vitro or in MBMMs. WT (squares) and cydC::Tn (circles) bacteria were grown in 7H9 broth (A–F) or in MBMMs (G and H), and OD₆₀₀ or cfus were measured at the indicated time points. (A) Bacteria were grown without INH. Bacteria in exponential phase (B and C) or stationary phase (D) were incubated with 1 μg/mL INH (B) or with 1 μg/mL INH plus 10 μg/mL EMB (C and D). Results are representative of four experiments. (E) WT (gray bars) and cydC::Tn (white bars) bacteria were treated with Diethylenetriamine/nitric oxide adduct (DETA-NO) for 4 h, followed by 1 μg/mL INH for 24 h. Results are representative of two experiments. (F) WT (□) and cydC::Tn (○) bacteria were treated with 10 μg/mL potassium cyanide (KCN) and 0.2 μg/mL INH. Results are representative of three experiments. WT (G) and cydC::Tn (H) bacteria were grown in MBMMs without (filled symbols) or with (empty symbols) 0.75 μg/mL INH added at 4 h postinfection. Intracellular cfus were measured at the indicated time points. Symbols represent mean ± SEM (n = 3). Results are representative of two experiments.

Fig. 4.

Host immunity modulates INH-mediated killing of cydC::Tn bacteria. (A–D) Mice were infected by aerosol with ∼200 cfus of WT (filled symbols and gray bars) or cydC::Tn (open symbols and white bars) bacteria. (A) Bacterial cfus were measured in the lungs of untreated C57BL/6 (■, □), NOS2^−/− (●, ○), and Irgm1^−/− (▲, △) mice at the indicated time points. Symbols represent mean ± SEM (n = 4). Groups of C57BL/6 (B), NOS2^−/− (C), and Igrm1^−/− (D) mice were treated with INH from 2 wk postinfection onward. Bacterial cfus were measured in the lungs at the indicated time points. Symbols represent mean ± SEM (n = 4). Results are representative of two experiments. Tukey's honestly significant difference test was used to compare the effectiveness of drug therapy (untreated vs. treated mice) against WT vs. mutant bacteria. P values are indicated for each time point where P < 0.05.