Mycobacterium tuberculosis Requires the Outer Membrane Lipid Phthiocerol Dimycocerosate for Starvation-Induced Antibiotic Tolerance

Abstract

Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (~1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug resistance. IMPORTANCE Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics. One factor that contributes to the length of TB treatment is M. tuberculosis phenotypic antibiotic tolerance, which allows the bacteria to survive prolonged drug exposure even in the absence of genetic mutations causing drug resistance. Here, we report a genetic screen to identify M. tuberculosis genes that promote drug tolerance during nutrient starvation. Our study revealed the outer membrane lipid phthiocerol dimycocerosate (PDIM) as a key determinant of M. tuberculosis antibiotic tolerance triggered by nutrient starvation. Our study implicates PDIM synthesis as a potential target for development of new TB drugs that would sensitize M. tuberculosis to existing antibiotics to shorten TB treatment.

Keywords: Mycobacterium tuberculosis; PDIM; Tn-seq; antibiotic resistance; drug tolerance; membrane permeability; nutrient limitation.

FIG 1

Growth of M. tuberculosis in MtbYM rich medium increases starvation-induced antibiotic tolerance. Wild-type M. tuberculosis Erdman was grown in 7H9 or MtbYM medium to stationary phase or starved of inorganic phosphate (P_i) for 72 h before adding the antibiotic ciprofloxacin (CIP, 8 μg/mL) plus isoniazid (INH, 0.2 μg/mL) (A) or rifampicin (RIF, 0.1 μg/mL) plus INH (0.2 μg/mL) (B). Cultures were incubated at 37°C with aeration for 9 days. Drug-tolerant bacteria were enumerated by serially diluting and plating on 7H10 agar at the indicated time points. The mean ± standard error of the mean from at least two independent experiments is shown.

FIG 2

Mutants with altered fitness upon drug treatment in P_i-limited MtbYM medium. (A to C) Volcano plots of TnseqDiff statistical analysis of Tn-seq data for P_i-starved Tn mutant pools treated with CIP+INH (A) or RIF+INH (B) or no-drug control (C) compared to input. Dashed lines indicate cutoffs for statistical significance of ±2 log₂ fold change and adjusted P value of <0.025. Tn mutants meeting significance are colored. (D and E) Venn diagrams displaying the number of Tn mutants with significant negative (D) or positive (E) fold changes in relative fitness.

FIG 3

Mutants with altered fitness upon drug treatment during stationary phase in MtbYM medium. (A to C) Volcano plots of TnseqDiff statistical analysis of Tn-seq data for Tn mutant pools grown to stationary phase in MtbYM and treated with CIP+INH (A) or RIF+INH (B) or no-drug control (C) compared to input. Dashed lines indicate cutoffs for statistical significance of ±2 log₂ fold change and adjusted P value of <0.025. Tn mutants meeting significance are colored. (D and E) Venn diagrams displaying the number of Tn mutants with significant negative (D) or positive (E) fold changes in relative fitness.

FIG 4

Individual Tn mutants identified in the P_i starvation Tn-seq screen exhibit reduced fitness during drug treatment. M. tuberculosis strains were P_i starved for 72 h in P_i-free MtbYM medium before addition of the drug combinations ciprofloxacin (CIP, 8 μg/mL) plus isoniazid (INH, 0.2 μg/mL) (A, C, and E) or rifampicin (RIF, 0.1 μg/mL) plus INH (0.2 μg/mL) (B, D, and F). Surviving bacteria were enumerated by plating serial dilutions on 7H10 agar. Data represent the average ± standard error of the mean from at least two independent experiments. Asterisks indicate statistically significant differences between Tn mutant and WT: *, P < 0.05; **, P < 0.005.

FIG 5

Loss of PDIM causes increased drug susceptibility of the clpS::Tn mutant. (A) Thin-layer chromatographic analysis of PDIM lipids extracted from [¹⁴C]propionate-labeled WT Erdman (lane 1), clpS::Tn ppsD Q291* (lane 2), clpS::Tn pMV-ppsD (lane 3), and ppgK::Tn ppsE W787S (lane 4). (B and C) M. tuberculosis strains were P_i starved for 72 h in P_i-free MtbYM medium before adding ciprofloxacin (CIP, 8 μg/mL) plus isoniazid (INH, 0.2 μg/mL) (B) or rifampicin (RIF, 0.1 μg/mL) plus INH (0.2 μg/mL) (C). Surviving bacteria were enumerated by plating serial dilutions on 7H10 agar. Data represent the average ± standard error of the mean from three biological replicates. Asterisks indicate statistically significant differences between WT and clpS::Tn (below points) or between clpS::Tn and clpS::Tn pMV-ppsD (brackets): *, P < 0.05; **, P < 0.005.

FIG 6

PDIM-deficient mutants are hypersusceptible to antibiotics in stationary- and exponential-phase MtbYM cultures. M. tuberculosis strains were grown to early stationary phase (SP) (A, B, and D) or exponential phase (Exp) (C and E) in MtbYM medium before adding ciprofloxacin (CIP, 8 μg/mL) plus isoniazid (INH, 0.2 μg/mL) or rifampicin (RIF, 0.1 μg/mL) plus INH (0.2 μg/mL), as indicated. Data represent the average ± standard error of the mean from at least two independent experiments (A to C) or the average ± standard error of the mean from biological triplicate cultures (D and E). Asterisks indicate statistically significant differences: *, P < 0.05; **, P < 0.005. ns, not significant.