Article Figures & Data
Figures
- FIG 1
RegX3-regulated genes are overexpressed in the ΔphoY1 ΔphoY2 mutant. RNA was isolated from M. tuberculosis WT, and the indicated deletion mutants grown to mid-exponential phase (OD600 0.5) in 7H9 medium. Expression of select transcripts was measured using quantitative reverse transcription-PCR, and the results normalized to the sigA transcript level. Data shown are the mean values ± standard deviations of three independent experiments. #, no detectable transcript. Asterisks indicate statistically significant transcript levels compared to the results for the WT, as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. (A) phoY1, phoY2, udgA, mgtA, and rv0784 transcripts were measured in M. tuberculosis WT and ΔphoY1, ΔphoY2, ΔphoY1 ΔphoY2, ΔphoY1 ΔphoY2/pMVphoY1, and ΔphoY1 ΔphoY2/pMVphoY2 mutants. (B) regX3, udgA, mgtA, and rv0784 transcripts were quantified in M. tuberculosis WT and ΔphoY1 ΔphoY2, ΔphoY1 ΔphoY2 ΔregX3, and ΔphoY1 ΔphoY2 ΔregX3/pNDregX3 mutants.
- FIG 2
Deletion of phoY1 and phoY2 causes a stationary-phase growth defect. M. tuberculosis WT and ΔphoY1, ΔphoY2, ΔphoY1 ΔphoY2, ΔphoY1 ΔphoY2/pMVphoY1, and ΔphoY1 ΔphoY2/pMVphoY2 mutants were grown to mid-exponential phase (OD600 of 0.5) in 7H9 medium, diluted to an OD600 of 0.05 in fresh 7H9 medium, and incubated at 37°C with shaking. (A and C) Optical density (OD600) was measured daily. (B and D) Viable CFU were enumerated by plating serially diluted cultures on 7H10 agar. For all panels, results shown are the average values ± standard deviations of three independent experiments.
- FIG 3
Deletion of phoY1 and phoY2 decreases persister frequency in M. tuberculosis. The indicated M. tuberculosis strains were grown in 7H9 medium to mid-exponential phase (OD600 0.5) and diluted to an OD600 of 0.2 before adding antibiotics. Cultures were incubated at 37°C with aeration, and viable CFU/ml were enumerated at the indicated times by plating serial dilutions of cultures on 7H10 agar. Results shown are the average values ± standard deviations of three independent experiments. Asterisks indicate statistically significant differences compared to the results for the WT, as follows: *, P < 0.05. (A and C) Ciprofloxacin (CIP) at 8 μg/ml and ethambutol (EMB) at 4 μg/ml. (B and D) Rifampin (RIF) at 0.1 μg/ml and EMB at 4 μg/ml.
- FIG 4
Deletion of regX3 suppresses the persister defect of the ΔphoY1 ΔphoY2 mutant. The indicated M. tuberculosis strains were grown in 7H9 medium to mid-exponential phase (OD600 0.5) and diluted to an OD600 of 0.2 prior to adding antibiotics. Cultures were incubated at 37°C with shaking, and viable CFU/ml were enumerated at the indicated times by plating serially diluted cultures on 7H10 agar. Results shown are the average values ± standard deviations of three or five independent experiments. (A) Ciprofloxacin (CIP) at 8 μg/ml and ethambutol (EMB) at 4 μg/ml. (B) Rifampin (RIF) at 0.1 μg/ml and EMB at 4 μg/ml. (C) CIP at 8 μg/ml and isoniazid (INH) at 0.1 μg/ml.
- FIG 5
Loss of pstA1 decreases persister frequency in M. tuberculosis. M. tuberculosis strains were grown to mid-exponential phase (OD600 0.5), diluted in fresh 7H9 medium to an OD600 of 0.2, and then treated with antibiotics. Cultures were incubated with aeration at 37°C, and viable CFU/ml were enumerated at the indicated times by plating serial dilutions on 7H10 agar. Results presented are the average values ± standard errors of three independent experiments. Asterisks indicate statistically significant differences from the results for the WT, as follows: *, P < 0.05; **, P < 0.005. (A) Ciprofloxacin (CIP) at 8 μg/ml and ethambutol (EMB) at 4 μg/ml. (B) CIP at 8 μg/ml and isoniazid (INH) at 0.1 μg/ml. (C) Rifampin (RIF) at 0.1 μg/ml and at EMB 4 μg/ml.
- FIG 6
PstA1 is required for M. tuberculosis persister formation during phosphate-limiting growth. M. tuberculosis strains were grown to mid-exponential phase (OD600 0.5) in complete 7H9 medium, washed twice in Pi-limiting 7H9 containing 2.5 μM Pi, and then diluted to an OD600 of 0.1 in Pi-limiting 7H9 containing 2.5 μM Pi. Cultures were incubated with aeration at 37°C in Pi-limiting 7H9 for 72 h prior to the addition of antibiotics. Viable CFU/ml were enumerated at the indicated times by plating serial dilutions on 7H10 agar. Results presented are the average values ± standard errors of three independent experiments. Asterisks indicate statistically significant differences from the results for the WT, as follows: *, P < 0.05; **, P < 0.005. (A and B) Ciprofloxacin (CIP) at 8 μg/ml and ethambutol (EMB) at 4 μg/ml. (C and D) CIP at 8 μg/ml and isoniazid (INH) at 0.1 μg/ml.
- FIG 7
Persistence of ΔphoY1 ΔphoY2 and ΔpstA1 mutants in mice treated with rifampin or isoniazid. C57BL/6 mice were aerosol infected with ~80 CFU of WT Erdman (A and D) and ΔpstA1 (C and F) strains or ~40 CFU of the ΔphoY1 ΔphoY2 mutant (B and E). Four weeks postinfection, mice were divided into no-drug control (closed squares), rifampin treatment (RIF; open squares), and isoniazid treatment (INH; open triangles) groups. At the indicated time points, groups of mice (n = 4) were sacrificed, and CFU were enumerated by plating serially diluted lung (A to C) and spleen (D to F) homogenates on 7H10 agar. Results presented are the mean values ± standard errors of the means. Asterisks indicate statistically significant differences compared to the results for the no-drug control, as follows: *, P < 0.05; **, P < 0.005. For the ΔpstA1 mutant, the results for both the rifampin and isoniazid treatment groups were significantly different from the results for the no-drug control group in the lungs at week 6 (P < 0.05).
Tables
- TABLE 1
MICs of antibiotics against M. tuberculosis wild-type and phosphate regulation mutants
Genotype MIC90 (μg/ml) ofa: CIP EMB INH RIF WT 0.2 0.5–1 0.05 0.050 ΔphoY1 0.2 1 0.025 0.050 ΔphoY2 0.2 0.5 0.025 0.050 ΔphoY1 ΔphoY2 0.1–0.2 0.5 0.025 0.0125 ΔphoY1 ΔphoY2/pMVphoY1 0.2 0.5 0.025 0.050 ΔphoY1 ΔphoY2/pMVphoY2 0.2 0.5 0.025 0.050 ΔphoY1 ΔphoY2 ΔregX3 0.2 1 0.025 0.025 ΔphoY1 ΔphoY2 ΔregX3/pNDregX3 0.2 1 0.025 0.0125 ΔpstA1 0.1–0.2 0.5 0.025–0.05 0.00625 ΔregX3 0.2 0.5–1 0.025–0.05 0.025–0.05 ΔpstA1 ΔregX3 0.2 0.5–1 0.025–0.05 0.025–0.05 ΔpstA1/pMVpstA1 — — — 0.025–0.05 ΔpstA1 ΔregX3/pNDregX3 — — — 0.00625 ↵ a MIC90 (μg/ml) is the minimum concentration required to inhibit 90% of growth compared to the results for the no-drug control. Results are from at least three independent experiments. Ranges are given for strains that exhibited variable MIC90s in two of four experiments. CIP, ciprofloxacin; EMB, ethambutol; INH, isoniazid; RIF, rifampin; —, MIC90 not determined.
- TABLE 2
Polyphosphate quantification in M. tuberculosis wild-type and Pst/SenX3-RegX3 mutant strainsa
Strain nmol polyP/mg total
protein (mean ± SD)bP value
(versus WT)WT 0.09 ± 0.02 ΔphoY1 0.13 ± 0.06 0.258 ΔphoY2 0.41 ± 0.17 0.010 ΔphoY2/pMVphoY2 0.19 ± 0.06 0.023 ΔphoY1 ΔphoY2 0.86 ± 0.21 0.0003 ΔphoY1 ΔphoY2/pMVphoY1 0.08 ± 0.02 0.413 ΔphoY1 ΔphoY2/pMVphoY2 0.07 ± 0.02 0.205 ΔphoY1 ΔphoY2 ΔregX3 0.64 ± 0.18 0.001 ΔphoY1 ΔphoY2 ΔregX3/pNDregX3 0.44 ± 0.10 0.0003 ΔpstA1 0.56 ± 0.15 0.001 ΔpstA1/pMVpstA1 0.21 ± 0.10 0.051 ΔregX3 0.07 ± 0.02 0.306 ΔpstA1 ΔregX3 0.09 ± 0.05 0.957 ΔpstA1 ΔregX3/pNDregX3 0.45 ± 0.22 0.016
FIG S1
Confirmation of phoY1 and phoY2 chromosomal deletions by Southern blotting. (A and B) Genomic DNA from M. tuberculosis Erdman (WT) and ΔphoY1, ΔphoY2, and ΔphoY1 ΔphoY2 mutants was digested with the indicated restriction enzymes, and bands that hybridized to a gene-specific probe were detected by chemiluminescence. Positions of molecular size markers are indicated. (A) PstI digest and phoY1 probe that hybridized to a 1.8-kbp fragment from the WT and a 1.1-kbp fragment from the ΔphoY1 mutants. (B) XhoI digest and phoY2 probe that hybridized to a 2.9-kbp fragment from the WT and a 2.3-kbp fragment from the ΔphoY2 mutants. (C and D) Maps of the phoY1 (C) and phoY2 (D) loci. Genes are indicted by gray arrows. Probes are indicated by red bars. Download FIG S1, EPS file, 0.7 MB.
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TABLE S1
Doubling times of phoY deletion mutants in Pi-rich 7H9 medium. Download TABLE S1, PDF file, 0.1 MB.
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FIG S2
The ΔphoY1 ΔphoY2 mutant is hypersensitive to cell wall and reactive oxygen stress. The indicated M. tuberculosis strains were grown to mid-exponential phase (OD600 0.5) in 7H9 medium, diluted to an OD600 of 0.05 in fresh 7H9 medium, and subjected to either sodium dodecyl sulfate (SDS; 0.125%) or hydrogen peroxide (H2O2; 3 mM) treatment for 24 h at 37°C with shaking. At 0 and 24 h, cultures were serially diluted and plated on 7H10 agar for CFU enumeration. Percent survival was calculated as follows: (CFU poststress)/(CFU prestress) × 100. Results presented are the mean values ± standard errors from three independent experiments. Asterisks indicate statistically significant differences in survival compared to the results for the WT, as follows: **, P < 0.005; ***, P < 0.0005. Download FIG S2, EPS file, 0.1 MB.
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FIG S3
Deletion of phoY1 and phoY2 decreases stationary-phase persister frequency. The indicated M. tuberculosis strains were diluted to an OD600 of 0.05 and grown for 10 days in 7H9 medium to stationary phase before adding 8 μg/ml rifampin. Cultures were incubated at 37°C without shaking. CFU were enumerated at 0, 3, and 9 days by plating serially diluted cultures on 7H10 agar. Percent survival was calculated as follows: (CFU poststress)/(CFU prestress) × 100. Results presented are the mean values ± standard errors from three independent experiments. Asterisks indicate statistically significant differences in survival compared to the results for the WT control, as follows: *, P < 0.05. Download FIG S3, EPS file, 0.1 MB.
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FIG S4
Increased envelope permeability of the ΔphoY1 ΔphoY2 mutant is caused by deletion of phoY2. M. tuberculosis mc27000, the ΔphoY1 ΔphoY2 mutant, and complemented strains were incubated with 2 μg/ml ethidium bromide and uptake rates were determined by measuring emission at 590 nm upon excitation at 544 nm. Data are expressed as relative fluorescence units (RFU) normalized to the fluorescence at 0 min and are the mean values ± standard errors of at least three independent experiments. Download FIG S4, EPS file, 0.1 MB.
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FIG S5
Loss of regX3 decreases M. tuberculosis persister frequency when exposed to rifampin and ethambutol. M. tuberculosis strains were grown to mid-exponential phase (OD600 0.5) and diluted in fresh 7H9 medium to an OD600 of 0.2 prior to the addition of antibiotics. Cultures were incubated with aeration at 37°C, and viable CFU/ml were enumerated at the indicated times by plating serial dilutions on 7H10 agar. Results presented are the average values ± standard errors of three independent experiments. Asterisks indicate statistically significant differences from the results for the WT, as follows: *, P < 0.05; **, P < 0.005. (A) Ciprofloxacin (CIP) at 8 μg/ml and ethambutol (EMB) at 4 μg/ml. (B) CIP at 8 μg/ml and isoniazid (INH) at 0.1 μg/ml. (C) Rifampin (RIF) at 0.1 μg/ml and EMB at 4 μg/ml. Download FIG S5, EPS file, 0.1 MB.
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FIG S6
The ΔphoY1 ΔphoY2 mutant is attenuated in vivo. C57BL/6 mice were aerosol infected with ~80 CFU of the WT Erdman or ΔpstA1 strains or ~40 CFU of the ΔphoY1 ΔphoY2 mutant. At the indicated time points, groups of mice (n = 4) were sacrificed and CFU were enumerated by plating serially diluted lung (A) and spleen (B) homogenates on 7H10 agar. Results shown are the mean values ± standard errors of the means. Asterisks indicate statistically significant differences between the results for the WT and ΔphoY1 ΔphoY2 or ΔpstA1 mutants, as follows: *, P < 0.05; **, P < 0.005. Download FIG S6, EPS file, 0.1 MB.
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TABLE S2
Percent survival of bacteria in lungs and spleens of antibiotic-treated mice relative to survival in no-drug control mice. Download TABLE S2, PDF file, 0.1 MB.
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FIG S7
ppk1 is overexpressed in the ΔphoY1 ΔphoY2 mutant. RNA was isolated from the indicated M. tuberculosis strains grown to mid-exponential phase (OD600 0.5) in 7H9 medium. Expression of ppk1, ppk2, ppx1, and ppx2 transcripts was measured using quantitative reverse transcription-PCR and normalized to the sigA transcript level. Data shown are the mean values ± standard deviations of three independent experiments. Asterisks indicate statistically significant differences in transcript abundance compared to the results for the WT, as follows: *, P < 0.05. Download FIG S7, EPS file, 0.2 MB.
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TABLE S3
Oligonucleotide primers used for cloning, strain construction, or real-time quantitative RT-PCR (qRT-PCR). Download TABLE S3, PDF file, 0.1 MB.
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