Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB), which kills more people than any other infection. The emergence of drug-resistant Mtb strains has exacerbated this already alarming epidemic. We have identified a small molecule, C10, that potentiates the activity of the frontline antibiotic isoniazid (INH) and prevents the selection for INH-resistant mutants. We find that C10 can even reverse INH resistance in Mtb. Therefore, our study reveals vulnerabilities that can be exploited to reverse INH resistance in Mtb.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

As the deadliest pathogen in the world, Mycobacterium tuberculosis (Mtb) causes infections responsible for 1.6 million deaths in 2017 (1). During infection, Mtb is exposed to an arsenal of host-derived stresses; however, it responds to stress with physiological changes that allow it to tolerate these immune stresses and persist (2). These same physiological changes result in antibiotic tolerance, in which Mtb is genetically susceptible to antibiotics but exists in a physiological state rendering it recalcitrant to therapy (3⇓⇓–6). As a result, long courses of antibiotic therapy are required to treat tuberculosis (TB) (7), leading to the emergence of drug-resistant mutant strains of Mtb. In 2017, out of the 10 million cases of TB, an estimated 19% of newly treated cases and 43% of previously treated cases exhibited resistance to at least one of the frontline antibiotics (1). Resistance to the frontline antibiotic isoniazid (INH) is the most common form of Mtb monoresistance and is associated with treatment failure, relapse, and progression to multidrug-resistant TB (1). Together, the problems of phenotypic tolerance and genetic resistance to antibiotics undermine current TB treatment options. There is an urgent need for new strategies that shorten the duration of treatment and target both drug-tolerant and genetically drug-resistant Mtb, which requires a better understanding of how Mtb survives exposure to immune defenses and antibiotic therapy.

Previous work has demonstrated that a number of stresses are capable of inducing the formation of drug-tolerant Mtb (8⇓–10). The most thoroughly studied inducer of drug tolerance is hypoxia. Exposure to hypoxic conditions has pleiotropic effects on the bacteria, including replication arrest (8), induced expression of dormancy-associated genes (11, 12), shifts in Mtb lipid composition (5, 13), and global shifts in Mtb metabolism and respiration (8, 14, 15). However, it remains unclear mechanistically how these changes in physiology confer tolerance to stress and antibiotics.

To address this gap in understanding, we developed a chemical screen to identify compounds that inhibit the development of hypoxia-induced stress and drug tolerance. Through this chemical approach, we identified a compound, C10, that inhibits the development of hypoxia-induced tolerance to oxidative stress and INH. In addition to blocking tolerance, C10 was found to prevent the selection for INH-resistant mutants and to resensitize an INH-resistant mutant to INH, providing evidence that INH resistance can be reversed in Mtb.

Results

C10 Potentiates Killing by INH and Prevents the Selection for INH-Resistant Mutants. The striking and specific effects of C10 on INH tolerance indicated that C10 uniquely potentiates INH. To test whether C10 has a general effect on INH sensitivity or whether it specifically blocks hypoxia-induced INH tolerance, we cultured Mtb in planktonic, aerated conditions in media containing C10 and/or INH, and monitored growth by changes in optical density (ODλ 600 ) (Fig. 2A). In these conditions, treatment with 5 μM C10 alone resulted in no difference in growth compared with the DMSO-treated control, and treatment with 25 μM C10 resulted in a 1.53-fold increase in Mtb doubling time (Fig. 2 A and B). Since we used an INH concentration above the minimum inhibitory concentration (0.02–0.04 μg/mL) (29), INH treatment inhibited Mtb growth with or without C10. We enumerated the surviving cfu after 10 d of treatment by plating the viable bacteria on agar media without drugs and found that the addition of C10 in combination with INH resulted in a significant and dose-dependent decrease in viable bacteria compared with INH alone. Therefore, C10 potentiates the bactericidal activity of INH against aerobically grown Mtb over a 10-d treatment period (Fig. 2C). Fig. 2. C10 potentiates killing by INH and prevents the selection of INH-resistant mutants. (A) WT Mtb was grown in aerated planktonic conditions in Sauton’s medium with 5 μM or 25 μM C10 ± 0.25 μg/mL INH, and ODλ 600 was measured over 10 d. Mean ± SEM is graphed; n = 3. (B) The doubling time ± SD of cultures in A was calculated between day 0 and day 4. This time frame was chosen because the DMSO cultures were in the exponential growth phase. N/A indicates that growth was inhibited, and the calculation of doubling time did not accurately represent the data, as determined by R2 value (R2 <0.98). (C) After 10 d of treatment, cfu/mL were enumerated from cultures in A. Mean ± SEM values are graphed. n = 3. (D) WT Mtb was plated onto Sauton’s agar containing 0.5 μg/mL INH ± 25 μM C10. Representative pictures from three independent experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s test. Complete statistical comparisons for all data are provided in SI Appendix, Table S1. To further study the impact of C10 on INH efficacy, we spread ∼8 × 107 cfu of Mtb on agar media containing C10 and/or INH so that the bacteria were continually exposed to the drugs, as opposed to the transient 10-d exposure in liquid culture. Mtb formed a lawn of bacterial growth on agar containing DMSO or 25 μM C10 (Fig. 2D). Growth of Mtb on agar containing 0.5 μg/mL INH was inhibited, with the exception of spontaneous INH-resistant colonies that emerged at an approximate frequency of 1 in 106, similar to previous reports (30). In contrast, when C10 was present in combination with INH, no resistant colonies grew, demonstrating that C10 blocked the selection for INH-resistant mutants (Fig. 2D).

C10 Resensitizes katG Mutant Mtb to Inhibition by INH. The majority of INH-resistant clinical isolates harbor mutations in katG (31), which encodes the sole catalase-peroxidase in Mtb and the enzyme that converts INH into its active form (32). We sequenced the katG gene from seven of the colonies that grew on agar containing INH (Fig. 2D) and identified katG mutations in all seven isolates; four harbored frameshift mutations, and three had missense mutations (SI Appendix, Table S2). Since no INH-resistant katG mutants grew when C10 was combined with INH (Fig. 2D), the growth of the katG mutants must be inhibited by either C10 alone or the C10-INH combination. To distinguish between these possibilities, we monitored the growth of an Mtb isolate with a frameshift mutation at amino acid 6 in katG (katGFS) in aerated, planktonic cultures in the presence of C10 and/or INH (Fig. 3A). As expected, the INH-resistant katGFS mutant was able to grow in media containing INH (doubling time 3.28 ± 0.20 d), albeit at a 1.23-fold slower rate than the DMSO-treated cultures (doubling time 2.66 ± 0.20 d) (Fig. 3B). The use of 5 μM C10 did not significantly affect the growth rate of the katGFS strain, and the use of 25 μM C10 increased the doubling time of the katGFS strain by 1.47-fold (Fig. 3B), which is comparable to the 1.53-fold increase in doubling time caused by 25 μM C10 in WT Mtb (Fig. 2B). Therefore, the katGFS strain was not significantly more sensitive than WT Mtb to treatment with C10 alone. However, the combination of C10 and INH significantly inhibited growth of the katGFS strain compared with INH or C10 alone (Fig. 3A). Fig. 3. C10 resensitizes katG mutants to inhibition by INH. (A) katGFS Mtb was grown in Sauton’s medium with 5 μM or 25 μM C10 ± 0.25 μg/mL INH, and ODλ 600 was measured over 10 d. Mean ± SEM is graphed. n = 3. (B) The doubling time of cultures in A was calculated between day 0 and day 4. This time frame was chosen to be consistent with that used in Fig. 2; however, the doubling time was similar when calculated over days 0–8. N/A indicates that growth was inhibited, and the calculation of doubling time did not accurately represent the data, as determined by R2 value (R2 <0.98). (C) After 10 d of treatment, cfu/mL were enumerated. Mean ± SEM values are graphed. n = 3. (D) katGFS Mtb was plated onto Sauton’s agar containing 0.5 μg/mL INH and/or 25 μM C10. Representative pictures from three independent experiments are shown. (E and F) Either katGA172T (E) or katGW328L (F) mutant Mtb was grown in Sauton’s medium with 5 μM C10 ± 0.25 μg/mL INH, and ODλ 600 was measured over 10 d. n = 2. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant by two-way ANOVA (A, E, and F) or one-way ANOVA (B and C) with Tukey’s test. Complete statistical comparisons for all data are provided in SI Appendix, Table S1. We enumerated viable bacteria from these cultures after 10 d of treatment by plating the surviving bacteria on agar media without drugs and found that although INH or C10 alone did not significantly decrease the number of surviving katGFS Mtb, the combination of C10 and INH resulted in a significant reduction in cfu (Fig. 3C), further demonstrating that the C10-INH combination inhibits the katGFS mutant. Similarly, the katGFS mutant grew on agar containing either C10 or INH alone, but not on agar containing the combination of both C10 and INH (Fig. 3D). Therefore, C10 restored the sensitivity of the katGFS mutant to INH. The absence of growth of any katG mutants on plates containing INH and C10 (Fig. 2D) suggests that C10 restores the sensitivity of all katG mutants that normally would be selected for in the presence of INH alone. To directly test whether the effect of C10 can be generalized to additional INH-resistant katG mutants, we measured the impact of C10 on INH sensitivity in two additional strains harboring mutations in katG at residues that were identified as mutated in INH-resistant clinical isolates: katGA172T and katGW328L (31, 33). When we treated the katGA172T and katGW328L Mtb mutants with 5 μM C10 and/or 0.25 μg/mL INH, we found that the combination of both C10 and INH resulted in significantly decreased growth compared with either treatment alone, similar to the katGFS mutant (Fig. 3 E and F). These studies provide evidence that INH resistance can be reversed in katG mutant strains of Mtb.

C10 Sensitizes Mtb to Acid Stress. Since respiration plays an important role in maintaining intrabacterial pH homeostasis (44⇓–46), we hypothesized that inhibition of respiration by C10 could compromise the ability of Mtb to survive exposure to acid stress. We tested whether C10 sensitizes Mtb to low pH by culturing Mtb aerobically in media at pH 7.0 or 5.5 and monitoring bacterial survival. In the absence of C10, Mtb cultured at pH 5.5 for 8 d showed no loss of viability. In contrast, in the presence of C10, the viability of Mtb cultured at pH 5.5 decreased by more than three orders of magnitude over 8 d (Fig. 5A), demonstrating that C10 sensitizes Mtb to low pH. In addition, C10 inhibited growth of Mtb on low-pH agar media (Fig. 5B), further demonstrating that C10 sensitizes Mtb to acid stress, consistent with our findings that C10 perturbs respiration. Fig. 5. C10 sensitizes Mtb to acid stress. (A) WT Mtb was cultured in Sauton’s media at pH 7.0 or 5.5 in the presence of DMSO or 50 μM C10, and viable bacteria were enumerated over time. n = 3. Mean ± SEM values are graphed. ****P < 0.0001; ns, not significant by two-way ANOVA with Tukey’s test. Relevant comparisons are indicated. Complete statistics are provided in SI Appendix, Table S1. (B) WT Mtb was cultured on Sauton’s agar, pH 5.5, with 25 μM C10 or DMSO, and pictures were taken at 43 d. Growth on pH 7.0 agar is shown in Fig. 2D.