Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity

Understanding how Mycobacterium tuberculosis has evolved into a professional pathogen is helpful in studying its pathogenesis and for designing vaccines. We investigated how the evolutionary adaptation of M. smegmatis mc251 to an important clinical stressor H2O2 allows bacteria to undergo coordinated genetic mutations, resulting in increased pathogenicity. Whole-genome sequencing identified a mutation site in the fur gene, which caused increased expression of katG. Using a Wayne dormancy model, mc251 showed a growth advantage over its parental strain mc2155 in recovering from dormancy under anaerobic conditions. Meanwhile, the high level of KatG in mc251 was accompanied by a low level of ATP, which meant that mc251 is at a low respiratory level. Additionally, the redox-related protein Rv1996 showed different phenotypes in different specific redox states in M. smegmatis mc2155 and mc251, M. bovis BCG, and M. tuberculosis mc27000. In conclusion, our study shows that the same gene presents different phenotypes under different physiological conditions. This may partly explain why M. smegmatis and M. tuberculosis have similar virulence factors and signaling transduction systems such as two-component systems and sigma factors, but due to the different redox states in the corresponding bacteria, M. smegmatis is a nonpathogen, while M. tuberculosis is a pathogen. As mc251 overcomes its shortcomings of rapid removal, it can potentially be developed as a vaccine vector.


INTRODUCTION
Tuberculosis (TB) is caused by the pathogen Mycobacterium tuberculosis and remains a public health threat, resulting in 1.4 million deaths in 2020 (WHO, 2021). The prevalence of multidrug-resistant M. tuberculosis and the rising cases of co-infection with HIV increase this health concern. The World Health Organization (WHO) has estimated that a quarter of the world's population is infected with M. tuberculosis (WHO, 2021). This latent state may be extended as long as the life of the infected host, but unfortunately, the reactive rate is approximately 5-10% of infected individuals (Flynn and Chan, 2001). TB is treated with chemotherapy, and the latent state of mycobacteria prolongs the time of treatment, which is one of the causes for the development of mycobacterial resistance.
As one of the world's most successful human pathogens, M. tuberculosis has evolved elegant strategies to escape the immune defensive system of the host. For example, D'Arcy et al. observed that M. tuberculosis-containing phagosomes do not fuse with the lysosome inside infected macrophages (Brown et al., 1969;Armstrong and Hart, 1971). Several studies have indicated that M. tuberculosis being an intracellular pathogen is partially due to its ability to survive and persist in macrophages, in hostile environments with oxidative stress, in low pH, and under starvation and other stresses (Cohen et al., 2018;Nauseef, 2019). When M. tuberculosis infects macrophages, mycobacteria must overcome exogenous reactive oxygen species (ROS), a classical innate defense mechanism against infection (Pieters, 2008). In addition, during latency, M. tuberculosis continues to be exposed to oxidative stress, and thus, the accumulation of mutations caused by oxidative DNA damage is predicted as a potential risk for resistance to antibiotics (Ford et al., 2011). Clinical investigation has shown that cells from TB patients produce less ROS than those of healthy individuals (Jaswal et al., 1992;Kumar et al., 1995). In addition, ROS can also be associated with the treatment of TB (Piccaro et al., 2014;Hu et al., 2021). To avoid ROS attack, an evolved detoxified system is essential for M. tuberculosis survival, persistence, and subsequent reactivation (Piccaro et al., 2014).
As a mycobacterial model of M. tuberculosis, M. smegmatis has contributed to understanding the functions of mycobacteria (Aldridge et al., 2012;Kieser et al., 2015;Gray et al., 2016). The essential genes in M. tuberculosis have a corresponding ortholog gene in M. smegmatis (Dragset et al., 2019;Judd et al., 2021). M. smegmatis mc 2 155 and M. tuberculosis H37Rv share 2547 mutually orthologous genes (Dragset et al., 2019). At least under certain conditions, M. tuberculosis and M. smegmatis have similar growth mechanisms. The assumed virulence factors identified in M. tuberculosis such as PhoPR and DosR/S/T (Gonzalo-Asensio et al., 2014;Mehra et al., 2015) are also present in M. smegmatis. However, compared to virulent M. tuberculosis, M. smegmatis is a nonpathogenic mycobacterium. M. tuberculosis can persist in the infected host, while the host can quickly remove M. smegmatis (Anes et al., 2003;Anes et al., 2006). Considering that M. tuberculosis with high resistance to hydrogen peroxide (H 2 O 2 ) persists in the lungs, while M. smegmatis with lower resistance to H 2 O 2 exists in the soil, we hypothesized that the redox state of M. tuberculosis and M. smegmatis may adapt to the corresponding redox environment. The differences between the two bacteria are due to their corresponding degree of resistance to H 2 O 2 . To test this hypothesis, we selected a series of H 2 O 2 -resistant mutant strains using a clinically important stressor H 2 O 2 and identified the highly H 2 O 2 -resistant mycobacterial strain mc 2 51 (Li et al., 2014b). Compared to the wild-type M. smegmatis mc 2 155 strain, the minimum inhibitory concentration (MIC) of H 2 O 2 was more than 80-fold higher, while the MIC of mc 2 51 to H 2 O 2 was 3.125 mM, similar to that of M. bovis BCG (0.625 mM) (Li et al., 2014a) and M. tuberculosis (0.625 mM), while that of mc 2 155 was 0.039 mM (Li et al., 2014b). mc 2 51 exhibited a slow growth rate similar to M. tuberculosis.
In this study, we first showed that H 2 O 2 -resistant mc 2 51 had a growth advantage both in mice and macrophages compared with wild-type mc 2 155, which indicated that the higher resistance to H 2 O 2 of mycobacteria is related to higher virulence. Similar to M. tuberculosis that can survive under hypoxia in the lungs and resuscitate under appropriate conditions, mc 2 51 presented a growth advantage to recover from dormancy using the Wayne dormancy model. Furthermore, we showed that Fur mutant carrying the A28V point mutation dysregulated katG levels, which was the main cause for resistance to H 2 O 2 and low levels of ATP. Additionally, the redox-related protein Rv1996, responsible for regulating gene expression, exhibited different phenotypes associated with isoniazid susceptibility in M. smegmatis mc 2 155 and mc 2 51, M. bovis BCG, and M. tuberculosis mc 2 7000. Thus, the same protein presents different phenotypes under different physiological conditions. Our results suggest that the difference in the corresponding redox status causes the difference in pathogenicity between M. tuberculosis and M. smegmatis.

RESULTS
The Mycobacterium tuberculosis-Like M. smegmatis Mutant Strain mc 2 51 Displayed Improved Virulence Previous studies showed that M. smegmatis with the gain of H 2 O 2 resistance, named mc 2 51, improves growth fitness in mycobacteria under stress (Table 1) (Li et al., 2014a;Li et al., 2014b). Prior to selecting H 2 O 2 -adapted mycobacterial mutants, we first measured the MIC of wild-type mc 2 155 to H 2 O 2 , which was 0.039 mM. We used 0.0293 mM H 2 O 2 for the initial screening, which was lower than the MIC of mc 2 155 to H 2 O 2 ( Table 1). The whole process was performed as follows: cultures were started from glycerol-frozen stocks and grown to log phase (OD 600 of 0.6-0.8). Then, the culture was diluted 1,000 times and grown under 0.0293 mM H 2 O 2 until the OD 600 reached logarithmic (log) phase. Cultures were then further diluted 1:1,000, and an additional 0.0293 mM of H 2 O 2 was added to the culture. This process was repeated until the H 2 O 2 concentrations reached 0.4395 mM. In further rounds of culture, H 2 O 2 was added in steps of 0.0879 mM instead of 0.0293 mM until a concentration of 1.5 mM was reached (Supplementary Figure S1; Supplementary Table S1). The actual MIC of H 2 O 2 of the mutant strain, named mc 2 51, selected at 1.5 mM, was 3.125 mM. In a previous study, the mutated mycobacterial strain, mc 2 51, evolved into an M. tuberculosis-like strain that presented slow growth and improved growth fitness under stress (Li et al., 2014b), which H 2 O 2 resistance in mycobacteria linked to virulence. Previous studies including ours have associated isoniazid (INH) with H 2 O 2 resistance (Timmins and Deretic, 2006;Hu et al., 2021). The MIC of isoniazid (INH) for mc 2 51 was dramatically reduced to~1% of the MIC for mc 2 155 (Table 1). To test our hypothesis that the H 2 O 2 -resistant strain mc 2 51 would be more persistent in the host than its parental strain mc 2 155, we performed non-invasive intranasal infections, which can induce respiratory mucosal immune responses and is a promising way of vaccination for respiratory infection diseases. As shown in Figure 1A, intranasal infection of C57BL/6 mice with~1 × 10 7 /50 μl and bacterial colony-forming units (CFUs) were counted 1 day after infection. Compared to mc 2 155 with the percentage of survival in the infected lung of 0.03653 ± 0.01462% (n = 3), the mc 2 51 strain with that of 0.7153 ± 0.2597% (n = 3) had a significantly higher survival percentage (p = 0.0451) ( Figure 1B). After infection with live mycobacteria, the bacilli enter alveolar macrophages (AMΦs) and persist within them. To exclude the possibility that the higher CFUs were caused by the larger number of infected bacilli being captured by AMΦs, macrophage-killing assays were explored using the THP-1 cell line. The survival percentage of mutant mc 2 51 is 0.3158 ± 0.1502 (n = 9), while the survival of wild-type mc 2 155 was 0.01921 ± 0.01223 (n = 6) (p = 0.0003) ( Figure 1C). These results indicated that mc 2 51 exhibited enhanced virulence.
Mutant fur Altered the Intracellular Redox State and Was Conductive to Latency and Resuscitation via Modulation of KatG Levels M. tuberculosis has evolved to survive in hypoxic conditions. Over more than 100 years of research, M. tuberculosis has been confirmed to be an obligate aerobic bacterium that cannot replicate under hypoxic conditions. However, M. tuberculosis has incredible survivability in long-term anaerobic environments.  The schematic diagram of the mice infection. C57BL/6 mice were intranasally infected as described in Materials and Methods with~1 × 10 7 /50 μl of M. smegmatis mc 2 155 or mc 2 51. At the 3rd day after infection, the whole lung homogenates were plated to determine bacterial numbers. (B) The H 2 O 2 -resistant mutant strain mc 2 51 has a significantly higher bacterial load in the infected lung. The lung burdens 3 days after infection with mc 2 155 (black circles) or mc 2 51 (black squares) were measured, and the survival percentage was calculated as described in Materials and Methods. Each group consisted of three mice. *p < 0.05. (C) The H 2 O 2 -resistant mutant strain mc 2 51 has a survival advantage over wild-type mc 2 155 in the macrophage-like cell line THP-1. THP-1 cells were infected with mc 2 155 (black circles) or mc 2 51 (black squares) at a multiplicity of infection of 1 h after infection, THP-1 cell lysates were collected, and the intracellular bacilli were measured, and the survival percentage was calculated as described in Materials and Methods. Each group consisted of nine repeats. ***p < 0.001.
Frontiers in Genetics | www.frontiersin.org March 2022 | Volume 13 | Article 758304 Evidence suggests that M. tuberculosis has the ability to reduce the respiratory system to low levels and maintain vitality (Loebel et al., 1933). Due to respiratory depression, ATP is maintained at low levels, which guarantees minimal metabolic activity to ensure membrane integrity under hypoxic conditions. We first compared the survival of mc 2 51 and mc 2 155 strains under hypoxic conditions. We established a Wayne dormancy model (Wayne and Hayes, 1996), consisting of mycobacterial strain cultures grown in the 7H9 medium to an OD 600 of 1.0, which were then transferred to anaerobic tubes containing 1 × 10 6 cells/ml with a headspace ratio of the culture system of 0.5. Methylene blue (1.5 mg/L) was added as an indicator of oxygen status. The OD 600 and CFUs at different indicated times and the color transition time were measured. The indicator of mc 2 51 had become colorless on the 6th day (141 h); on the contrary, the indicator of mc 2 155 was still in blue at this time ( Figure 2A). For mc 2 155, the indicator became colorless in about 8 days (189 h), indicating that it has entered an anaerobic state. The mc 2 51 entered the anaerobic state faster in the later stages, and the number of viable bacteria in the anaerobic state was significantly higher than mc 2 155 ( Figures 2B,C).
For the activation experiment, bacteria cultured in the anaerobic conditions were collected and diluted to 1 × 10 6 cells/ml in 7H9 and the brain-heart infusion medium and were then grown under aerobic conditions. The three independent mc 2 51 clones were set, and each clone set up three replicates, all of which were grown better than the three independent clones of mc 2 155 ( Figure 2D). Thus, mc 2 51 had the growth advantage of recovering from the dormancy of the anaerobic state over mc 2 155. We also measured the intracellular ATP levels of mc 2 51 and mc 2 155. As expected, The oxygen tension indicator methylene blue of mc 2 51 culture (left) changes to colorless on day 6, while the indicator of mc 2 155 culture (right) stays blue in the Wayne dormancy model. The cultures were initially inoculated in anaerobic tubes at an OD 600 of 0.01 with a headspace ratio of 0.5. The cultures were stirred at 120 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. Methylene blue changes from blue to colorless under reducing conditions. Data present results of three biological replicates. Growth rates of strains mc 2 155 (navy blue) and mc 2 51 (brown) were measured by measuring the OD 600 (B) and by determination of CFUs (C) after plating on 7H10. Data are presented as the mean ± standard deviation of three independent replicates. **p < 0.01. (D) The H 2 O 2 -resistant mutant strain mc 2 51 shows a growth advantage of recovering from dormancy in an anaerobic state over mc 2 155. After the indicator methylene blue in the culture of the Wayne dormancy model became colorless, cultures of mc 2 155 (navy blue) or mc 2 51 (brown) were collected and reinoculated into 7H9 combined with the heart infusion medium at an OD 600 of 0.01 and aerobic shaken at 200 rpm. The CFUs were determined 20 h after inoculation. Data are presented as the mean ± standard deviation of three independent replicates. ****p < 0.0001.
Frontiers in Genetics | www.frontiersin.org March 2022 | Volume 13 | Article 758304  the abundance of ATP in mc 2 51 (0.1885 ± 0.0481 μM/mg) was lower than that of mc 2 155 (0.8138 ± 0.1324 μM/mg) ( Figure 3B). We previously performed whole-genome sequencing to compare differences in mc 2 155 and mc 2 51 at the genome level. Whole-genome sequencing revealed that there were 29 single-nucleotide polymorphisms (SNPs) in mc 2 51. All 29 SNPs were cloned, and each was transformed into mc 2 155 strains, and only the fur (msmeg_3460), located upstream of msmeg_3461, encoding KatG ( Figure 4A), could restore the resistance phenotype of H 2 O 2 (Li et al., 2014b). The furencoded protein Fur negatively regulated katG expression (Pym et al., 2001). The A28V Fur mutation (mFur) in mc 2 51 may also affect the expression of katG. To verify this hypothesis, we examined the binding of mFur to the target DNA (the promoter region of the fur) using electrophoretic mobility shift assays (EMSAs). Compared to wild-type Fur protein, EMSA showed that mFur decreased DNA binding ( Figure 4B), which resulted in the katG transcription dysregulation by mFur. In addition, the RNA of mc 2 155 and mc 2 51 was extracted and quantified. The results showed that compared to the mc 2 155 strain, the expression of the catalase-peroxidase (KatG) encoding gene katG of the mc 2 51 strain was significantly upregulated tõ 61.82-fold that of the wild-type strain. Taken together, mFur increases the KatG protein level in mc 2 51. KatG is a dual enzyme for catalase and peroxidase, which hydrolyzes ROS (Ng et al., 2004). Thus, the mc 2 51 may maintain ATP at lower levels through KatG, compared to mc 2 155 levels; that is, the abundance of KatG may affect the mycobacterial redox state and, thus, change the susceptibility to H 2 O 2 . We then constructed the ΔkatG (mc 2 155 with knockout katG) and pMV261-katG/ mc 2 155 (mc 2 155 with overexpression of katG) strains, and their respective ATP content was tested. As shown in Figure 3A, the KatG level negatively correlated with the ATP level. Furthermore, we constructed the specific site mutant of the fur gene (mfur) in wild-type mc 2 155 causing an amino acid change of A28V of Fur ( Figures 5A,B) by using recombination protein gp61 from Che9c mycobacteriophage (van Kessel et al., 2008), to construct mc 2 155mfur ( Table 1). As we expected, the Fur mutation at A28V induced high resistance to H 2 O 2 with the MIC of H 2 O 2 in mc 2 155-mfur being 0.64 mM. We showed that the point mutation of fur dysregulation of katG expression is a major factor leading to the phenotype H 2 O 2 resistance.

The Same Protein Performs Different Functions in Different Redox States
As a successful human pathogen, M. tuberculosis has unique respiration properties. M. tuberculosis excretes alkaline supernatants, which is in contrast to other strains that excrete acidic supernatants (Merrill, 1930). The difference between secreted compounds with different acid-base properties suggested that M. tuberculosis has a distinctive redox state. As shown in Table 2, the comparative genomic analysis shows that PhoPR and DosR/S/T, identified as virulence factors of M. tuberculosis, are present in M. smegmatis. The signaling transduction systems such as the two-component systems and the sigma factors of M. tuberculosis are homologous in M. smegmatis (Table 3). Different phenotypes might be due to different redox states. Thus, we considered that the same redox-regulated related protein might perform different functions in different redox states. To test this hypothesis, we examined the biological function of a universal stress protein Rv1996 that increases the expression of KatG, in various mycobacterial strains (Hu et al., 2015). Both previous studies and our studies have linked isoniazid action with redox states, and H 2 O 2 resistance is negatively correlated with INH susceptibility in mycobacteria (Bhaskar et al., 2014;Hu et al., 2015;Vilcheze et al., 2017). We used INH as a chemical probe for monitoring mycobacterial redox states and measured the MICs of INH to the corresponding mycobacterial strains ( Figure 6). As predicted, the MICs of INH differed across the tested mycobacterial strains: the MIC of INH in pMV261-rv1996/mc 2 7000 was equal to that of pMV261/mc 2 7000; the MIC of INH in pMV261-rv1996/BCG was lower than that in pMV261/BCG; the MIC of INH in pMV261-rv1996/mc 2 51 was equal to that of pMV261/mc 2 51; and in mc 2 155, the opposite results were observed with the MIC of

DISCUSSION
Understanding how M. tuberculosis evolved into a professional pathogen is of benefit to the study of its pathogenesis and design of vaccines. The combination of experimental evolution and whole-genome sequencing provides a powerful method for identifying adaptive mutations and elucidating the specific genotype-phenotype relationship (Elena and Lenski, 2003;Lenski, 2017). Historically, the most successful example of continuous selective cultures is M. bovis BCG, the only anti-TB vaccine, which was attenuated after 13 years of continuous in vitro passages of M. bovis BCG. We previously used a similar adaptive evolution strategy to select H 2 O 2 -resistant M. smegmatis strains by using a clinically key stressor H 2 O 2 (Li et al., 2014b). Preliminary results showed that the mc 2 51 strain was highly resistant to H 2 O 2 and had greater susceptibility to INH, compared to mc 2 155. The mc 2 51 phenotype showed an M. tuberculosis-like M. smegmatis phenotype. Altogether, the mutant M. smegmatis mc 2 51 exhibited higher virulence.
The whole-genome sequencing showed the presence of gene mutations in fur, and the mutant Fur resulted in katG levels ( Figure 4B). In the Wayne dormancy model, mc 2 51 shows a growth advantage of recovering from dormancy under anaerobic conditions over mc 2 155 ( Figure 2B). In parallel, a high level of katG in mc 2 51 is accompanied by lower ATP levels, which implied mc 2 51 exhibited at a lower level of respiration ( Figure 3B). Moreover, we showed that a redox-related protein Rv1996 exhibits a different phenotype under different specific redox states in M. smegmatis mc 2 155 and mc 2 51, M. bovis BCG, and M. tuberculosis mc 2 7000 ( Figure 6). This study indicated that the same genotype presents different phenotypes under different physiological conditions. We at least partially explain why M. smegmatis and M. tuberculosis have similar virulent factors, including a two-component system and sigma factors (Tables 2, 3), but M. smegmatis is a nonpathogen and M. tuberculosis is a pathogen.
M. tuberculosis is a successful human pathogen. It is considered to be derived from the environment (Gutierrez  (2007) Msm, Mycobacterium smegmatis; Mtb, Mycobacterium tuberculosis CDC1551. +Genes encoding the sensor kinase and the response regulator are present and genetically linked.
-Genes encoding both the sensor kinase and the response regulator are absent. *Genes encoding the two sensor kinases have been fused, and the gene encoding this fused sensor kinase is genetically linked to the response regulator.  , 2007) and has adapted to the immune environment of the human body through long-term evolution. Its successfully established infection is partially attributed to its survival capacity and persistence in macrophages (Podinovskaia et al., 2013). To defend against mycobacterial infection, the host produces ROS, as an important innate defense mechanism. Consequently, M. tuberculosis has evolved a hierarchy and unique antioxidant function and maintains a low level of respiration, manifested by slow growth and persistence in the host. In contrast, M. smegmatis is present in the soil, which is a totally different environment from the host (Zhang and Furman, 2021). In Table 2, we show that M. tuberculosis and M. smegmatis have similar genotypes; however, they show different phenotypes, in terms of INH susceptibility, H 2 O 2 resistance, and virulence. We believe that this striking difference is due to H 2 O 2 resistance. The selected resistance to H 2 O 2 of mc 2 51 shows improved virulence in both the macrophage-killing assay and in an animal model (Figure 1). In fact, several studies have shown that abiotic stress can improve the virulence phenotype of bacterial pathogens (Sundberg et al., 2014;Li et al., 2021). Our study also supports the sit-and-wait hypothesis (Wang et al., 2017), that is, bacterial environmental abiotic stress and virulence evolution. In addition, this study also suggests that we can use mc 2 51 as a model strain, replacing mc 2 155, to study the regulation of redox homeostasis of M. tuberculosis. We previously sequenced the whole genome of mc 2 51 strain (Li et al., 2014a) and identified 29 SNPs, compared to mc 2 51. Confirmed with our previous study (Li et al., 2014b), we found that only the fur gene can partially complement the resistant phenotype. This suggested that the fur mutation facilitated elevated H 2 O 2 resistance, although it was not entirely responsible for the high resistance observed in mc 2 51. We also mutated the fur in wild-type mc 2 155 by genome editing and produced a phenotype similar to mc 2 51, which is highly resistant to H 2 O 2 ( Table 1). Large-scale whole-genome sequencing studies on the evolutionary history of tuberculosis also show that key tract mutations at the transcription site will have a critical impact on the particular phenotype (Gagneux, 2018). For example, the change of PhoP in BCG allows infection with bovine pathogenic bacteria capable of infecting humans (Gonzalo-Asensio et al., 2014;Broset et al., 2015). This study reminds us that when designing vaccines, greater attention should be paid to regulators, which may be more efficient targets. M. smegmatis is an effective vaccine for TB and HIV (Sweeney et al., 2011;Kim et al., 2017). The disadvantage of M. smegmatis as a vaccine vector is its transient infection and difficulty to establish a persistent infection and produce adaptive immunity. The M. tuberculosislike mutant M. smegmatis mc 2 51 may be developed as a vaccine vector.
By comparing the survival of mc 2 155 and mc 2 51 in the Wayne dormancy animal model, we also found that low respiratory levels are beneficial for survival under anaerobic conditions and resurrection ( Figure 2). In the future, we plan to use these strains to compare physiological indicators such as NADH/ NAD + , NADPH/NADP + , and ATP, to further understand the mechanisms underlying M. tuberculosis resurrection. This study provides insight into H 2 O 2 -resistant mechanisms in mycobacteria and has important implications for linking mycobacteria redox capacity and persistence infection in mice.

Strains and Growth Conditions
The H 2 O 2 -resistant Mycobacterium smegmatis strain mc 2 51 was screened in the Mi lab (Li et al., 2014b). Mycobacterium tuberculosis ΔpanCD (named mc 2 7000)  was kindly gifted by J Deng. The M. smegmatis wild-type mc 2 155, mutant strain mc 2 51, M. bovis BCG Pasteur, and M. tuberculosis mc 2 7000 were cultured in Middlebrook 7H9 (Becton Diskinson Sparks, MD, United States) supplemented with ADS (10% albumin, dextrose, and saline), 0.05% Tween 80 (Sigma, St. Louis, MO, United States), and 0.5% glycerol (Beijing Modern Eastern Fine Chemical Co., Ltd., Beijing, China) for liquid culture and Middlebrook 7H10 (Becton Diskinson Sparks, MD, United States) supplemented with ADS for bacterial colony culture. The colony-forming units (CFUs) of mycobacterial strains were determined by plating serial dilutions of cultures on Middlebrook 7H10 agar plates and incubating at 37°C in an atmosphere of 5% CO 2 for the indicated time. For mc 2 7000 culture, panthothenate (24 mg/L) was added. When required, kanamycin (25 mg/L, Amresco, United States) and hygromycin (50 mg/L, Sigma, United States) were added. All bacterial strains used in this study are listed in Table 1.

Determination of MIC to Isoniazid and H 2 O 2
The susceptibility of isoniazid (INH) or H 2 O 2 of mycobacteria was evaluated using the modified broth microdilution method Frontiers in Genetics | www.frontiersin.org March 2022 | Volume 13 | Article 758304 8 (Franzblau et al., 1998). In brief, INH or H 2 O 2 was serial diluted using the 7H9 medium. The diluted fold was 1.25-or 2-fold, when required. Then, 40 μl of diluted INH or H 2 O 2 was mixed with 40 μl of mycobacterial suspension with 1 × 10 7 cells/ml in each well of 96-well microtiter plates and then incubated at 37°C for the indicated days. As an indicator, 0.02% resazurin was added to individual samples, and the color switches from blue to pink were recorded after 4 h. All the experiments were performed in triplicate. The abundance of the cultures was measured using a microplate reader (FLUOstar OPTIMA, BMG Labtech). A difference of two serial dilutions or more indicated a significant difference in the INH or H 2 O 2 susceptibility of bacterial strains.

Mice Infection
Female pathogen-free C57BL/6 mice (aged 6-8 weeks) were purchased from Vital River (Beijing, China). For the intranasal infection of M. smegmatis, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg), and~10 7 CFU/50 µl PBS of mc 2 51 or mc 2 155 was introduced dropwise through the nostril of each mouse. The bacterial burden throughout the infection was monitored by collecting whole lung tissue at the indicated times after the mice were euthanized, and serial dilutions were then plated on 7H10 agar plates. The dose of infection was confirmed on day one after infection by plating whole lung homogenates from three mice on 7H10 agar. The percentage of survival was calculated as (CFUs after infection/ CFUs before infection) × 100%.

Macrophage-Killing Assay
Human-derived cell line THP-1 (ATCC TIB-202) was cultured in RPMI medium with 10% fetal bovine serum (FBS, GIBCO, United States). THP-1 cells were activated with 100 ng/ml phorbo-12-myristate-13-acetate (PMA, Sigma, United States) overnight. Infection was carried out at a multiplicity of infection (MOI) of 10 for 1 h at 37°C and 5% CO 2 atmosphere. The infected THP-1 cells were washed with RPMI 3 times and then chased for 1 h. The cells with intracellular bacilli were then washed and lysed in sterile cold PBST (PBS with 0.05% Tween 20). Lysates were then vortexed, diluted, and plated on 7H10 agar plates as previously described (Chan et al., 1992). The percentage of survival was calculated as (CFUs after infection/CFUs before infection) × 100%.

Wayne Dormancy Model and Dormancy Exit
Mycobacterial strains were cultured under hypoxic conditions as described by Wayne and Hayes (Wayne and Hayes, 1996). In brief, cultures were initiated at an OD 600 of~0.01 (1 × 10 6 ) and incubated in anaerobic tubes with sealed caps. The headspace ratio of the cultures was 0.5. The cultures were stirred using an 8 mm Teflon stir bar (Fisher Scientific, United States) at 200 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. It changes in color from blue to colorless under low oxygen tension. The color transition time was recorded. All experiments were performed in triplicate. Growth was monitored by measuring the OD 600 and by determination of CFUs after plating on 7H10.
The indicator methylene blue in the culture became colorless, indicating that the bacteria entered anaerobic conditions. Then, after collecting the bacteria in the anaerobic tube, they were washed with the culture medium or PBS three times and resuspended in a culture medium (7H9 and brain-heart infusion medium), the concentration was adjusted to the same amount of OD 600 (OD 600 of~1.0) and diluted for the CFU count, 1:100 or 1:50 into a fresh culture medium was shaken at 37°C, to monitor the status of the bacteria, and the OD 600 was measured. Three independent mycobacterial strain clones were set, and each clone set up three replicates.

Measurement of Intracellular ATP
The ATP Assay Kit was purchased from Beyotime Biotechnology (Beijing, China). The intracellular ATP assay was performed following the protocol provided by the manufacturer. In brief, the sample measurements were prepared as follows: cultures of indicated mycobacteria were obtained to an OD 600 of 0.8. Bacteria were collected by low-temperature centrifugation at the maximum speed, and the pellet was washed with precooled PBS buffer 3 times. A 300 μl volume of ATP detection lysate and 0.5 ml volume of glass beads were added for cell lysis. The lysate obtained was centrifuged at low temperature for 5 min, and the supernatant was placed on ice for later use. The preparation of the standard solution of gradient concentration ATP was performed as follows: the ATP standard solution was serially diluted into 7 concentrations of 10, 3.333, 1.111, 0.37, 0.1234, 0.04115, and 0.01371 μM and stored on ice for later use. The preparation of the working solution for the detection of ATP was performed as follows: an appropriate amount of ATP detection reagent was prepared according to the number of samples, and then, a 90% final volume of ATP detection reagent diluent was added. The prepared working fluid was placed on ice for further use. The determination of the ATP level was performed as follows: 1) the prepared ATP detection working solution was dispensed into 1.5 ml centrifuge tubes, 100 μl per tube, and was allowed to incubate at room temperature for 5 min to allow full reaction of the ATP in the centrifuge tube; 2) during the test, 20 μl of each sample (standard or total protein sample) was added to a 1.5 ml centrifuge tube containing 100 μl of ATP detection working solution and was mixed quickly with a pipette and incubated for 2 s to complete the reaction before using a Cytation 3 Cell Imaging Multi-Mode Reader to determine the relative light unit (RLU); 3) a standard curve was constructed to measure and determine the concentration of the sample by converting the RLU into an ATP concentration; and 4) in order to eliminate the error caused by the difference in the amount of protein during sample preparation, the BCA protein concentration determination kit produced by Beyotime Biotechnology (Beijing, China) was used to determine the protein concentration in the sample. The ATP concentration was converted to μM/mg protein.

Electrophoretic Mobility Shift Assay
The coding regions of fur and mfur were amplified from mc 2 155 and mc 2 51 genomic DNA and cloned into the Escherichia coli expression vector pET23b (+) (Novagen, Madison, WI, United States) in-frame fused with a C-terminal His 6 -tag sequence to construct the plasmids pET23b-fur and pET23b-mfur. The final constructs were transformed into BL21 (DE3) for expression, and recombinant Fur/mFur proteins were purified using Ni-NTA agarose (Qiagen, California, United States). The proteins were induced by the addition of 1 mM IPTG at 16°C for 12 h. Protein purification was performed as described previously (Li et al., 2014c). The protocols of the recombinant protein purification are available on request. The recombinant protein MSMEG_2415 was purified as described previously (Li et al., 2014c) and used as a negative control for EMSA, while MSMEG_2415 is irrelated to Fur. The DNA fragment containing the promoter region of fur for gel shift experiments was amplified by PCR with specific primers (forward: 5′-CGTTGGAAAACAACCATTGCAAG-3′, reverse: 5′-CATCCGCAGTTGGGCTTCGAAC-3′). Binding reaction mixtures in 20 μl of binding buffer (20 mM Tris HCl pH 8.0, 1 mM dithiothreitol (DTT), 50 mM KCl, and 5 mM MgCl 2 ) containing 0.15 pmol of the DNA fragment were incubated with purified Fur/mFur protein (0.5 nmol) for 30 min at 30°C. Reaction mixtures were loaded on a 4-20% polyacrylamide gel containing 0.5 × TBE. Gels were run at 70 V at 4°C for 3 h. The gel was stained with Good-view and photographed for the image.

Generation of the katG Knockout and KatG Overexpression Strains
The knockout katG strain was constructed using mycobacteriophage-based specialized transduction (Bardarov et al., 2002;Li et al., 2014c). The upstream and downstream sequences of katG were amplified from M. smegmatis genome DNA. The knockout vector was constructed using phAE159 (Hsu and Jacobs, unpublished data). The mycobacteriophage used for knockout was obtained using MaxPlax packaging extract (Epicentre Biotechnologies, Madison, WI, United States), and a katG knockout strain was obtained by phage transduction, named ΔkatG. The KatG overexpression strain was constructed using pMV261 to yield pMV261-katG, and the constructed plasmid was electroporated into mc 2 155, yielding pMV261-katG/mc 2 155. The detailed information for construction of all the mycobacterial strains and primers for plasmid construction is available on request.

Generation of Fur Point Mutation on the Chromosome in M. smegmatis
The single-strand (ss) DNA oligonucleotides used for recombineering were ordered from Genewiz (Suzhou, China). The site-directed mutagenesis of fur was obtained using Phage Che9c gp61-mediated recombination (van Kessel et al., 2008). The detailed information on primers for construction of the fur mycobacterial strain (named mc 2 155-mfur) is available on request. The coding region containing fur point mutation in the genome (encoding mFur) was amplified and sequenced by Genewiz (Suzhou, China).

Statistical Analysis
Each experiment was carried out at least twice with three-nine mice or samples per group. The CFUs and OD 600 were analyzed using an unpaired t-test (Version 8.0 for Windows GraphPad Software). The ATP content was analyzed using ANOVA tests (Version 8.0 for Windows GraphPad Software). ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Animal Ethics
This study was performed in strict accordance with the recommendations of the Ethics Committee established in the Guide for the Care and Use of Laboratory Animals of the Institute of Microbiology, Chinese Academy of Sciences (IMCAS). The protocol was approved by the Committee on the Ethics of Animal Experiments of the IMCAS. The mice were bred under specific pathogen-free conditions at the IMCAS laboratory animal facility. All animal experiments were conducted under isoflurane anesthesia, and all efforts were made to minimize suffering.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the following: BioProject: PRJNA233977; BioSample: SAMN02951866, https://www.ncbi. nlm.nih.gov/nuccore/JAJD00000000.