Tubercle bacilli depend on the uptake, not synthesis, of vitamin B12
In spite of being considered highly clonal, the MTBC contains genetic polymorphisms which hypothetically have contributed to shape its evolution and host preference. Specifically, of the 16 cob genes putatively involved in Cbl synthesis in Mycobacterium12, we found non-synonymous mutations, insertions and deletions in 10 cob genes, which affect different branches of the MTBC (Fig. 1a). We confirmed genomic data by Sanger sequencing of specific clinical isolates belonging to the different MTBC lineages affected by cob mutations (Fig. 1a and S1). Of these polymorphisms, it is remarkable the presence of cobF and RD9 genomic deletions since they are large polymorphisms affecting several arms of the MTBC. The cobF gene is absent in all MTBC lineages, except lineage 813; and consequently, the most widespread lineages of M. tuberculosis, M. africanum lineages 5 and 6, and animal-adapted lineages, carry this deletion. Similarly, RD9, which eliminates the 5’-terminus of the cobL gene, is absent in M. africanum and animal-adapted mycobacteria (Fig. 1a).
a Identification of mutations (red dots) and genomic deletions (red boxes) affecting the de novo B12 biosynthesis in MTBC strains and in its last known common ancestor (M. canettii). The bottom left box shows the validation by Sanger sequencing of one of these mutations. Additional Sanger sequencing confirmations are provided in Fig. S1. b B12-levels synthesized by MTBC species (color codes matches those in panel A), in M. canettii (dark gray) and in environmental and opportunistic mycobacteria (yellow). “n.d.” denotes non detected. Bars and error bars are the average and SD from three biological replicates. c Cyanocobalamin (CNB12) and adenosylcobalamin (AdoB12) scavenged by MTBC members in cultures containing B12 grown until exponential growth-phase. Data are the mean and standard deviation from three biological replicates. d Development of mouse B12 anemic models and confirmation of decreased serum B12-levels in the treated groups. Graph data are mean ± SD of n = 6 and n ≥ 3 biological replicates of C57BL/6 and SCID mice, respectively. Statistical analysis was performed using unpaired t-test. e Experimental set-up of the survival and virulence experiments in SCID and C57BL/6 anemic mice infected with the wild type strains of M. tuberculosis H37Rv and M. canettii C59. f Survival rates from SCID mice fed with each diet and inoculated by the intranasal route with M. tuberculosis H37Rv or M. canettii C59. Each curve represents the pool from 2 independent experiments (n = 12). Statistical analysis was performed using Log-rank (Mantel-Cox) test. g Bacterial loads in the lungs of control and anemic C57BL/6 mice after 24 h, 4 weeks, or 8 weeks infected with M. tuberculosis H37Rv or M. canettii C59. Data represent mean ± SD of n ≥ 6 biological replicates per mice group. Statistical analysis was performed using unpaired t-test. p-values are as follows: ****0.0001 > p; ***0.001 > p > 0.0001; **0.01 > p > 0.001; ns not significant, p ≥ 0.05.
Overall, these data indicate that the MTBC have evolved a genomic decadence in B12 synthesis genes. However, being aware that non-synonymous mutations do not necessarily abrogate enzyme activity, and that gene deletions could be compensated by accessory genes, we assayed B12 production in representative Mycobacterium strains. None of the MTBC strains tested, (M. tuberculosis strains from lineages 2 and 4; M. africanum from lineages 5 and 6; and the animal-adapted M. bovis), produced detectable levels of B12 (Fig. 1b). In contrast, the MTBC progenitor, M. canettii showed an optimal B12 endogenous synthesis (Fig. 1b). Further, we also demonstrate that environmental (M. smegmatis), and opportunistic (M. avium, M. abscessus, M. fortuitum, M. gordonae, M. mucogenicum, M. xenopii) mycobacteria produce variable B12 levels (Fig. 1b). This latter result is in agreement with a recent study demonstrating B12 production in various non-tuberculous mycobacteria, but not in M. tuberculosis14.
Once demonstrated that B12 synthesis is abrogated in the MTBC, we reasoned that the presence of B12-dependent enzymes in these pathogens12 could imply the existence of a B12 uptake mechanism. Therefore, upon incubation of M. tuberculosis, M. africanum and M. bovis with exogenous B12, we confirmed the capacity of these bacteria to internalize the molecule (Fig. 1c). All MTBC species tested were able to uptake two B12 isoforms, namely cyano-Cbl and adenosyl-Cbl, in either exponential (Fig. 1c), and stationary growth phases (Fig. S2). The evolutionary preservation of a mechanism for B12 transport, irrespective of a decayed B12 biosynthetic pathway, emphasizes the relevance to scavenge this molecule for the physiology of the MTBC.
M. tuberculosis exhibits reduced virulence in B12 anemic mouse models
Our observation that MTBC members have retained the ability to scavenge exogenous B12, despite lacking endogenous synthesis, led us to investigate whether B12 could represent a host factor which modulate M. tuberculosis virulence. To prove this hypothesis, we first established B12 anemic mouse models based on feeding rodents during 8 weeks with a B12-deficient diet15. We confirmed that immunocompetent C57BL/6, and immunocompromised SCID mice fed without B12 showed 6.5- and 3.4-fold less B12 serum levels, respectively, than their counterparts fed with a normal diet (Fig. 1d). Rodents fed with both diets showed signs of animal welfare, which is in agreement with previous findings that a moderate depletion in B12 serum levels does not impact on the physiological status of mice16. Then, we infected B12-defective, and control mice, by the intranasal route with M. tuberculosis H37Rv and we evaluated either SCID mice survival times, or bacterial loads in C57BL/6 organs (Fig. 1e). We observed that anemic SCID mice survived significantly better after the M. tuberculosis infection than the control group (Fig. 1f and S6). This denotes that, in the absence of adaptive immunity, the virulence of M. tuberculosis is reduced under suboptimal B12 serum levels, and viceversa.
This observation was confirmed after examination of bacterial loads in lungs and spleen of C57BL/6 immunocompetent animals. To rule out that differences in bacterial loads were due to an in vitro B12-dependent fitness, we first confirmed equivalent M. tuberculosis growth rates in laboratory media supplemented with or without B12 (Fig. S3). Depleted B12 serum levels in C57BL/6 mice resulted in significantly lower bacterial replication in the lungs, and lower dissemination to the spleen, relative to the control group (Fig. 1g and S7); reinforcing the hypothesis that M. tuberculosis needs optimal host B12 serum levels to develop a complete virulence. Then, we tested a M. canettii infection in our mouse anemic models, because unlike M. tuberculosis, this ancestor mycobacterium is able to endogenously produce its own B12 (Fig. 1b). First, we optimized the inoculum of M. canettii since this bacterium is known to be less persistent and less virulent than M. tuberculosis9. In order to obtain equivalent survival in SCID mice, it was required from 100–1000-fold higher M. canettii inoculum relative to M. tuberculosis (Fig. S4). Once established the infection conditions, we found equivalent survival times in SCID mice infected with M. canettii, independently of the B12 serum levels of mice (Fig. 1f and S6). Further supporting the independence of M. canettii virulence and host B12 status, non-significant differences in lungs and spleen bacterial loads were observed in C57BL/6 mice fed with normal or B12-depleted diets upon infection with the ancestor M. canettii (Fig. 1g and S7). Post-mortem analysis of M. tuberculosis– and M. canettii-infected mice confirmed that B12 serum levels remained low in anemic animals relative to control groups throughout the curse of the experiments (Fig. S5). To confirm the link between M. canetti virulence and its intrinsic ability to produce B12, we constructed a M. canettii ΔcobMK mutant unable to synthesize this molecule (Figs. S8 and S15). This M. canettii B12 mutant was used to infect B12-deficient mice and we found that, unlike the wild type, the mutant resulted strongly attenuated as measured by longer SCID survival, and significantly lower bacteria in C57BL/6 organs (Fig. S8). Together, results indicate that host B12 levels directly correlate with M. tuberculosis virulence, but this association does not apply to the ancestor of the MTBC due to its specific ability to synthesize B12.
B12 supplementation of anemic mice recovers M. tuberculosis virulence
Results described above led us to speculate that restoring healthy B12 serum levels in anemic mice might impact on the virulence of M. tuberculosis in infected animals. Accordingly, 4 groups of SCID mice with different B12 treatments were established: a standard diet control, 2 groups fed with independent B12-deficient diets, and another group receiving weekly subcutaneous B12 supplementation after being fed with a B12-deficient diet (Fig. 2a). Following each B12 treatment, all animal groups were intranasally infected with M. tuberculosis and we interrogated survival times and B12 serum levels at sacrifice. We found that both B12-defective diets resulted in significantly reduced serum B12 levels relative to the control diet. Further, supplementation with B12 resulted in efficient restoration of serum levels of this vitamin, comparable to those of the control mice (Fig. 2b). Survival times of each mice group remarkably correlated to their respective B12 serum levels. Interestingly, the gradual decrease in B12 serum levels observed after treatment with the different diets translated into longer survival times and consequently into reduced M. tuberculosis virulence (Fig. 2b, c). Confirming this observation, restoration of healthy B12 levels in anemic mice by subcutaneous supplementation resulted in a M. tuberculosis virulence indistinguishable from the mice group fed with a standard B12-containing diet (Fig. 2c). Together, this confirms that sub-physiological levels of B12 in mice causes M. tuberculosis attenuation, and highlights the importance to assimilate B12 from the host for this pathogen.
a Experimental design and timelines of the different B12 treatments, B12 subcutaneous supplementation, and M. tuberculosis infection, in each SCID mice group. b B12 serum levels in animal groups described in panel (a) after sacrifice at the humane endpoint. B12 levels remained significantly decreased in animals treated with B12 restricted diets relative to the control group throughout the curse of the experiment. Note that weekly B12 supplementation restored standard B12 serum levels in mice. Graph data are mean ± SD of n = 6 biological replicates. Statistical analysis was performed using One-Way ANOVA and Tukey post-test. c Survival rates from groups of 6 SCID mice for each type of treatment after infection by the intranasal route with M. tuberculosis. Statistical analysis was performed using Log-rank (Mantel-Cox) test. p-values are as follows: ****0.0001 > p; **0.01 > p > 0.001; *0.05 > p > 0.01; ns not significant, p ≥ 0.05.
The B12-dependent transcriptome in Mycobacterium establishes a link with methionine metabolism
Having demonstrated that MTBC bacteria scavenge B12 from the host to modulate virulence, we sought to identify the molecular mechanism(s) responsible for this phenotype. We studied bacteria from the most representative lineages causing TB in humans and animals. Specifically, M. tuberculosis GC1237 and H37Rv were selected as representative of human-adapted strains from lineages 2 (sublineage Beijing) and 4, respectively; and M. bovis (strain AF2122) was selected as a representative animal-adapted strain. Replicates from each strain were grown in the presence, or absence, of exogenous B12, bacterial RNA was sequenced by RNA-seq, and differential gene expression between both conditions was interrogated (Supplementary Data 1–3). This analysis produced three differential expression sets, one per each MTBC strain analyzed, which were merged to gain insight into the B12-dependent transcriptome in the MTBC (Fig. 3a). We identified 7 genes organized into 2 genome clusters, which showed a robust downregulation in the presence of B12 (Fig. 3b and S9). All these genes exhibited B12-dependent repression across replicates in all Mycobacterium strains tested, which probes that the identified B12-dependent transcriptome is reproducible and extensive to the MTBC (Fig. 3a, b, S9 and Supplementary Data 1–3). To additionally confirm the B12 regulation of the identified genes, we corroborated downregulated expression of Rv1129c, prpD, metE, PPE2, and cobQ1 measured by quantitative-PCR in M. tuberculosis H37Rv cultures incubated with B12 relative to controls (Fig. 3c and S9). Further, we conducted a Multiple Reaction Monitoring (MRM-MS) proteomic approach to measure PrpD, PrpC and MetE protein levels in M. tuberculosis GC1237 and H37Rv cultures treated with or without B12. Results demonstrated a complete absence of PrpD and PrpC, and an average 9.8-fold reduction of MetE protein, when B12 is added to the cultures (Fig. 3d and S10). In a previous independent study with the H37Rv strain, the authors also identified PrpR, PrpDC, Rv1132 and MetE as B12-regulated genes, providing robustness and cumulative knowledge about the B12 regulatory network in M. tuberculosis17
a Experimental procedure for RNA-seq analysis of differentially expressed (DE) genes in response to B12 supplementation of MTBC cultures. Venn diagrams show the resulting DE genes from two independent replicates of M. tuberculosis H37Rv (L4), M. tuberculosis GC1237 (L2), and the animal-adapted M. bovis AF2122. Those DE genes common to the three strains are indicated as the “core B12-regulon”, and consist on 7 downregulated genes in the presence of B12. b RNA-seq profiles showing downregulated gene expression of the gene cluster Rv1129c (prpR)-prpD–prpC-Rv1132-metE in M. tuberculosis H37Rv in response to exogenous B12 supplementation (light blue) relative to the experimental control without B12 (dark blue). Note that in order to visualize expression differences, each genetic section has been represented with its corresponding scale. Expression profiles of the remaining B12-dependent genes are indicated in Fig. S9. c qRT-PCR measures of Rv1129c (prpR), prpD and metE in M. tuberculosis H37Rv cultures grown with or without B12. Relative quantity refers to the differential expression of the selected genes in the presence of B12 in comparison with its expression in the absence of B12. Each gene was normalized against sigA expression in each sample. Graphs represents mean ± SD from three biological replicates. Expression of additional B12-dependent genes is shown in Fig S9. d Quantification of PrpD, PrpC and MetE protein levels of M. tuberculosis H37Rv both in presence and absence of B12 by MRM-MS. Bars represent the area under the curve for every transition from a specific peptide of each protein in both experimental conditions. Equivalent results were obtained for the rest of the analyzed peptides (Fig. S10) in two biological replicates of H37Rv and GC1237 strains of M. tuberculosis.
We then focused on the genome cluster containing 5 out of 7 genes of the B12-dependent transcriptome Rv1129c (PrpR), the prpC–prpD–Rv1132 operon, and the metE gene (Fig. 3B). Provided that the role of PrpR, and PrpDC, has been studied in detail18,19,20,21,22 as will be discussed below, we decided to focus our study in the B12-regulated metE gene, whose implications in the virulence of M. tuberculosis are less understood.
A M. tuberculosis metE mutant is predominantly attenuated in anemic mice
The final step of methionine synthesis in M. tuberculosis is catalyzed by two complementary methionine synthases: MetE or MetH. The activity of both proteins is regulated by B12, albeit by different mechanisms. Expression of metE is regulated by a B12 riboswitch located upstream the metE coding sequence, while MetH requires B12 as cofactor23. Our transcriptomic and proteomic results demonstrate decreased metE mRNA and protein expression when B12 is present (Fig. 3b–d), which is in agreement with the regulatory mechanism exerted by the riboswitch, and with previous observations23. We constructed a ΔmetE mutant in M. tuberculosis H37Rv (Fig. S15) to understand the role of this gene in virulence modulation by B12. This ΔmetE mutant is still able to synthesize methionine by the B12-dependent MetH, and consequently the ΔmetE mutant is predicted to strongly depend on B12 supplementation (Fig. 4a). We confirmed a total absence of in vitro growth, in liquid and solid media, of the ΔmetE mutant relative to M. tuberculosis H37Rv wild type in the absence of B12 (Fig. 4b, c). Raising B12 concentrations to 0.01 μg/mL partially recoved growth of the ΔmetE mutant, and 0.1 μg/mL resulted in a complete growth rescue on agar plates (Fig. S11). In addition, we wondered whether M. tuberculosis might scavenge L-methionine from the environment. Cultivation of the M. tuberculosis ΔmetE mutant in L-methionine-containing plates without B12 neither resulted in appreciable growth (Fig. 4c), indicative of lack of exogenous L-methionine foraging. To further confirm these phenotypes, we also generated ΔmetE mutants in wild type M. canettii and in a M. smegmatis strain defective in endogenous B12 synthesis by deletion of the cobLMK operon (Fig. S15). Both mutants exhibited the strong dependency on exogenous B12 to restore in vitro growth to wild type levels previously observed in M. tuberculosis (Fig. S12), indicative that methionine synthesis by MetH is evolutionary conserved in Mycobacterium. Surprisingly, the growth defect in the absence of B12 showed by ΔmetE mutants of M. canettii and B12-deficient M. smegmatis, was successfully rescued in the presence of exogenous L-methionine on agar plates (Fig. S12). This indicates that ancestor and environmental mycobacteria have retained a mechanism for L-methionine transport when growing on solid media, which is lost in M. tuberculosis. However, a previous study demonstrated the ability of a M. tuberculosis ΔmetA auxotroph to recover growth in liquid media after supplementation with L-methionine24. Based on this observation, we also confirmed the ability of the ΔmetE mutant to recover planktonic growth upon supplementation with L-methionine (Fig. S13). Overall, this dependency on exogenous L-methionine of the ΔmetE mutant when growing on solid, but not in liquid media, is intriguing. We can hypothesize that either L-methionine bioavailability is higher in liquid media, facilitating its assimilation by M. tuberculosis; or that M. tuberculosis expresses L-methionine transport mechanisms exclusively under planktonic growth. Thus, to gain insight into the virulence implications of metE inactivation, and to evaluate whether M. tuberculosis can directly access host B12, we evaluated the in vivo phenotype of this mutant under a physiological scenario.
a Expected phenotypes of the M. tuberculosisH37Rv ΔmetE knockout compared to its wild type strain in presence and absence of B12. b and c in vitro growth in liquid (boxed images) and in solid media of the M. tuberculosis H37Rv wild type strain (b) and its ΔmetE mutant (c) in the absence or presence of exogenous B12 and/or L-methionine. d and f Survival rates from SCID mice inoculated by the intranasal route with M. tuberculosis H37Rv and H37Rv ΔmetE fed with normal diet (d) and a B12-deficient diet (f). Each curve represents the pool from 2 independent experiments (n = 12). Statistical analysis was performed using Log-rank (Mantel-Cox) test. e and g Bacterial loads in the lungs of normal (e) and anemic (g) C57BL/6 mice after 24 h, or 4 weeks infected with M. tuberculosis H37Rv and its ΔmetE mutant. Data are mean ± SD of n ≥ 6 biological replicates per mice group. Statistical analysis was performed using unpaired t-test. p values are as follows: ****0.0001 > p; ***0.001 > p > 0.0001; p ≥ 0.05; ns: not significant. Results show that inactivation of the methionine synthase MetE attenuates the M. tuberculosis virulence more markedly in B12 anemic mice relative to controls.
We found a significant attenuation of the mutant measured by improved survival times (Fig. 4d), and lower bacterial loads (Fig. 4e and S7), in SCID and C57BL/6 mice fed with a conventional diet, respectively. This attenuation was much more noticeable upon infection of anemic mice, according to the B12 requirements of the M. tuberculosis ΔmetE strain (Fig. 4f). Comparatively, median survival times of the mutant were 30 days longer than the wild type in SCID anemic mice, compared to 8 days in non-anemic mice fed with routine diet. With regards to lung bacterial loads in C57BL/6 mice, the mutant showed lower bacterial burden when infecting B12-deficient animals (Fig. 4g and S7). These results support the M. tuberculosis dependency on host B12 for L-methionine synthesis mediated by MetH. In addition, the remnant attenuation of the M. tuberculosis ΔmetE mutant in mice fed with a conventional B12 diet (Fig. 4d, e), might indicate that in vivo L-methionine synthesis by MetH is far from optimal, due to either insufficient B12 scavenging from the host, or to inadequate enzymatic activity of MetH. Additionally, the overt attenuation of the M. tuberculosis ΔmetE strain in vivo resembles its growth defects on solid media. Having demonstrated the lack of L-methionine transport on agar plates (Fig. 4c), it is tempting to speculate that either M. tuberculosis exhibits suboptimal L-methionine uptake from the host, or that L-methionine intracellular concentration is too low to support optimal growth of M. tuberculosis.
Suppressive mutations in the B12-dependent metE riboswitch relieve the attenuation of a M. tuberculosis metH mutant in non-anemic mice
For a complete understanding of the implications of the B12-dependent methionine metabolism in M. tuberculosis, we also studied the phenotype of a M. tuberculosis ΔmetH mutant (Fig. S15). This mutant still maintains L-methionine production by MetE, and accordingly, it is expected to have growth defects in the presence of B12 due to the inhibition of the metE riboswitch (Fig. 5a)23. Once constructed, the M. tuberculosis ΔmetH mutant was inoculated in laboratory media with or without B12 confirming absent, or reduced growth, in liquid and solid media, respectively, when B12 is present (Fig. 5b, c). Next, we confirmed that concentrations of 0.01 μg/mL B12 were sufficient to partially inhibit the growth of M. tuberculosis ΔmetH on solid plates, otherwise indicative of inhibition of the metE riboswitch (Fig. S11). We also used this mutant to confirm the absence of exogenous L-methionine uptake in M. tuberculosis on agar plates, since supplementation of B12 plates with L-methionine did not restore bacterial growth to wild type levels (Fig. 5b, c). In contrast, a ΔmetH mutant constructed in M. canettii (Fig. S15), grew equivalently to its parent strain in solid media supplemented with B12 and L-methionine (Fig. S12), confirming that L-methionine uptake from solid media is evolutionarily maintained in ancestor mycobacteria, but not in M. tuberculosis. As previously demonstrated with the ΔmetE mutant, we confirmed that assimilation of exogenous L-methionine in the ΔmetH mutant occurs under planktonic growth, but not during growth on agar plates (Fig. S13). In vivo evaluation of the M. tuberculosis ΔmetH mutant showed reduced virulence in SCID mice (Fig. 5d and S7), and reduced lung bacterial loads in C57BL/6 mice (Fig. 5e) under a conventional diet. In contrast, no differences in bacterial virulence were observed when animals were foraged with a B12 deficient diet (Figs. 5f, g and S7), indicating that suboptimal B12 serum levels are insufficient to repress the metE riboswitch in vivo. We also sequenced the metE riboswitch region from 22 M. tuberculosis ΔmetH colonies grown on solid media supplemented with B12, and 30 colonies from lungs of mice infected with this strain and fed with normal diet. As expected, we found that 17/22 of the colonies grown on solid media contained mutations in the riboswitch (Fig. 5h and S14). Surprisingly, only a single colony from the mouse lungs contained mutations in this region, which might indicate that physiological levels of B12 do not impose a selective pressure as high as that observed in vitro. Mapping of the mutations to the predicted structure of the riboswitch demonstrated that these polymorphisms were regularly distributed, and different polymorphisms affecting invariant residues of B12 riboswitches25 arose independently in independent colonies (Fig. 5i). This result suggests that suppressor mutations in the B12 riboswitch could alleviate the B12 repression of the metE gene, and favor the appearance of M. tuberculosis ΔmetH escape mutants when B12 is present, preferentially during growth in vitro.
a Expected phenotypes of the M. tuberculosis H37Rv ΔmetH knockout compared to the wild type strain in presence and absence of B12. b and c in vitro growth in liquid and in solid media of the M. tuberculosis H37Rv wild type strain (b) and its ΔmetH mutant (c) in absence or presence of exogenous B12 and/or L-methionine. d and f Survival rates from groups of SCID mice fed with normal diet (d) and B12-deficient diet (f), and inoculated by the intranasal route with M. tuberculosis H37Rv and the ΔmetH mutant. Each curve represents the pool from 2 independent experiments (n = 12). Statistical analysis was performed using Log-rank (Mantel-Cox) test. e and g Bacterial loads in the lungs of control (e) and B12 anemic (g) C57BL/6 mice after 24 h, or 4 weeks infected with M. tuberculosis H37Rv and its ΔmetH mutant. Data are mean ± SD of n ≥ 6 biological replicates per mice group. Statistical analysis was performed using unpaired t-test. p-values are as follows: ***0.001 > p > 0.0001; ns not significant, p ≥ 0.05. Results show that inactivation of the MetH isoform attenuates the mutant exclusively in animals with normal serum B12-levels (h) 5’ UTR sequences of the metE B12-Riboswitch. Negative numbers indicate nucleotides immediately upstream of the metE initiation codon. Bases showing mutations in M. tuberculosis H37Rv ΔmetH colonies grown with B12 are highlighted in red in the Sanger chromatograms. Aditional mutations are shown in Fig. S14. i Secondary structure prediction of the metE B12-riboswitch. Location of the identified mutations in the B12-resistant M. tuberculosis H37Rv ΔmetH colonies are indicated by red arrows. Invariant residues across ~200 B12-riboswitches are indicated in capital letters. Lowercase letters indicate M. tuberculosis specific residues. The conserved B12-box is highlighted in yellow. The metE 5’UTR region is shown in blue letters with the possible RBS underlined. The start of the metE CDS is shown with a purple arrow and the initiation codon is highlighted in red.
This finding led us to hypothesize that introduction of a B12-independent metE gene into the M. tuberculosis ΔmetH mutant should rescue the B12 growth defects of this strain without the need to evolve suppressor mutations in the metE riboswitch (Fig. 6a). We placed the Ag85A promoter (Pr-Ag85A) immediately upstream of the metE coding region, and this construction was introduced in the chromosome of the M. tuberculosis ΔmetH (Fig. 6a and S15). Improved expression of the Pr-Ag85A-controlled copy of metE was confirmed with respect to M. tuberculosis wild type and its metH mutant (Fig. 6b). Then, we confirmed that the Pr-Ag85A-metE copy enabled the M. tuberculosis ΔmetH to grow in liquid and solid media containing B12, in contrast to the parent M. tuberculosis ΔmetH strain (Fig. 6c). The Pr-Ag85A-metE complemented strain was tested in vivo in mice fed with a B12 containing diet, to confirm that survival in SCID mice and bacterial loads in C57BL/6 lungs were equivalent to those of the wild type strain (Figs. 6d, e and S7). This later result was also reproduced in animals fed with a B12-deficient diet (Fig. 6f, g and S7). Together, our results confirm the role of B12 suppressor mutations in the metE riboswitch to relieve the pressure imposed by B12 over the M. tuberculosis ΔmetH strain in vivo.
a Expected phenotypes of the M. tuberculosis H37Rv ΔmetH PrAg85ametE::Kan complemented strain compared to the parental ΔmetH strain in presence and absence of B12. b Validation by qRT-PCR of the metE complementation and its stable expression in the complemented mutant M. tuberculosis H37Rv ΔmetH PrAg85ametE::Kan compared to the ΔmetH mutant and the wild type strains. Relative quantity refers to the differential metE gene expression in each strain in comparison with its expression in the wild type H37Rv. Each gene was normalized against sigA expression in each sample. Graphs represents mean ± SD from one experiment with three replicates. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons post-test. p-values are as follows: ****0.0001 > p; ***0.001 > p > 0.0001; *0.05 > p > 0.01. c Experimental growth in liquid (boxed images) and in solid media of the M. tuberculosis H37Rv ΔmetH and the ΔmetH PrAg85ametE::Kan strains in presence and absence of B12. d and f Survival rates from groups of SCID mice fed with normal diet (d) or B12-deficient diet (f), and inoculated by intranasal route with M. tuberculosis H37Rv, its ΔmetH mutant or the ΔmetH PrAg85ametE::Kan complemented strain. Each curve represents the pool from 2 independent experiments (n = 12). Statistical analysis was performed using Log-rank (Mantel-Cox) test. e and g Bacterial loads in the lungs of normal (e) and B12 anemic (g) C57BL/6 mice after 24 h, or 4 weeks infected with M. tuberculosis H37Rv, the ΔmetH knockout, or the ΔmetH PrAg85ametE::Kan Data are mean ± SD of n ≥ 6 biological replicates per mice group. Statistical analysis was performed using unpaired t-test. p-values are as follows: ***0.001 > p > 0.0001; *0.05 > p > 0.01; ns: not significant, p ≥ 0.05. Results show that complementation with a B12-independent metE restores virulence in the M. tuberculosis ΔmetH mutant independently of the B12 status of the host.
