Aideen C Allen

Wladimir Malaga

Cyril Gaudin

Arnaud Volle

Flavie Moreau

Ali Hassan

Catherine Astarie-Dequeker

Antonio Peixoto

Rudy Antoine

Alexandre Pawlik

Wafa Frigui

Céline Berrone

Roland Brosch

Philip Supply

Christophe Guilhot
email: christophe.guilhot@ipbs.fr

Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The success of Mycobacterium tuberculosis (MTB), the primary aetiological agent of human tuberculosis (TB), is largely attributed to its ability to evade host defences and thus persist for months within the lungs. Indeed, it is estimated that one quarter of the world's population has been infected with this pathogen and a substantial fraction still carries viable bacilli [START_REF] Behr | Revisiting the timetable of tuberculosis[END_REF][START_REF]Global tuberculosis report[END_REF] . The adaptations that allowed the ancestor of MTB to colonise humans and evolve into the persistent pathogen that now causes an estimated 1.5 million deaths each year remain mostly uncharacterised [START_REF]Global tuberculosis report[END_REF][START_REF] Chiner-Oms | Genomic determinants of speciation and spread of the Mycobacterium tuberculosis complex[END_REF] .

In contrast to MTB, Mycobacterium canettii are TB bacilli of only marginal epidemiological importance.

These rare human isolates can cause disease clinically indistinguishable from classical TB, however epidemiological data indicate that they are not fully adapted for colonisation of the human host [START_REF] Blouin | Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti[END_REF][START_REF] Koeck | Clinical characteristics of the smooth tubercle bacilli 'Mycobacterium canettii' infection suggest the existence of an environmental reservoir[END_REF][START_REF] Fabre | Molecular characteristics of "Mycobacterium canettii" the smooth Mycobacterium tuberculosis bacilli[END_REF] .

Consistently, M. canettii strains are less able to persist and cause disease in animal models than MTB strains [START_REF] Lopez | A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes[END_REF][START_REF] Dormans | Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in BALB/c mouse model[END_REF][START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF] . During evolution, these strains diverged early from MTB and they are now believed to have an environmental reservoir [START_REF] Koeck | Clinical characteristics of the smooth tubercle bacilli 'Mycobacterium canettii' infection suggest the existence of an environmental reservoir[END_REF][START_REF] Boritsch | A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent[END_REF] .

The current model is that MTB evolved by clonal expansion from an M. canettii-like progenitor through adaptations that rendered them persistent in mammalian hosts [START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF][START_REF] Fabre | High genetic diversity revealed by variable-number tandem repeat genotyping and analysis of hsp65 gene polymorphism in a large collection of "Mycobacterium canettii" strains indicates that the M. tuberculosis complex is a recently emerged clone of "M. canettii[END_REF][START_REF] Gutierrez | Ancient orgin and gene mosaicism of the progenitor of Mycobacterium tuberculosis[END_REF] . However, virtually all identified MTB factors important for establishing chronic infection are also present in M. canettii [START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF] , and few genes and loci are specific to MTB. It is therefore likely that the strict pathogenic phenotype differentiating MTB from facultative human pathogens like M. canettii and other environmental mycobacteria is due to modification of shared pathways. The M. canettii strains are therefore an outstanding resource for investigating the molecular mechanisms driving enhanced persistence in tuberculosis bacilli, and for gaining new insights into the evolutionary events involved in MTB emergence. Following aerosol or intravenous (i.v.) infection of mice, M. canettii and MTB strains multiply in the lungs similarly during the acute phase of infection (first 4 weeks). In the chronic phase of infection, MTB persists and the bacterial load is comparable to that achieved during the acute phase. In sharp contrast, the lung bacterial load for M. canettii strains declines substantially during the chronic infection phase, and for STB-K, the most attenuated M. canettii isolate in this model, becomes undetectable after 30 weeks of infection. This lower persistence is associated with less pathology [START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF] , possibly explaining why there is no direct transmission in humans reported for M. canettii [START_REF] Blouin | Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti[END_REF][START_REF] Koeck | Clinical characteristics of the smooth tubercle bacilli 'Mycobacterium canettii' infection suggest the existence of an environmental reservoir[END_REF][START_REF] Fabre | Molecular characteristics of "Mycobacterium canettii" the smooth Mycobacterium tuberculosis bacilli[END_REF] .

Our objective for this study was to select M. canettii mutants with enhanced persistence in vivo in order to identify what adaptations are associated with this phenotypic gain. We first implemented a positive selection strategy based on unbiased experimental evolution of 8 M. canettii populations in a mouse model. Then we used a combination of genome sequencing and phenotypic characterisation of multiple evolved clones to identify and describe the genomic and functional adaptations associated with enhanced persistence of M. canettii. Here we show that increased resistance to host-derived stresses, and especially nitric oxide (NO), is a key adaptation required by M. canettii to survive for several months in vivo.

Results

Unbiased in vivo experimental evolution of M. canettii selects mutants with enhanced persistence

It has previously been established that M. canettii are less persistent than MTB in different mouse models [START_REF] Dormans | Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in BALB/c mouse model[END_REF][START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF] . We therefore set up an experimental evolution procedure in mice to select for M. canettii mutants with an improved ability to persist, thus resembling MTB strains. These mutants should outcompete parental M. canettii strains in competitive experiments in mice.

Our experimental evolution strategy consisted of serial passages in BALB/c mice (Fig. 1a) starting from two distinct M. canettii isolates; the STB-K and STB-D strains which are phylogenetically farthest from and closest to MTB respectively 9 (Supplementary Fig. 1). For both STB-K and STB-D, we generated four evolution lineages (A, B, C and D) that evolved independently and were never mixed. We performed 15 cycles for STB-K and 6 cycles for STB-D. At each cycle, we evaluated the lung and spleen bacterial loads at days 1, 28 and 56 post-infection, and we calculated a persistence index corresponding to the log ratio of lung bacterial burden at day 56 vs day 28 (Fig. 1b). For both STB-K and STB-D, we found that the persistence index increased throughout the cycles, suggesting that mutants with enhanced abilities to persist emerged. To confirm this, we randomly collected clones from agar plates used to evaluate the bacterial burden in the lungs during the experimental evolution cycles, and intranasally (i.n.) infected BALB/c mice with 24 individual clones recovered after cycles 6, 9, 12 and 15 for strains derived from STB-K, or cycle 6 for STB-D. We found that all the tested clones caused an increased bacterial burden in the lungs and spleen relative to their parental strains (Fig. 1c,d and Supplementary Fig. 2). This was most pronounced at later time points, indicating that mutants with enhanced persistence were selected during experimental evolution of both M. canettii STB-K and STB-D.

Small numbers of mutations were fixed in the populations during the experimental evolution

To identify the mutations that arose in STB-K and STB-D during experimental evolution in mice, we sequenced the whole genomes of 100 individual clones derived from STB-K and 40 clones derived from STB-D, and searched for differences relative to their parental strains.

In the STB-K series, we found that mutations in two loci were fixed independently in the four evolution lineages. An identical nucleotide substitution C>T at nucleotide position 1582889, corresponding to the gene BN42_21248, was found in all clones recovered after cycle 6 (Supplementary Table 1). This mutation changed a single amino acid P96L in the protein it encodes, which is the orthologue of Rv1339, a MTB H37Rv protein, which was recently shown to encode a phosphodiesterase degrading cyclic-AMP (cAMP) [START_REF] Thomson | Modulation of the cAMP levels with a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria[END_REF] (Fig. 2a). In the second locus, three mutations became additionally fixed independently in evolution lineages A, B and C: two single nucleotide insertion/deletions (indels) and a 3073 bp deletion. The two small indels at positions 2112495 and 2113560 induce frameshifts in two adjacent genes, pe18 and ppe26 respectively. The large deletion resulted in removal of the genes pe18 and ppe26, and fusion of the 5'-terminal part of ppe25 with the 3'-terminal part of ppe27, probably due to homologous recombination (Fig. 2b).

In the STB-D series, the analyses revealed a more complex situation. Indeed, we identified single nucleotide polymorphisms (SNPs) present at relatively high frequency in the inoculum used for the first cycle, suggesting three genetic profiles (Supplementary Table 2). Other SNPs were fixed independently in these three different genetic backgrounds in orthologues of the genes rv0200, rv0246 and rv2990c in evolution lineages A, B and C respectively (Supplementary Table 2). No mutation was clearly selected in the evolution lineage D.

These findings indicated parallel evolution in the different lineages derived from STB-K, whereas in the STB-D background, such patterns were not visible in the framework of our experimental setting. We decided to focus our studies on STB-K clones, given the limited number of mutations identified and their apparent strong selective advantage. We first analysed the appearance and kinetics of mutation selection in the rv1339 orthologue and pe18-ppe26 locus. Whole-genome sequencing (WGS) of the bacterial populations from each evolution lineage and each cycle revealed that the rv1339 orthologue mutation was fixed very early, before cycle 4, in the four evolution lineages (Fig. 2c). In contrast, mutations at the pe18-ppe26 locus became fixed in the population later, between cycles 9 and 13 (Fig. 2d). These later mutations were only selected after fixation of the rv1339 orthologue mutation. Rapid emergence and parallel fixation of an identical SNP in the rv1339 orthologue in the four evolution lineages suggested that it may have pre-existed at low frequency in the initial STB-K culture. However, deep sequencing did not detect the mutation above background variant frequency (below 0.1%) in the starting STB-K inoculum (Fig. 2c). As our experimental evolution strategy included a 14 day in vitro culture between each cycle, we hypothesised that the rv1339 orthologue mutation might have provided a selective advantage for growth in vitro. However, WGS of cultures subjected to 6 cycles of in vitro experimental evolution showed that this mutation was not enriched in the population during these passages (Fig. 2e). Thus we concluded that the mutation in the rv1339 orthologue provides a strong in vivo selective advantage in the STB-K genetic background, whereas mutations at the pe18-ppe26 locus may provide an additional advantage only when rv1339 is mutated.

The persistence phenotypes of rv1339 and pe18-ppe26 STB-K mutants are not specific to the mouse strain or route of infection

To further investigate the effects of the selected mutations in the STB-K background, we chose two STB-K-derived variants isolated after cycle 9: KC9A1 which only has the mutation in the rv1339 orthologue (hereafter referred to as the rv1339 mutation), and KC9C1 which has both the pe18-ppe26 deletion and the rv1339 mutation. To examine whether the persistence phenotypes of these strains were specific to the BALB/c mouse genetic background, we infected C3HeB/FeJ mice, which are very susceptible to TB infection [START_REF] Kramnik | Susceptibility to tuberculosis as a complex genetic trait: analysis using recombinant congenic strains of mice[END_REF][START_REF] Kramnik | Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis[END_REF] , with either the parental STB-K strain, KC9A1, KC9C1, or MTB H37Rv.

The bacterial burden in the lungs and spleen of C3HeB/FeJ mice was consistent with an enhanced persistence phenotype for KC9A1 and KC9C1 when compared to STB-K 77 days after i.n. infection (Fig. 3a,b), indicating that this phenotype was not restricted to a single mouse genetic background. Furthermore, we found that this phenotype was also not specific to the route of infection: C3HeB/FeJ mice infected i.v. with KC9A1 and KC9C1 also had substantially more bacteria in the lungs and spleen at 76 days post-infection than had mice infected with STB-K (Fig. 3c,d), despite exhibiting similar bacterial loads at 21 days post-infection (Supplementary Fig. 3). C3HeB/FeJ mice have been reported to develop well-organised and necrotic granulomas in the lungs following aerosol infection with MTB H37Rv [START_REF] Lanoix | Heterogeneous disease progression and treatment response in a C3HeB/FeJ mouse model of tuberculosis[END_REF][START_REF] Harper | Mouse model of necrotic tuberculosis granulomas develops hypoxic lesions[END_REF] . Therefore, we examined if there was a difference in granuloma formation 76 days after i.n. infection with STB-K, KC9A1, KC9C1, and H37Rv. We found that H37Rv-infected mice had twice as much lung inflammation as had mice infected with the M. canettii strains (Fig. 3e), and only H37Rvinfected mice had mature granulomas in the lungs (Supplementary Fig. 4). There was, however, no significant difference in lung inflammation between the three M. canettii strains (Fig. 3e). Analyses of the cell types present in granulomas showed no difference between KC9A1 and STB-K, but KC9C1 exhibited a pattern more similar to H37Rv, i.e. higher percentages of CD3 + cells and neutrophils, and lower percentages of B220 + cells than for STB-K (Supplementary Fig. 5). Similar concentrations of IFN-γ, TNF and IL-10 were found in the organ homogenates from mice 21 and 76 days after i.v. infection with the different strains (Supplementary Fig. 6). These results showed a convergence in the immune response induced by the more persistent STB-K-derived mutant, KC9C1, and H37Rv. In contrast, the persistence gain of KC9A1 was not associated with a change in inflammatory response.

The pe18-ppe26 and rv1339 mutations confer enhanced persistence

To demonstrate that the pe18-ppe26 deletion is directly responsible for the difference in persistence between KC9A1 and KC9C1, we complemented KC9C1 (which has both the pe18-ppe26 deletion and the rv1339 mutation) with a plasmid containing the ppe25-ppe27 region of STB-K (KC9C1::K), thus rendering the strain genetically similar to KC9A1. We also constructed strains containing empty vectors (EV), KC9A1::EV and KC9C1::EV, to control for potential effects of the plasmid on strain fitness.

We infected BALB/c mice i.n. with these strains and found that at 70 days post-infection, KC9C1::K persisted in the lungs to the same extent as KC9A1 and KC9A1::EV (Fig. 4a). In contrast, mice infected with KC9C1 or KC9C1::EV had approximately 10-fold more bacteria in the lungs (Fig. 4a). This trend was also reflected in the spleen (Fig. 4b). These data indicate that the pe18-ppe26 deletion is responsible for the enhanced persistence phenotype of KC9C1 relative to KC9A1.

To extend these findings, we examined if the pe18-ppe26 deletion selected after experimental evolution of STB-K could also confer enhanced persistence to another M. canettii strain. To do so, we deleted the ppe25-ppe27 region in STB-D and complemented this strain with the ppe25-ppe27 regions from either STB-D (::D), STB-K (::K), or KC9C1 (::C1) (Supplementary Fig. 7). BALB/c mice infected i.n. with these strains had very similar levels of bacteria in the lungs 70 days post-infection (Supplementary Fig. 8), indicating that either the pe18-ppe26 deletion confers a persistence advantage in STB-K only, or that it requires an existing mutation in rv1339 to have a significant impact.

To demonstrate that the rv1339 mutation is directly responsible for the gain of persistence in KC9A1 and to examine if the gain associated with this mutation is specific to STB-K, we used the STB-D strain in which the rv1339 orthologue is 100% identical in sequence to that in STB-K. First we knocked out the rv1339 orthologue of STB-D (DΔrv1339), and then we complemented this strain by introducing a plasmid containing either the wild-type rv1339 orthologue (::rv1339K), or the mutated rv1339 identified in KC9A1 (::rv1339A1) (Supplementary Fig. 9). BALB/c mice infected i.n. with DΔrv1339 or ::rv1339A1 had substantially more bacteria in their lungs 77 days post-infection than had mice infected with STB-D or ::rv1339K (Fig. 4c). A similar phenomenon was also seen for ::rv1339A1 in the spleen (Fig. 4d). These data confirm that the rv1339 mutation is responsible for the enhanced persistence phenotype of KC9A1 relative to STB-K, and suggest that it is a loss-of-function mutation.

They also demonstrate that although the rv1339 mutation arose during experimental evolution of STB-K, it can enhance persistence of the STB-D strain and is therefore possibly a general mechanism for increasing the persistence of M. canettii strains.

The rv1339 mutation confers enhanced resistance to host-derived stresses

As seen above, the enhanced persistence of KC9A1 relative to STB-K does not seem to relate to a profound alteration of the immune response. Due to lymphocyte recruitment and phagocyte activation with IFN-γ, bacilli must withstand various stresses [START_REF] Singh | Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response[END_REF][START_REF] Vandal | A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis[END_REF][20][START_REF] Albrethsen | Proteomic profiling of Mycobacterium tuberculosis identifies nutrientstarvation-responsive toxin-antitoxin systems[END_REF] . These include low pH, in the range of pH 4.5 -5.5 [START_REF] Vandal | A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis[END_REF][START_REF] Schaible | Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages[END_REF][START_REF] Macmicking | Immune control of tuberculosis by IFNgamma-inducible LRG-47[END_REF] , nutrient restriction [START_REF] Neyrolles | Mycobacteria, metals, and the macrophage[END_REF][START_REF] Rifat | Phosphate depletion: a novel trigger for Mycobacterium tuberculosis persistence[END_REF][START_REF] Rengarajan | Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages[END_REF] , metal intoxication [START_REF] Neyrolles | Mycobacteria, metals, and the macrophage[END_REF][START_REF] Wolschendorf | Copper resistance is essential for virulence of Mycobacterium tuberculosis[END_REF][START_REF] Botella | Mycobacterial P1-Type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF] , or physical attacks by oxygen or nitrogen radicals [START_REF] Macmicking | Identification of nitric oxide synthase as a protective locus against tuberculosis[END_REF][START_REF] Nicholson | Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis[END_REF][31] and antimicrobial peptides [START_REF] Martineau | Neutrophil-mediated innate immune resistance to mycobacteria[END_REF][START_REF] Martineau | IFN-gamma-and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37[END_REF][START_REF] Liu | Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin[END_REF][START_REF] Stenger | An antimicrobial activity of cytolytic T cells mediated by granulysin[END_REF] . Therefore, we examined if the selected mutants have an enhanced capacity to survive these various bactericidal mechanisms. Growth of STB-K, KC9A1 and KC9C1 at various neutral or acidic pH and in phosphate-limited media was similar for the three strains (Supplementary Fig. 10). H37Rv was more resistant to H 2 O 2 -induced stress than the three M. canettii strains, which resembled each other in terms of resistance (Supplementary Fig. 10). In contrast, both KC9A1 and KC9C1 were more tolerant than STB-K to high concentrations of copper and to a lesser extent, zinc, two metals identified as important effectors of nutritional immunity against MTB [START_REF] Neyrolles | Mycobacteria, metals, and the macrophage[END_REF][START_REF] Wolschendorf | Copper resistance is essential for virulence of Mycobacterium tuberculosis[END_REF][START_REF] Botella | Mycobacterial P1-Type ATPases mediate resistance to zinc poisoning in human macrophages[END_REF] (Fig. 5a,b). Furthermore, both KC9A1 and KC9C1 were significantly more resistant to the NO donor DETA/NO than was STB-K (Fig. 5c).

To link increased resistance to stress with enhanced persistence in vivo, we examined the persistence of STB-K and KC9A1 in NOS2 -/-mice which are deficient for NO production, and in bone-marrow derived macrophages (BMDMs) stimulated with IFN- and LPS and treated with L-NAME to inhibit NO production (Supplementary Fig. 11). We found that, at 56 days post-infection, KC9A1 had higher bacterial loads in the lungs and spleens of NOS2 -/-mice than in control mice. A similar trend was observed for STB-K in the spleen. Consistently in BMDMs, treatment with L-NAME substantially increased the survival of both STB-K and the two mutants KC9A1 and KC9C1. Together these findings demonstrate that NO is an important factor reducing STB-K and KC9A1 persistence.

As KC9A1 and KC9C1 had similar levels of resistance to NO and tolerance to copper and zinc in vitro, we hypothesised that these phenotypes were associated with their common mutation in the rv1339 orthologue. To test this, we treated STB-D, DΔrv1339, ::rv1339K, and ::rv1339A1 with DETA/NO for 48 hours. We found that DΔrv1339 and ::rv1339A1 were significantly more resistant to killing by NO than ::rv1339K and STB-D (Fig. 5d), demonstrating that the rv1339 mutation is responsible for this phenotype.

As the first barrier to exogenous toxic compounds is the bacterial cell envelope, we examined whether the rv1339 mutation impacts its permeability. To address this question, we used the non-pathogenic Mycobacterium smegmatis model, which carries a rv1339 orthologue (MSMEG_4902). First, we disrupted this gene to generate smegΔrv1339 (Supplementary Fig. 12). Then we complemented this mutant strain by introducing a plasmid containing either the wild-type rv1339 orthologue from STB-K (smeg::rv1339K) or the mutated rv1339 from KC9A1 (smeg::rv1339A1). Next, we performed a permeability assay using ethidium bromide (EtBr) as a reporter molecule and found that both the smegΔrv1339 mutant and the smeg::rv1339A1 complemented strain incorporated less EtBr in comparison to the wild-type and smeg::rv1339K strains (Fig. 5e).

Together, these findings indicate that the rv1339 mutation decreases the cell envelope permeability and confers increased resistance to several stresses encountered by the pathogen during infection.

MTB exhibit higher NO resistance than M. canettii and this gain is important for persistence

Persistent bacteria arising from experimental evolution of STB-K had mutations in rv1339, whereas the persistent bacteria that evolved from the STB-D strain did not. As tuberculosis bacilli have acquired multiple mechanisms to resist host-derived stress [START_REF] Singh | Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response[END_REF][START_REF] Vandal | A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis[END_REF][20][START_REF] Buchmeier | A parallel intraphagosomal survival strategy shared by Mycobacterium tuberculosis and Salmonella enterica[END_REF][START_REF] Ng | Role of KatG catalaseperoxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst[END_REF][START_REF] Primm | The stringent response of Mycobacterium tuberculosis is required for longterm survival[END_REF] , we examined the sensitivities of several persistent STB-D mutants selected after cycle 6 to NO as a representative stress. Two out of four persistent mutants tested, DC6C1 and DC6D1, were substantially more resistant to DETA/NO treatment than STB-D, and closely resembled MTB H37Rv (Fig. 6a). This indicated that phenotypic convergence occurred during experimental evolution of the genomically distant STB-K and STB-D strains, and highlighted that increased resistance to NO, and possibly to other stresses, are key mechanisms driving persistence in vivo.

To assess the ability of phylogenetically diverse M. canettii strains (Supplementary Fig. 1) to resist NO, we exposed five strains, STB-K, -D, -A, -J and -L, to DETA/NO for 48 hours. All the strains tested were significantly more sensitive to killing by NO than MTB H37Rv (Fig. 6b). We also tested the susceptibilities of three other MTB strains from various lineages to DETA/NO and found that there was no significant difference in the resistance of MTB strains to NO with the exception of the lineage 1 strain IO367, which was slightly more susceptible to killing than H37Rv only (Fig. 6c). These results thus demonstrate that MTB are more resistant than M. canettii to stresses, such as NO and indicate that increased NO resistance is associated with enhanced persistence in vivo.

Discussion

Parallel molecular evolution and phenotypic convergence are canonical signatures of genes and processes under strong adaptive selection, such as those related to pathogen adaptation to host environments [START_REF] Lieberman | Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes[END_REF][START_REF] Lenski | Chance and necessity in the evolution of a bacterial pathogen[END_REF][START_REF] Stern | The genetic causes of convergent evolution[END_REF][START_REF] Didelot | Within-host evolution of bacterial pathogens[END_REF][43] . Here, the identification of parallel and convergent evolution during experimental evolution of ancestor-like MTB strains in mice highlights that a key patho-adaptation differentiating modern MTB from their environmental ancestor is enhanced resistance to stress, especially NO. NO and other reactive nitrogen intermediates are produced by macrophages upon activation by T cellderived IFN-γ, and are major effectors in the immune response to TB [START_REF] Macmicking | Identification of nitric oxide synthase as a protective locus against tuberculosis[END_REF]31,[START_REF] Chan | Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages[END_REF][START_REF] Flesch | Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates[END_REF] . These radicals are believed to protect TB-infected hosts through direct bacterial killing [START_REF] Long | Mycobacteriocidal action of exogenous nitric oxide[END_REF][START_REF] Nozaki | Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in human alveolar macrophages[END_REF] , and inhibition of granulocytic inflammation and immunopathology [START_REF] Mishra | Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis[END_REF][START_REF] Mishra | Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1beta[END_REF] . Enhancing resistance to these compounds, and possibly other toxic molecules generated during the immune response such as metal ions, appears to be required to increase persistence of ancestor-like MTB strains in the lungs, and was probably a major step in the emergence of MTB.

In this study, we found that diverse mutations acquired by M. canettii STB-K and STB-D through experimental evolution produce this phenotype. One of these mutations was in the rv1339 orthologue, and it conferred both STB-K and STB-D strains with enhanced resistance to NO. The gene rv1339 is highly conserved in TB bacilli (Supplementary Fig. 13) and other mycobacteria and a recent study revealed that the Rv1339 protein degrades cAMP, an important signalling molecule controlling peptidoglycan metabolism [START_REF] Thomson | Modulation of the cAMP levels with a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria[END_REF] . Inactivation of rv1339 or expression of the mutated rv1339 allele from KC9A1 increased stress resistance in M. canettii STB-D and STB-K and reduced envelope permeability in M. smegmatis. Consistently MTB strains containing point mutations or transposon insertions in rv1339 have increased resistance to antitubercular compounds [START_REF] Chandrasekera | Improved phenoxyalkylbenzimidazoles with activity against Mycobacterium tuberculosis appear to target QcrB[END_REF][START_REF] O'malley | Imidazopyridine Compounds Inhibit Mycobacterial Growth by Depleting ATP Levels[END_REF] and a reduced capacity to import fatty acids during macrophage infection [START_REF] Nazarova | The genetic requirements of fatty acid import by Mycobacterium tuberculosis within macrophages[END_REF] . Therefore, the likely mechanism of enhanced persistence associated with the rv1339 mutation is a decrease in envelope permeability allowing bacilli to withstand toxic stresses such as NO or metals. The other mutations selected in the STB-K-derived persistent clones concerned pe18 and ppe26 genes located within the esx-5 locus, which encodes one of the five type VII protein secretion systems of MTB. Interestingly, clinical MTB isolates successfully spreading in Tunisia harbour a single pe-ppe gene pair in the esx-5 locus that likely arose by homologous recombination between ppe25-ppe27 genes [START_REF] Karboul | Frequent homologous recombination events in Mycobacterium tuberculosis PE/PPE multigene families: potential role in antigenic variability[END_REF] as in the mutant KC9C1. Furthermore, MTB strains from the Beijing family exhibit esx-5 locus mutations disrupting secretion of a large number of PE/PPE proteins and enhancing their virulence 54 . This convergence between experimental evolution of ancestor-like MTB strains and the natural evolution of modern TB strains suggests that the loss of certain PE/PPE proteins is another method exploited by TB bacilli to better survive and enhance their virulence in mammalian hosts. The precise function of PE18-PPE26 is unknown but PE/PPE proteins were recently found to mediate solute-specific import across the MTB outer membrane [START_REF] Nazarova | The genetic requirements of fatty acid import by Mycobacterium tuberculosis within macrophages[END_REF][START_REF] Wang | PE/PPE proteins mediate nutrient transport across the outer membrane of Mycobacterium tuberculosis[END_REF] . Therefore, pe18-ppe26 mutations in STB-K might also limit the penetration of toxic molecules allowing the M. canettii persistent mutants to better survive the immune response. However, we found no evidence of enhanced resistance to stress associated with the pe18-ppe26 mutation. Therefore, an alternative explanation might relate to the difference in immune cell recruitment observed in mice infected with the KC9C1 mutant that more closely resembles the situation in H37Rv-infected mice.

In this study, we identified a major driving force for patho-evolution of TB bacilli and illustrated that the required adaptations can be acquired by exploiting diverse bacterial pathways. This supports the concept that phenotypic convergence can be achieved through diverse genetic modification due to historical contingency. The associated loss-of-function mutational patterns apparent in STB-K further highlight the key role of gene loss in driving evolutionary adaptation [START_REF] Helsen | Gene loss predictably drives evolutionary adaptation[END_REF] , in this case towards enhanced persistence of a major pathogen. The rapid selection of M. canettii mutants with similar persistence characteristics to MTB and diversity of mutations leading to a similar gain-of-function is surprising, considering the accepted model that MTB emerged only once from an environmental ancestor [START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF] . Longterm association with the human host requires both the ability to persist, and to transmit to uninfected people. M. canettii strains have never been reported to be transmitted between humans [START_REF] Blouin | Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti[END_REF] . which remains to be identified. 305

Materials and methods

Mycobacterial strains and culture conditions

The M. canettii, MTB and M. smegmatis strains used in this study are listed in Supplementary Table 3. All strains were cultured in Middlebrook 7H9 broth (BD Difco) supplemented with 0.2% glycerol (w/v) and 10% Middlebrook albumin-dextrose-catalase enrichments (ADC; BD BBL), with 0.05% Tween 80 (Sigma) (7H9/ADC/Tween) unless otherwise described. To grow recombinant strains, the antibiotics hygromycin (Euromedex) and kanamycin (Euromedex) were added as required at final concentrations of 50 µg/ml and 40 µg/ml respectively. Cultures were agitated approximately twice per week and maintained for 3-4 weeks at 37ᵒC before use. During the experimental evolution cycles, the antibiotic mixture PANTA (BD BBL) was added at a dilution of 25 µl for 10 ml 7H9/ADC/Tween for in vitro cultures. For growth on solid medium, 7H11 agar base (BD BBL) was supplemented with 0.5% glycerol and 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC; BD BBL). Counting of CFUs on 7H11 agar plates was performed after 2, 3 and 4 weeks of incubation at 37ᵒC.

Experimental evolution strategy

The experimental evolution procedure consisted of independent serial passages of strain STB-K (phylogenetically farthest from MTB) and strain STB-D (phylogenetically closest to MTB) in BALB/c mice. For both STB-K and STB-D, four independent cultures were passaged corresponding to evolution lineages A, B, C and D, and they were never mixed. At cycle 1, 12 mice were infected i.n. with 10 4 CFU of STB-K or STB-D. STB-infected mice were then euthanised after 1 and 28 days of infection (4 mice per time point) and the CFU in the lungs and spleens were enumerated to evaluate the initial bacterial load and the multiplication during the acute phase of the infection. The 4 remaining STB-infected mice were kept until 56 days post-infection and then sacrificed to determine CFU at the chronic infection stage. The bacterial load in the organs was evaluated by plating part of the lung and spleen homogenates on solid medium in order to get information on the entire population. Part of the lung homogenates recovered from the 4 mice 56 days post-infection was also used to initiate four separate cultures (evolution lineages A, B, C and D) which were allowed to grow for 14 days at 37ᵒC before being used as the new inocula for the next cycle. Each subsequent cycle was initiated by i.n. infecting For whole genome sequence analyses, CLC Genomics Workbench, version 20.0 (QIAGEN, Aarrhus) was utilised for sequence mapping, using the global alignment option and ignoring reads with more than one match, and variant calling, using the basic variant calling mode with a minimum frequency filter set to 85%, and other parameters set to default. Mapping was performed against previously assembled reference genomes of STB-K and STB-D (accession number FO203509.1 and NC_019950.1 respectively), and a newly generated dataset obtained by PacBio for STB-K. WGS data generated from STB-K and STB-D parental strain batches, used for the first cycle of experimental evolution, were used to correct for SNPs that were pre-existing in the genome assemblies (i.e. before experimental evolution). Manual curation was subsequently performed to discard probable false positive variants typically identified in repetitive regions (e.g. PE/PPE protein-encoding genes), including variants found within a 12 bp-distance range. The false positive variant calling rate with this analysing pipeline was verified by mapping sequence reads from MTB H37Rv genomic DNA against a previously assembled reference genome obtained after PacBio sequencing of the same genomic DNA preparation, resulting in absence of any detectable SNP or indel.

For the large deletion identified in the pe18-ppe26 gene region of STB-K evolution lineage C, a specific read mapping analysis was performed against the parental reference sequence using bowtie2 version 2.3.5 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] . Proportions of bacterial wild-type versus mutant (i.e. carrying this deletion) subpopulations over the different cycles were determined after normalisation of the coverage depth detected around the deleted region, by dividing this coverage depth by the mean genome wide coverage depth obtained for the cycle considered. The proportion detected in the parental strain was also set at 100%, to better visualise the progressive fixation of the deletion, starting from the initial population.

To search for rv1339 variants among TB bacilli isolates, we analysed a collection of 6167 genome assemblies available as of January 2020 from the NCBI by BLASTN search-based screening and we looked at the distribution of mutations fixed in the rv1339 gene in isolates belonging to lineages 1-8 or the animal lineages of the MTB complex.

Strain and plasmid construction

The plasmids and primers used in this study are listed in Supplementary Tables 4 and5, respectively.

For the construction of the BN44_11505 (rv1339 orthologue) mutant in M. canettii STB-D, a 2kb DNA fragment overlapping the BN44_11505 gene was amplified by PCR using primers 39a and 39b and cloned into the plasmid pJET (ThermoFisher Scientific) to give plasmid pWM355. A 0.8kb PCR fragment containing the kanamycin (km) gene was amplified from plasmid pET26b (Addgene) using primers km1 and km2 and inserted into the EcoRV site of plasmid pWM355, which is located inside the BN44_11505 gene, to generate plasmid pWM359. The substrate for allelic exchange was produced by PCR amplification of a 2.8kb fragment, carrying the BN44_11505 gene disrupted by the km cassette, from pWM359. This DNA fragment was electroporated in a recombinant M. canettii STB-D expressing the recombineering system [START_REF] Van Kessel | Mycobacterial recombineering[END_REF] from plasmid pJV53H and transformants were selected on km-containing plates. Ten km-resistant clones were randomly picked and analysed by PCR using primers 39c / kmF, 39d / kmR and 39e / 39f. A clone giving the expected PCR profile (Supplementary Fig. 9) was selected for further experiments and named PMM305 (or DΔrv1339).

For the construction of the DΔppe25-ppe27 mutant (also named PMM304), three PCR fragments were amplified and fused. First, a 1kb fragment upstream of ppe25 was generated from STB-D genomic DNA by PCR amplification using primers esx1 and esx2 which carry DraIII restriction site at their 5'-end. A second 1kb fragment downstream of ppe27 was produced using primers esx5 and esx6 which also carry DraIII restriction sites at their 5'-end. Finally, the third fragment corresponding to the km gene was amplified using primers k1 and k2 which add DraIII sites on both side of the km gene. The three PCR fragments were digested using the restriction enzyme DraIII for 1h at 37°C. The three fragments were then ligated and the allelic exchange substrate corresponding to the ligated PCR fragments was amplified by PCR as a 2.8kb product using primers esx1 and esx6. This DNA product was transferred by electroporation into STB-D:pJV53H after induction of phage recombinase using acetamide as previously described [START_REF] Van Kessel | Mycobacterial recombineering[END_REF] . Transformants were selected on km-containing plates. Ten colonies were randomly picked and analysed by PCR using primers esx7 / kmR, esx8 / kmF and 26a / 26b. A clone giving the expected PCR profile (Supplementary Fig. 7) was selected for further experimentation and named PMM304 (or DΔppe25-ppe27).

For the construction of the MSMEG_4902 (rv1339 orthologue) mutant in M. smegmatis mc 2 155, two 1kb DNA fragments located upstream and downstream of MSMEG_4902 gene were amplified by PCR using either primers 4902A and 4902B, or primers 4902C and 4902D, and then purified. The two fragments were ligated before amplification using primers 4902E and 4902F to generate a 2 kb DNA fragment. This fragment was cloned into the plasmid pJET. A 0.8kb PCR fragment containing the km gene was amplified from plasmid pET26b using primers km1 and km2 and inserted into the EcoRV site located at the junction of the fused 1kb fragments to generate plasmid pAH06. The substrate for allelic exchange was produced by PCR amplification of a 2.8kb fragment, carrying the MSMEG_4902 gene disrupted by the km cassette, from pAH06. This DNA fragment was ligated into the EcoRV site of plasmid pJQ200 [START_REF] Pelicic | Generation of unmarked directed mutations in mycobacteria, using sucrose counterselectable suicide vectors[END_REF] to generate plasmid pAH10. This plasmid was electroporated into M. smegmatis mc 2 155 and transformants were selected on km-containing plates. One transformant was used to grow a culture and then plated on solid medium containing km and sucrose 5%. Ten km-resistant clones were analysed by PCR using primers 4902D / kmF, 4902A / kmR, and 4902A / 4902G. One clone giving the expected PCR profile (Supplementary Fig. 12) was selected for further experiments and named PMM280 (or smeg Δrv1339).

The BN42_21248 gene from M. canettii STB-K (orthologue of rv1339 in MTB H37Rv) or the mutated allele from KC9A1, were amplified from the corresponding strains using primers 1339G and 1339H.

They were then inserted between the Nde1 and Spe1 sites of a derivative of the integrative plasmid pMV361 [START_REF] Stover | New use of BCG for recombinant vaccines[END_REF] carrying a hygromycin resistance gene (hyg) and the mycobacterial promotor pBlaF* [START_REF] Dantec | Genomic sequence and transcriptional analysis of a 23-kb mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems[END_REF] , to give the complementation plasmids pAH01H (BN42_21248 mutated) and pAH02H (BN42_21248 wildtype). In pAH01H and pAH02H, the wild-type BN42_21248 or the mutated variant were expressed under the control of the heterologous promotor pBlaF*. These plasmids were transferred independently into the strain PMM305 by electroporation to give PMM305::pAH01H (abbreviated ::rv1339A1) and PMM305::pAH02H (abbreviated ::rv1339K). Plasmid pAH01 (carrying the gene BN42_21248 mutated from KC9A1) or pAH02 (carrying the wild-type gene BN42_21248 mutated from STB-K) were also transferred independently into the strain PMM280 by electroporation to give PMM280::pAH01H (abbreviated smeg::rv1339A1) and PMM280::pAH02H (abbreviated smeg::rv1339K).

Similarly, the ppe25-ppe27 genes from M. canettii STB-K or the corresponding allele from KC9C1 (carrying the pe18-ppe26 deletion) were produced by PCR amplification of the corresponding genomic region using primers esxA and esxB. These regions were then inserted between the XbaI/HindIII restriction sites from the pMV361 derivative carrying the hyg gene to give the complementation plasmids pWM361 (ppe25-ppe27 region from STB-K) and pWM386 (ppe25-ppe27 region from KC9C1).

The corresponding genomic regions from STB-D were amplified using primers esxC and esxD and cloned in the same vector to generate pWM385 (ppe25-ppe27 region from STB-D). These three complementation plasmids, or the corresponding empty vectors (EV), were transformed into strains KC9A1, KC9C1, PMM304, or STB-D.

Animal infection studies

The persistence capacity of the M. canettii mutants and recombinant strains was assessed in four different mouse models. 6-8 week old female BALB/c mice (Janvier; 3/4 mice per group per time point) were infected i.n. with approximately 10 3 CFU per strain. 6-10 week old female C3HeB/FeJ mice (bred in-house in the IPBS animal facility; 4/5 mice per group per time point) were infected either i.n. with approximately 200 CFU per strain or i.v. with approximately 10 3 CFU per strain. All mice were anaesthetised prior to infection: BALB/c mice were injected intraperitoneally (i.p.) with a combination of valium, atropine and imalgene, and C3HeB/FeJ mice were injected i.p. with a ketamine-xylazine mixture for i.n. infection, or were exposed to isoflurane for i.v. infection. For experiments with NOS2 - /-mice, 7-15 week old NOS2 -/-mice (gift from E. Russo and P. Bousso, Institut Pasteur) or 6-8 week old C57BL/6 control mice (Janvier) were infected via aerosol with approximately 100 CFU per strain.

Mice were housed under specific pathogen-free conditions. Mice displaying signs of health deterioration were euthanised by cervical dislocation. At the indicated time points, mice were sacrificed by cervical dislocation and the lungs and spleens were isolated and homogenised using a GentleMACS Dissociator (Miltenyi Biotec) and gentlemacs M tubes. Bacterial load in the organs was estimated by plating serial dilutions of the lung and spleen homogenates on solid medium. For inoculum preparation, 3-4 week old bacterial cultures were re-cultured for 10 days in 7H9/ADC/Tween. 10 day old cultures were washed twice in PBS/0.05% Tween 80 and adjusted to the final desired concentration in PBS/0.05% Tween 80 for infection. Inoculum accuracy was verified by plating serial dilutions on 7H11 agar plates supplemented with 10% OADC.

Histopathology

For histopathological evaluation, C3HeB/FeJ mice were sacrificed by i.p. injection of Dolethal at 11 weeks post-infection, and lungs were isolated and fixed in 10% neutral buffered formalin for 48 hours.

Following fixation, lungs were transferred into 70% ethanol and then delivered to the Experimental Histopathology service in Purpan Hospital, Toulouse (Anexplo). The lungs were embedded in paraffin for 5 μm sectioning and sections were stained with hematoxylin and eosin. Stained samples (one entire lung section for each mouse) were analysed using Pannoramic Viewer software to quantify the percentage lung inflammation per sample. For immunohistochemistry analyses, the epitopes were unmasked by HIER treatment for 20 minutes at 95°C, either in citrate buffer pH6 for B cell and neutrophil labelling or in citrate buffer pH9 for T-cell labelling. For macrophages, regeneration was performed by PIER treatment for 10 minutes at room temperature. Endogenous peroxidase and endogenous biotin were blocked by treatment with blocking solutions (S2023 Agilent for peroxidase and NB306 Clinisciences for biotin) for 10 minutes and 20 minutes respectively. Then the tissue sections were incubated with goat serum for 30 minutes. Specific labelling was performed by incubation for 1 hour with primary antibodies for macrophages (F4/80, MCA497G, Serotec; 1/100 dilution), B lymphocytes (B220/CD45r, MCA1258G, Serotec; 1/400 dilution) and T lymphocytes (CD3, ab16669, Abcam; 1/100 dilution) as well as overnight at 4°C for neutrophils (Neutrophil marker, sc-71674, Santa Cruz biotechnology; 1/200 dilution). The lung sections were then incubated for 30 minutes with either biotinylated goat anti-rat secondary antibody for neutrophils, macrophages and B cells or goat anti-rabbit biotinylated antibody for T cells and then incubated with horseradish peroxidase (R.T.U., PK-7100, Vector Laboratories) for 30 minutes. The labelling was revealed in Diaminobenzidine (DAB) with a counter-stain of modified hematoxylin. Quantification of positive cells for F4/80, CD3, B220 and neutrophils markers per lesion was performed using the QuPath software [START_REF] Bankhead | QuPath: Open source software for digital pathology image analysis[END_REF] .

More precisely, regions of interest were drawn for each individual lung lesion using the wand annotation tool. Next, cell detections were performed within each region of interest using hematoxylin staining to detect the nucleus of individual cells combined with cell expansion option to segment the whole cell. Finally, the mean intensity of DAB staining of cells from infected lung sections stained with biotinylated secondary antibody only was used to define DAB intensity thresholds for each marker and applied to quantify the percentage of positive cells within each lesion.

Cytokine quantification in organ homogenates

The lungs and spleens of infected mice were isolated and homogenised as described above. Samples were centrifuged at 1800 rpm for 10 minutes at 4ᵒC to pellet the remaining tissue, and supernatants were filtered twice using 0.2 µm Millex-GP polyethersulfone filters (Merck Millipore). The cytokine concentrations in the lung and spleen supernatants were quantified using standardised ELISA kits according to the manufacturer's instructions; IL-10 (R&D), TNF (Invitrogen) and IFN-γ (BD).

Bone-marrow-derived macrophage studies

Bone-marrow derived macrophages were obtained from the femur, tibia, and hips from bones from BALB/c mice. Bone marrow cells were cultured at 37°C and 5% CO 2 for 6 days in BMM medium to obtain BMDMs. BMM medium consisted of high glucose DMEM medium with no glutamine or phenol red (ThermoFisher) supplemented with 10% heat-inactivated fetal calf serum (Sigma), 20% M-CSF (Generous gift from Dr Etienne Meunier, Toulouse), 1% Hepes (Gibco), 200 mM Glutamine (ThermoFisher), 100 mM Sodium pyruvate (Gibco) and 0.5% Penicillin/Streptomycin (Sigma), which was filtered before use. After 6 or 7 days of culture, BMDMs were washed, counted and plated in flatbottomed 24 well cell culture plates at 2.5 x 10 5 cells per well in BMM containing no antibiotics.

BMDMs were then either left unstimulated, or activated by treatment for 16 hours with IFN- (100 U/ml, Biolegend) + LPS (100ng/ml, Sigma) to induce the production of NO, or activated for 16h with IFN- (100 U/ml) + LPS (100ng/ml) and treated L-NAME (4mM, Sigma) to inhibit iNOS. In these conditions, we detected no NO in the supernatant of unstimulated cells, between 10 and 20 µM NO in the supernatant of activated cells, and less than 5 µM NO in activated cells treated with L-NAME, using the Griess assay (Sigma). Following stimulation, BMDMs were washed and then incubated with STB-K, KC9A1, KC9C1 or MTB H37Rv at MOI 0.5 bacteria per cell for 2 hours before washing again. To evaluate bacterial counts just after infection on day 0 or 3 days after infection, BMDMs were lysed using 0.1% Triton X-100 in H 2 O and cell lysates were plated on 7H11 agar plates.

In vitro sensitivity testing

Before performing susceptibility testing to NO, 3-4 week old bacterial cultures were re-cultured for 10 days in 7H9/ADC/Tween to ensure bacteria were in the exponential phase. 10 day old cultures were washed twice in PBS/0.05% Tween 80 and diluted into 7H9/ADC/Tween at a calculated final OD 600nm of 0.0005, corresponding to 10 5 CFU/ml, and incubated in the presence of 5 mM DETA/NO (Sigma).

The number of viable bacteria in treated and non-treated control cultures was determined by CFU plating at the times indicated in the figure legends.

To evaluate bacterial growth at neutral pH, 3-4 week old cultures were diluted 1 in 100 into medium composed of 7H9/ADC/Tween at pH 6.8. For the acidic pH growth assay, 3-4 week old cultures were diluted 1 in 100 into 7H9 supplemented with ADC and 0.05% Tyloxapol (Merck), which had been adjusted to pH 5, 5.5, 6 or 6.6. 100 mM MOPS buffer (Sigma) was used to keep the pH stable in medium adjusted to pH6.6, and 100 mM MES buffer (Sigma) was used for pH5, 5.5 and 6. Tyloxapol was used as the dispersal agent for this experiment as Tween 80 was previously shown to be toxic to MTB at acidic pH [START_REF] Vandal | A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis[END_REF] . Growth was monitored by measuring OD 600nm at various intervals. Results are represented as the log 10 OD 600nm and are representative of three independent experiments.

To evaluate bacterial growth in phosphate-limiting conditions, strains were grown for 3-4 weeks in 7H9/ADC/Tween, pelleted, washed three times in Sauton's medium minus phosphate and then diluted to approximately OD 600nm 0.05 in Sauton's medium containing the standard concentration of phosphate (2870 µM) or low-phosphate concentrations (0 µM, 50 µM). The composition of Sauton's medium minus phosphate was 4g/L L-asparagine, 2g/L citric acid monohydrate, 0.05g/L ferric ammonium citrate, 1% zinc sulfate, 0.5g/L magnesium sulfate heptahydrate and 60ml/L glycerol in H 2 O. Phosphate was added as required in the form of potassium phosphate dibasic trihydrate. Strain growth was evaluated by monitoring the OD 600nm . Data are represented as the mean and standard deviation from three independent experiments.

To evaluate bacterial tolerance to zinc and copper, 3-4 week old bacterial cultures were re-cultured for 7 days in 7H9/ADC/Tween to ensure bacteria were in the exponential phase. 7 day old cultures were diluted into 4 ml 7H9/ADC/Tween at a calculated final OD 600nm of 0.005 in McFarland tubes, and incubated in the presence of various concentrations of zinc (ZnSO 4 , Sigma), or copper (CuCl 2 , Sigma).

At the indicated time points, each tube was vortexed before evaluation of turbidity using a McFarland densitometer. Each measure was performed in triplicate and the experiment was performed four times independently. Data are represented as the mean and standard deviation of the triplicates from one experiment.

Permeability assay

To assess membrane permeability of the wild-type M. smegmatis (WT), PMM280 (smegΔrv1339), and the complemented strains PMM280::pAH01H (smeg::rv1339A1) and PMM305::pAH02H (smeg::rv1339K), an EtBr uptake assay on intact mycobacteria was performed, as previously described [START_REF] Rodrigues | Measuring efflux and permeability in mycobacteria[END_REF] . Briefly, logarithmic-phase strains grown in 7H9/ADC/Tween were centrifuged, normalised to OD 600nm of 0.8 in PBS buffer containing glucose 0.4%. 100 μl of bacterial suspension was added per well into 96-well microplates. 10 µl EtBr (25 µM) was added to the bacterial suspension and the mix was incubated at 37°C. Fluorescence, associated with accumulation of EtBr, was measured for 1 hour at 3 minute intervals with an excitation wavelength of 525 nm and an emission wavelength of 615 nm.

The experiment was performed twice in quintuplicate for the WT and smegΔrv1339 strains and once in quintuplicate for the smeg::rv1339A1 and smeg::rv1339K strains.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism 5 and 7. One or two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test was used to analyse the statistical significance between three or more groups. P values less than 0.05 were considered to be statistically significant.

When bacteria were undetectable, the CFU value was set at the limit of detection (log 10 1.7 for in vivo studies and log 10 1.3 for in vitro studies , with the exception of the study involving C57BL/6 and NOS2 - /-mice where the limit was log 10 2) for the purpose of statistical analysis. For comparison of cell types in the lesions, the Brown-Forsythe and Welch ANOVA test with the Games-Howell post hoc test was used.

Ethics statement

All studies with M. canettii and M. tuberculosis strains were performed in biosafety level 3 (BSL3) conditions, strictly following all relevant biosafety guidelines for experimental work with wild-type and For both strains, 4 independent cultures were passaged corresponding to evolution lineages A, B, C and D, and were never mixed. At the beginning of each cycle, 12 mice were infected i.n. with 10 4 CFU.

CFU numbers in the lungs and spleen were evaluated at 1, 28 and 56 days post-infection (4 mice per time point) by plating. Part of the lung homogenates recovered from the mice sacrificed at 56 days post-infection was also used to initiate 4 separate cultures. These cultures were grown for 14 days at 37ᵒC and were then used as the new inocula for the next cycle. 15 cycles of experimental evolution were performed for STB-K and 6 cycles were performed for STB-D. This diagram was created using images from Servier Medical Art.

b) The persistence index was evaluated for each cycle in the experimental evolution by calculating the log ratio of bacterial burden in the lung at day 56 vs day 28. Data are shown as log 10 CFU D56/D28 for each lineage and the mean. For c), and d) whole -genome sequence data were generated from the bacterial populations corresponding to the inoculum and to each evolution lineage recovered after cycles 1 to 15. Positions corresponding to the mutation in the rv1339 orthologue or the deletions in the ppe25-ppe27 regions were analysed to determine the distribution of wild-type and mutant alleles in the population.

For e), we performed 6 cycles of growth of STB-K in 7H9/ADC/Tween supplemented with panta (as for the in vitro cultures between each mice infection cycle). Whole -genome sequence data were generated from the bacterial populations corresponding to the inoculum and bacterial populations recovered after cycles 3 and 6. For the position corresponding to the C>T in the rv1339 orthologue, the coverage depth was x98, x124 and x159 for the inoculum, cycle 3 and cycle 6 populations respectively.

the STB-K-derived populations from each evolution lineage after each cycle by WGS. 642 The experiment was performed twice in quintuplicate for the WT and smegΔrv1339 strains and once in quintuplicate for the smeg::rv1339A1 and smeg::rv1339K strains. In these experiments, stimulation of BMDMs with IFN- and LPS reduced the survival of the 4 strains and this effect is partially suppressed by inhibiting iNOS. b) PCR profiles corresponding to a mutant interrupted for the rv1339 orthologue (PMM280), or the wild-type M. smegmatis mc 2 155 strain (WT), when the indicated primer pairs were used for amplification.

Cycle

  12 mice with approximately 10 4 CFU of the new STB-K or STB-D inocula prepared from cultures of bacterial populations obtained 56 days post-infection in the previous cycle. This process of infection and CFU monitoring was repeated for 15 cycles for STB-K and 6 cycles for STB-D. At cycles 3, 6, 9, 12 and 15 of STB-K, 20 individual colonies were picked up, briefly cultivated and stored at -80°C (corresponding to 100 clones total). 20 of these individual clones (8 selected at cycle 6 and 4 selected at cycles 9, 12 and 15) were characterised individually for their persistence in animal models. At cycles 3 and 6 of STB-D, 20 individual colonies were similarly picked up, briefly cultivated and stored at -80°C (corresponding to 40 clones total). 4 of these individual clones (selected at cycle 6) were characterised for their persistence in animal models.Sequencing and sequence analysesWe performed Illumina-based WGS on 100 individual clones derived from STB-K (recovered at cycles 3, 6, 9, 12 and 15) and 40 clones derived from STB-D (recovered at cycles 3 and 6). Genomic DNA from each clone was extracted from stationary phase culture using either the DNeasy Blood & Tissue kit (Qiagen) or the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research) according to the manufacturer protocols.

Figure 1 :

 1 Figure 1: Experimental evolution in mice selects M. canettii clones with enhanced persistence.

c)

  Persistence characteristics of 4 individual clones recovered after cycle 9 from STB-K evolution lineages A, B, C and D. Mice were i.n. infected with 10 3 CFU of the parental STB-K strain, mutants KC9A1, KC9B1, KC9C1, KC9D1, or MTB H37Rv and the bacterial burden in the lungs and spleen was assessed at days 1, 28, 77 and 140 post-infection. Data are shown as log 10 CFU for individual mice and the mean from an experiment performed with 3/4 mice per group. d) Persistence phenotypes of 4 individual clones recovered after cycle 6 from STB-D evolution lineages A, B, C and D. Mice were i.n. infected with 10 3 CFU of the parental STB-D strain, mutants DC6A1, DC6B1, DC6C1, DC6D1 or MTB H37Rv and the CFU numbers in the lungs and spleen were assessed at days 1, 29, 70 and 141 post-infection. Data are shown as log 10 CFU for individual mice and the mean from an experiment performed with 3/4 mice per group. When bacteria were undetectable, the CFU value was set at the limit of detection (log 10 1.7).

29

 29

Figure 2 :

 2 Figure 2: Mutations in the rv1339 orthologue and the ppe25-ppe27 region of STB-K mutants were

Figure 3 :

 3 Figure 3: The persistence phenotypes of rv1339 and pe18-ppe26 STB-K mutants are not specific to

Figure 4 :

 4 Figure 4: The pe18-ppe26 and rv1339 mutations confer enhanced persistence.

Figure 5 :

 5 Figure 5: The rv1339 mutation contributes to enhanced resistance to host-derived stresses.

Figure 6 :

 6 Figure 6: MTB exhibit higher nitric oxide resistance than M. canettii.

FO203509. 1 ,

 1 indicates the mutated position in reference to the NCBI GenBank sequence 883 Ref and Alt refer to the wild-type and mutated alleles detected respectively, and Ki00 884 refers to the frequency of the mutated allele detected in the inoculum. Table cells coloured in grey 885 indicate the deleted region identified in evolution lineage C after cycle 6. The variants identified in this 886 region are presumably false positives due to the recombination of ppe25 and ppe27. Column four 887 mentions mean depth calculated for the 20 sequenced clones from the corresponding cycle at the 888 indicated position. 889 890

NC_019950. 1 ,

 1 indicates the mutated position in reference to the NCBI GenBank sequence 898 Ref and Alt refer to the wild-type and mutated alleles detected respectively, and Di00 899 refers to the frequency of the mutated allele detected in the inoculum. The light orange and green 900 boxes indicate SNPs segregated together in different evolution lineages. Most of these SNPs were 901 detected above background in the inoculum supporting the conclusion that the STB-D inoculum, used 902 to start the experimental evolution, contains at least three genetic profiles. 903

Animal studies were performed in agreement with European and French guidelines (EC Directive 2010/63/UE and French Law 2013-118 issued on February 1, 2013) after approval by the relevant Ethics Committee (Comité d'éthique en experimentation animale N°01 and N°89) and the French Ministry for Higher Education and Research after ethical evaluation (number 201508271122464 v2 (APAFIS#1535) and number 2018060717283847 v1 (APAFIS#15409)). C3HeB/FeJ mice were i.v. infected with approximately 10 3 CFU of STB-K, KC9A1, KC9C1, or MTB H37Rv, and IL-10, IFN-γ and TNF concentrations in the lung and spleen homogenates were quantified at a) 21 days and b) 76 days post-infection by ELISA. Mean cytokine concentrations are shown, in addition to the concentrations for individual mice (4/5 per group). *P < 0.05 by one-way ANOVA with the Bonferroni post hoc test.

Acknowledgements

The authors thank T. Seemann for the initial help with the network phylogeny analysis. The authors are grateful to Erica Russo and Philippe Bousso (Institut Pasteur, France) for the gift of NOS2 -/-mice.

The authors acknowledge support from the GenoToul (Toulouse, France) Bioinformatics and GeT platforms for sequencing, computing and storage resources, and the Anexplo platform for animal experimentation. The authors thank Samantha Milia for technical assistance at the Experimental Histopathology Facility of INSERM / UPS US006 CREFRE, Toulouse Purpan, France. The authors also thank Life Science Editors for their professional editing assistance during the preparation of the manuscript. This project has received funding from the European Respiratory Society and the European Union's H2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 713406 awarded to A.C.A. This work was also supported by grants from the French National Research Agency (TBemerg contract N°ANR-16-CE35-0009 awarded to R.B., P.S. and C.G.; , Equipex ANINIMIP ANR-11-EQUIPEX-0003 awarded to C.G.; Labex ANR-10-LABX-62-IBEID awarded to R.B.), by a grant from the European Union (PathoNgenTrace contract N°FP7-278864 awarded to P.S.), and by grants from the Fondation pour la Recherche Médicale (Equipe FRM 2016 DEQ20160334879), the Fondation Bettencourt Schueller and the Fondation MSD Avenir awarded to C.G.

Data availability

The sequence data were deposited in the NCBI repository under project PRJNA662472, PRJNA664307 and PRJNA665366 accession codes for Illumina-derived genome sequences of STB-K-and STB-Dderived clones and populations and PacBio-derived genome sequence of STB-K respectively. All other data that support the findings of this study are available from the corresponding author upon request.

Supplementary Figure 1: Phylogenetic tree showing the distribution of M. canettii and MTB strains.

Network phylogeny inferred among 28 genomes by NeighborNet analysis, based on pairwise alignments of whole genome SNP data from a selection of strains (17 M. canettii, 8 MTB from various phylogenetic lineages and 3 strains from the M. africanum and M. bovis lineages), depicted by the SplitsTree software [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF] . The whole SNP dataset was originally generated for Supplementary Figure 1 of reference [START_REF] Boritsch | A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent[END_REF] , using genome data described by [START_REF] Blouin | Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti[END_REF][START_REF] Supply | Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis[END_REF]65 When bacteria were undetectable, the CFU value was set at the limit of detection (log 10 1.7). Non-significant by one-way ANOVA with the Bonferroni post hoc test. When bacteria were undetectable, the CFU value was set at the limit of detection (log 10 1.7). Using this route of infection and the C57BL/6 mouse strain, the bacterial burden in the lungs and spleen at day 56 post-infection was consistent with the enhanced persistence phenotype of KC9A1 when compared to STB-K. In NOS2 -/-mice, the persistence of STB-K in the spleen and KC9A1 in both organs were increased showing that NO is an important factor reducing STB-K and KC9A1 persistence.

Supplementary

Supplementary

However, the difference between STB-K and KC9A1 was maintained in NOS2 -/-mice, and even Supplementary Figure 12 These analyses identified about 30 different non-synonymous mutations in rv1339 in this dataset, which were phylogenetically linked in all cases, i.e. found in one (sub)lineage only. Of note, two frameshift-causing indels and one non-sense mutation were detected, which were however represented only in 2 isolates at most. These results indicate a strong conservation of the rv1339 gene among MTB isolates.

Supplementary Table 1: Mutations identified in STB-K 866

Cycle 3 867 STB-D deleted for the ppe25-ppe27 region DΔppe25-ppe27::EV PMM304 complemented with a derivative of the integrative plasmid pMV361 DΔppe25-ppe27::D PMM304 complemented with plasmid pWM385 containing ppe25-ppe27 of STB-D DΔppe25-ppe27::K PMM304 complemented with plasmid pWM361 containing ppe25-ppe27 of STB-K DΔppe25-ppe27::C1 PMM304 complemented with plasmid pWM386 containing ppe25-ppe27 of KC9C1

Conflict of interest

The authors declare no competing financial interests.