New Results

A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region

Jean Claude Semuto Ngabonziza, Chloé Loiseau, Michael Marceau, Agathe Jouet, Fabrizio Menardo, Oren Tzfadia, Esdras Belamo Niyigena, Wim Mulders, Kristina Fissette, Maren Diels, Cyril Gaudin, Stéphanie Duthoy, Willy Ssengooba, Emmanuel André, Michel K Kaswa, Yves Mucyo Habimana, Daniela Brites, Dissou Affolabi, Jean Baptiste Mazarati, Bouke Catherine de Jong, Leen Rigouts, Sebastien Gagneux, Conor Joseph Meehan, Philip Supply
doi: https://doi.org/10.1101/2020.01.20.912998

Abstract

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have clonally expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug-resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

Introduction

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTBC), is among the most ancient scourges of humankind1, and remains the leading cause of mortality globally due to an infectious disease2. Intense research has been dedicated to decipher the evolutionary history of the MTBC and to understand the causes underlying the worldwide spread of TB3–5. Current genome data show that the MTBC is comprised of two main branches, one including the five human-adapted lineages representing M. tuberculosis sensu stricto (L1– 4, and L7), and the second branch comprising two other human-adapted lineages traditionally referred to as M. africanum (L5-6) and at least nine animal-adapted lineages6. Africa is the only continent where all MTBC lineages are present, suggesting that the MTBC emerged from a common ancestor therein and then clonally expanded to the rest of the world following human migrations3, 7, 8. However, the genomic traits of this common ancestor and the region from which this expansion took place in Africa remain unknown. Whole genome sequencing (WGS) analyses showed that rare human TB bacilli with a smooth colony morphotype, highly restricted to the Horn of Africa and named Mycobacterium canettii (alias smooth tubercle bacilli or STB) represent early evolutionary branching lineages that predate the emergence of the most recent common ancestor (MRCA) of the MTBC (or of the rest of the MTBC, if M. canettii is considered to be part of the complex)4, 9, 10 Indeed, whereas known MTBC strains differ by no more than ∼2,000 Single Nucleotide Polymorphisms (SNPs)11, M. canettii strains are 10 to 25-fold more genetically diverse and separated by at least 14,000 SNPs from the hitherto known MTBC MRCA4, 10. Moreover, M. canettii strains are less virulent and possess highly mosaic genomes, possibly reflecting primal adaptation to an environmental reservoir favouring active lateral gene flow4, 12, 13. These biological differences support the existence of intermediate lineages in the evolution from M. canettii towards the obligate MTBC pathogens. Here, we describe two exceptional strains representing a new, third main branch, diverging before the MRCA of the other MTBC lineages. These two strains, isolated in Rwanda and Uganda, respectively, were discovered in two independent analyses, and were both multidrug-resistant (MDR; i.e. resistant to at least rifampicin and isoniazid). We used PacBio and Illumina WGS to reconstruct the full circular genome and reconstitute the phylogeny of this novel lineage, which we named Lineage 8 (L8), and further investigate molecular and evolutionary events associated with the emergence of the MTBC.

Results

L8 related TB patient in Rwanda

The strain in Rwanda was isolated from a male patient, aged 77 years, HIV-negative, resident of Rulindo district bordering with the Southwest of Uganda, and who had lived in Uganda previously. The patient was diagnosed with rifampicin-resistant TB and the Xpert MTB/RIF assay (Xpert; Cepheid, Sunnyvale, CA, USA) showed a rare delayed probe B reaction (∼3% prevalence in Rwanda)14, later confirmed (see below) to be due to the Asp435Tyr mutation in the rpoB gene15, 16.

Per routine practice, the patient was initiated on standard short-course MDR-TB treatment17. However, the patient developed hypotension, and eventually died due to probable cardiac failure, after 20 days of treatment. Phenotypic drug-susceptibility testing (DST) confirmed resistance to both rifampicin and isoniazid, and susceptibility to other anti-TB drugs including ethambutol, fluoroquinolones, and second-line injectables.

Growth characteristics and biochemical properties of the Rwandan strain

The strain from Rwanda was grown in 12.5 days on Mycobacterial Growth Indicator Tubes. Colonies were observed on the fifth week after initial inoculation on Löwenstein-Jensen medium, indicating a slow grower phenotype with rough colonies (Figure 1). The strain also displayed archetypal biochemical characteristics of M. tuberculosis sensu stricto, including niacin production combined with urease hydrolysis (Table 1).

Figure 1.
Figure 1.

Microscopic image of L9 on Dubos agar medium showing typical rough colonies (read at 100x).

View this table:
Table 1.

Standard biochemical characteristics of selected mycobacterial species or M. tuberculosis complex lineages/subspecies versus L8

Genotypic resistance and SNP profile of the Rwandan strain by Deeplex Myc-TB

Following the MDR-TB diagnosis, the strain was included in the first set of tests for an ongoing MDR-TB diagnostic trial “DIAgnostics for MDR-TB in Africa (DIAMA) Clinicaltrials.gov, NCT03303963”, evaluating a new targeted deep sequencing assay, called Deeplex-MycTB (GenoScreen, Lille, France). Deeplex-MycTB testing confirmed the presence of the rpoB Asp435Tyr mutation conferring rifampicin resistance, along with the inhA Ser94Ala mutation conferring isoniazid resistance, consistently with the MDR phenotype identified by phenotypic DST (Figure 2). This strain also harboured two alleles in phylogenetic positions in embB (Ala378) and gidB (Ala205) not associated with resistance to ethambutol or streptomycin, which were both shared by several MTBC lineages (L1, 5, 6, 7, and animal lineages) and M. canettii18. In addition, nine other - so far uncharacterized - SNPs were identified in six of the 18 gene targets interrogated by the assay (Figure 2). Moreover, this test detected an atypical spoligotype pattern, 1111100000000000000000000000000001110000000 (Figure 2), which was further confirmed by conventional membrane-based spoligotyping testing. This spoligotype pattern was unique in the global spoligotype database that comprises 111,637 MTBC isolates from 131 countries19.

Figure 2.
Figure 2.

Deeplex-MycTB results identifying a multidrug resistant strain from Rwanda with an atypical genotypic profile in the M. tuberculosis complex. Target gene regions are grouped within sectors in a circular map according to the tuberculous drug resistance with which they are associated. The two sectors in red indicate regions where rifampicin and isoniazid resistance associated mutations are detected. The multiple sectors in blue refer to regions where as yet uncharacterized mutations are detected, while sectors in green indicate regions where no mutation or only mutations not associated with resistance (shown in gray around the map) were detected. Green lines above gene names represent the reference sequences with coverage breadth above 95%. Limits of detection (LOD) of potential heteroresistance (reflected by subpopulations of reads bearing a mutation), depending on the coverage depths over individual sequence positions, are indicated by grey (LOD 3%) and orange zones (variable LOD >3%–80%) above the reference sequences. Information on an unrecognized spoligotype, an equivocal SNP-based and on mycobacterial species identification, based on hsp65 sequence best match, are shown in the centre of the circle. *AMI, amikacin; BDQ, bedaquiline; CAP, capreomycin; CFZ, clofazimine; EMB, ethambutol; ETH, ethionamide; FQ, fluoroquinolones; KAN, kanamycin; LIN, linezolid; INH, isoniazid; PZA, pyrazinamide; RIF, rifampin; SM, streptomycin; SIT, spoligotype international type.

WGS analysis and phylogenetic position of the Rwandan and Ugandan strains

Results from WGS analysis of the Rwandan strain using Illumina sequencing confirmed all Deeplex-MycTB findings.

The strain isolated in Uganda was discovered independently upon screening global, publicly available genome datasets, where it was misclassified as M. bovis isolated from a human patient20. These WGS data revealed a similar spoligotype 1111100000000000000000000000000001111000111, characterized by the presence of spacers 1 to 5 and 34 to 37 (vs 34-36 in the Rwandan strain) with all intervening spacers missing. Moreover, the Ugandan strain also shared the same rpoB Asp435Tyr and inhA Ser94Ala mutations and the same sequence alleles in embB and gidB. The Ugandan strain contained an additional katG Ser315Thr mutation conferring isoniazid resistance, as well as the embA C-11A and embB Asp328Tyr mutations, and two pncA missense mutations, predictive of pyrazinamide resistance. Moreover, only three of the nine aforementioned uncharacterized SNPs detected by Deeplex-MycTB were shared between both strains.

To further assess the relationships between both strains and in comparison to other MTBC strains, a maximum likelihood phylogeny was inferred from 241 MTBC genomes, including representatives of all known human- and animal- adapted lineages6 and using a M. canettii strain as an outgroup. This reconstruction revealed a unique phylogenetic position of the two new genomes from Rwanda and Uganda (Figure 3), representing a newly characterised monophyletic clade in which none of the known MTBC genomes are contained. Based on the phylogeny, this clade shares a MRCA with the rest of the MTBC, thus representing a new sister clade to the known MTBC, which we named Lineage 8 (L8). Comprehensive SNP analysis identified a total of 189 SNPs separating both genomes, which is within the range of zero to 700 SNPs found between any two strains within any of the lineages 1 to 7 of the MTBC11. The absence of any matching pattern in the global spoligotype database, as well as the lack of detection of this clade in previous large WGS datasets of MTBC strains from global sources, indicate that L8 is generally rare and geographically restricted to the African Great Lakes region. Specifically, the L8 spoligotype signature and the 3 SNPs specifically shared by both L8 strains were not detected in any of 115 MTBC genomes from a previous drug resistance survey in Uganda21, nor in 380 rifampicin-resistant strains from Rwanda collected between 1991 and 2018, from routine drug resistance surveillance as well as various drug resistance surveys22–24. Furthermore, among 14 other isolates out of 27 from Uganda and Rwanda tested by Gene Xpert MTB/RIF that showed the same delayed probe B as L8, none displayed the L8 signatures when tested by Deeplex Myc-TB or by classical spoligotyping. Likewise, none of > 1,500 clinical samples from TB patients tested by Deeplex-MycTB from a recent nationwide drug resistance survey performed in Democratic Republic of Congo displayed the L8 spoligotype signature or the specific SNPs (data not shown).

Figure 3.
Figure 3.

Maximum likelihood phylogeny of 241 MTBC genomes, inferred from 43,442 variable positions. The scale bar indicates the number of substitutions per polymorphic site. Branches corresponding to human-adapted strains are coloured and branches corresponding to animal-adapted strains are depicted in black. The phylogeny is rooted on M. canettii and bootstrap values are shown for the most important splits.

Defining features of a complete L8 genome

To further assess the sister position of L8 and split from the remaining MTBC inferred from SNP analysis, the Rwandan strain was subjected to long read-based PacBio sequencing. Comparison of the obtained assembly with 36 available complete genomes of MTBC members, comprising L1-L4 (including H37Rv), M. africanum (L6) and M. bovis strains, showed a highly syntenic organization, with no major structural rearrangement between both groups. Although the assembled L8 genome of 4,379,493 bp was within the 4.34-4.43 Mb size range of the other MTBC genomes, it was 30 kb smaller than the 4.41-Mb mean size of genomes of M. tuberculosis sensu stricto25. However, the largest part of this gap was accounted by the absence of three genomic regions in L8, corresponding to regions of difference (RDs) known to be variably present or absent in other MTBC (sub)lineages26, 27(Supplementary Table 2). These include a 9.3-kb PhiRv1 prophage region (RD3), as well as 10.0-kb and 8.5-kb segments corresponding to RD14 and RD5, comprising the plcABC gene cluster and the plcD gene regions, respectively26. In L8, each of the two latter regions only contained one copy of the IS6110 insertion sequence, devoid of direct repeats (DRs) that normally flank IS6110 copies after transposition, indicating that these deletions in L8 resulted from recombination between two adjacent IS6110 copies with loss of the intervening sequences28. These mobile DNA-related deletions, which also arose independently in several other MTBC branches26, 29, probably occurred after the divergence of L8 from the other MTBC lineages.

Conversely, a particular 4.4 kb genome region was present in both genomes of L8 and M. canettii, but absent in all other known members of the MTBC (Supplementary Table 3). This region comprises the cobF gene (Figure 4), encoding the precorrin 6A synthase involved in the cobalamin/vitamin B12 synthesis, along with two other genes, respectively encoding a PE-PGRS protein family member and a protein of unknown function. This region is present in the M. canettii genomes, as well as in the phylogenetically proximal non-tuberculous mycobacterial species M. marinum and M. kansasii (Supplementary Table 3). This ancestral locus was thus most likely lost in the MRCA of the other MTBC lineages, after its divergence from L8. However, none of the almost 900 other genes specifically identified in the M. canettii genomes, and absent in the other MTBC genomes, were found in the L8 genome, supporting the close relationship with the previously known MTBC branches indicated by the SNP-based phylogeny.

Figure 4.
Figure 4.

Aligned genome segments showing the cobF gene region in M. tuberculosis L8, M. canettii CIPT140010059 (alias STB-A), M. kansasii ATCC12478 and M. marinum M strains, and the corresponding deletion in M. tuberculosis H37Rv, M. bovis AF2122/97, and M. africanum GM041182. Coding sequences of this region are shown in green, and flanking coding sequences in red. Sequences flanking the deletion point in truncated genes in M. tuberculosis, M. africanum and M. bovis, and in the cobF region present in M. canettii, M. kansasii and M. marinum are indicated in red and black, respectively. Dashed lines correspond to missing segment parts relatively to the longest segment found in M. marinum.

Further evidence for the early branching of L8 relative to the rest of the MTBC comes from examination of interrupted coding sequences (ICDSs), putatively reflecting molecular scars inherited during progressive pseudogenization of the MTBC genomes30, 31. Four orthologues of MTBC ICDSs were previously found to be intact in the genomes of M. canettii strains, as well as in M. marinum and M. kansasii 4. One of these four orthologues (pks8), which belongs to a multigene family encoding polyketide synthases involved in the biosynthesis of important cell envelope lipids32, was also intact in the genomes of both L8 strains (Figure 5 and Supplementary Table 4). Moreover, we found an additional orthologue of MTBC ICDSs (i.e. rv3899c-rv3900c), coding for a conserved hypothetical protein, which was intact in the genomes of M. canettii, M. kansasii, M. marinum and both L8 strains (Supplementary Table 4). These two molecular scars were also likely acquired by the other MTBC lineages after their divergence from the common progenitor shared with L8.

Figure 5.
Figure 5.

Aligned genome segments showing the interrupted coding sequences pks8/17 in M. tuberculosis H37Rv and M. bovis AF2122/97 gene region, and complete pks8 genes in L8, M. canettii CIPT140010059 (alias STB-A) and M. kansasii ATCC12478. pks8/17 and pks8 coding sequences are shown in green, and flanking genes in red. Sequences flanking the 1-nucleotide deletion and resulting in a frameshift in M. tuberculosis complex strains are indicated. Dashed lines correspond to missing segment parts relatively to the longest segment found in M. canettii.

The assembled L8 genome also included 48 out of 50 genes (the exception are rv3513c encoding the probable fatty-Acid-Coa ligase FadD18 and the PhiRv1 region; see above) present in MTBC members but not found in any of the STB genomes, including a number of genes putatively acquired through horizontal gene transfer by the common ancestor of the MTBC after its separation from M. canettii4 (Supplementary Table 5). Likewise, consistent with the rough colony morphotype of the Rwandan strain, both L8 strains displayed the single polyketide-synthase-encoding pks5 gene configuration shared by all MTBC members, instead of the dual pks5 conformation found in M. canettii strains involved in the smooth colony phenotype of the latter strains12. Thus, the recombination between the two pks5 genes and the loss of the intervening pap gene, thought to have resulted in surface remodelling and incremental gain of virulence after the phylogenetic separation from M. canettii12, already existed in the common progenitor of L8 and the rest of the MTBC. Moreover, both L8 strains also contained the intact TbD1 and RD9 regions, shared by the other “ancestral” M. tuberculosis lineages (L1, L7) but subsequently lost by the so-called “modern” lineages of M. tuberculosis (TbD1 lost in L2-4), M. africanum (L5 and L6) and the animal lineages (RD9)26.

In contrast to the highly clonal structure of the MTBC, M. canettii strains are highly recombinogenic, as apparent from mosaic sequence arrangements in their genomes and functional DNA transfer between M. canettii strains mediated by a distributive conjugal transfer (DCT)-like mechanism4, 33. However, no significant genome-wide recombination signal was detected by ClonalFrameML analysis34 between L8 and other MTBC strains (data not shown).

Discussion

The discovery of L8 provides unique insights into an ancestor of the MTBC that existed after the pks5-recombination-mediated surface remodelling, which occurred after separation of the MTBC MRCA from the M. canettii clade, but preceded the loss of the cobF region and gene interruptions in a later common ancestor of the other MTBC lineages. The seeming restriction of this lineage to the African Great Lakes region represents new evidence supporting an origin for the MTBC in the eastern part of the African continent. These findings reinforce results from previous work suggesting an East-rather than a West African origin of the MTBC3, 4, 8, 9, 35.

A distinct ecological niche, linked to a potential environmental reservoir, has been hypothesized to explain the marked geographic restriction of M. canettii strains to the Horn of Africa, the lower persistence of these strains in infection models as well as their genome mosaicism implying multiple DNA recombination events within the M. canettii strain pool4, 10. However, our results indicate L8 is as clonal as the rest of the MTBC3, 13, 29, 36. Moreover, multi-drug resistance in both L8 isolates, and their detection in human patients in both cases (with reported absence of previous TB history for the Rwandan patient), suggests prolonged exposure to antibiotic treatment, and human-to-human transmission of a drug-resistant strain, rather than infection from a non-human source. While based on only two initial strains, these results are consistent with the presumed scenario of a human rather than a zoonotic origin for the TB disease26, 37.

The observation that both L8 strains share two uncommon rifampicin- and isoniazid-resistance conferring mutations in rpoB and inhA suggests that multidrug resistance was already acquired in their common ancestor. Isoniazid and rifampicin were introduced in TB treatments in Rwanda and Uganda in the late fifties and early nineties, respectively. Therefore, the ∼100 SNPs distance separating these two strains from their MRCA would imply a rapid molecular clock for L8, above the upper limit of 2.2 SNPs/genome/year most recently estimated for other MTBC clades38. However, this mutation rate cannot be confirmed until additional L8 samples are uncovered.

Remarkably, the absence of other L8 strains in datasets from Uganda, Rwanda and DRC, together comprising more than 2,000 strains, suggests that L8 is rare even within the African Great Lakes region. Such scarcity is compatible with selective sweeps of later branching MTBC strains, introduced more recently into the region. Similar scenarios have also been proposed to explain the slow apparent replacement of MTBC L5 and L6 by L4 in West Africa39–41 and the restriction of L7 to Northern Ethiopia42.

Loss-of-function linked to the deletion of cobF is a plausible candidate molecular event involved in such a replacement scenario for L8. Indeed, loss-of-function appears to be an important mechanism driving the pathoadaptive evolution of the TB pathogen, as shown for the role of the loss of lipo-oligosaccharide production (via recombination in the pks5 locus)12 in the evolution towards increased virulence from M. canettii to MTBC strains. Likewise, loss of secretion of PPE-MPTR and PE_PGRS proteins by the type VII secretion system ESX-5 (via mutations of the ppe38 locus) has been involved in the hypervirulence of recent branches of L2 (alias “modern” Beijing) strains43. The loss of the cobF region in the other MTBC lineages, inferred from comparative genomics with M. canettii and non-tuberculous mycobacteria4, was previously hypothesised to reflect enhanced adaptation to an intracellular parasitic lifestyle44. Indeed, the cobalamin/vitamin B12 synthesis pathway, of which the cobF-encoded precorring-6a synthase is a component, represents a highly complex and energy consuming process with about 30 enzymatic steps45. While the absence of this component may not entirely ablate cobalamin biosynthesis46, 47, its loss might have resulted in gain of fitness and reflect enhanced pathogenic professionalisation, by economical reliance upon the mammalian host environment as source of vitamin B12. If true, more recently emerged or introduced, cobF-deleted strains might conceivably have largely outcompeted L8 strains.

Our genomic data, on an as-yet-unknown ancestral stage between the MTBC and the putative progenitor pool of M. canettii-like mycobacteria, suggest further experiments to examine candidate molecular events potentially involved in the pathoadaptive evolution of M. tuberculosis. The discovery of such rare strains raises the possibility for the existence of further extant strains, especially in Eastern Africa, representing other clades further closing the biological gap between the MTBC and M. canettii.

Methods

Phenotypic characterization

We studied conventional mycobacterial growth and biochemical characteristics including colony morphology, niacin production, nitrate reduction, p-nitro benzoic acid growth inhibition, catalase production, urea hydrolysis, tween 80 hydrolysis, and thiophene carboxylic acid hydrazide growth inhibition48. For comparative purpose, a reference set of the seven known human-adapted MTBC lineages49, together with M. canettii (BCCM/ITM2018-C02321), M. bovis (BCCM/ITM960770), M. bovis BCG (BCCM/ITMM006705), and M. orygis (BCCM/ITM2018-01492) strains were processed with the novel strain isolated in Rwanda. Moreover, phenotypic drug susceptibility testing to first- and second-line anti-TB drugs was done using the proportion method50.

Targeted and whole genome sequencing

For targeted sequencing using the Deeplex-MycTB assay16 and short-read Illumina-based WGS, a bead beating method was used to extract DNA from colonies (Supplementary method 1). Libraries of Deeplex-MycTB amplicons or genome fragments were constructed using the Nextera XT kit and sequenced on an Illumina MiSeq platform with paired end, 150-bp read lengths (Illumina, CA, USA). DNA extraction suitable for PacBio SMRT sequencing was performed using the Genomic DNA Buffer Set (Qiagen Inc, Germantown, Maryland, USA) (Supplementary method 2). Sequencing was performed on a PacBio RS II using the SMRT technology.

Deeplex-MycTB analysis and spoligotyping

Analysis of the Deeplex-MycTB sequencing data, including SNP calling and spoligotype identification, was performed by read mapping on M. tuberculosis H37Rv sequence references, using a parameterized web application (GenoScreen)16. Membrane-based spoligotyping was performed as described previously51.

Illumina whole genome sequencing analysis

Raw genomic reads from the newly sequenced L8 genome from Rwanda and the L8 genome from Uganda (SAMN02567762) were processed as previously described52. Briefly, the reads were trimmed with Trimmomatic v0.33.2253 and reads larger than 20 bp were kept. The software SeqPrep (https://github.com/jstjohn/SeqPrep) was used to identify and merge any overlapping paired-end reads. The resulting reads were aligned to the reconstructed ancestral sequence of the MTBC54 using the mem algorithm of BWA v0.7.1355. Duplicated reads were marked using the MarkDuplicates module of Picard v2.9.1 (https://github.com/broadinstitute/picard) and local realignment of reads around InDels was performed using the RealignerTargetCreator and IndelRealigner modules of GATK v3.4.056. SNPs were called with Samtools v1.2 mpileup57 and VarScan v2.4.158 using the following thresholds: minimum mapping quality of 20, minimum base quality at a position of 20, minimum read depth at a position of 7X, maximum strand bias for a position 90%.

The spoligotype pattern of the strain from Uganda was extracted in silico from the raw reads using kvarQ59.

Phylogenetic reconstruction

The maximum likelihood phylogeny was inferred with RAxML v.8.2.860 using an alignment containing only polymorphic sites and the branch lengths of the tree were rescaled using invariant sites (rescaled_branch_length = (branch_length * alignment_length) / (alignment_length +invariant_sites))38, 61.

A position was considered polymorphic if at least one genome had a SNP at that position. Deletions and positions not called according to the minimum threshold of 7x were encoded as gaps. We excluded positions with more than 20% missing data, positions falling in PE-PGRS genes, phages, insertion sequences and in regions with at least 50 bp identity to other regions in the genome. We also excluded variable positions falling in drug resistance-related genes. The phylogeny was computed using the general time-reversible model of sequence evolution (-m GTRCAT -V options), 100 bootstrap inferences and M. canettii (SRR011186) was used as an outgroup to root the phylogeny.

Whole genome de novo assembly, annotation and comparative genomics

Raw PacBio reads obtained from the Rwandan strain were assembled with Canu v1.662, using default settings and an expected genome size of 4.4 Mbp, typical of MTBC strains. After discarding 60,272 reads below minimal quality parameters, 106,681 reads were used for the assembly, resulting in mean coverage of 186x, 39x and 38x, after read correction, trimming, and unitigging, respectively. The obtained unique contig of 4,387,285 bp was circularized with Circlator v1.5.563 using default settings, resulting in an assembly of 4,379,493 bp. Additional sequence verification and correction was then performed by mapping Illumina reads obtained from the same strain, using pacbio-utils version 0.264 (https://github.com/douglasgscofield/PacBio-utilities) and snippy version 4.465 (https://github.com/tseemann/snippy). Alignments of the final assembly were performed against an ensemble of complete genome sequences available from 38 strains of tubercle bacilli. This set included 34 M. tuberculosis strains from lineages 1, 2, 3 and 4 (comprising H37Rv), M. africanum L6 GM041182, M. bovis AF2122/97, as well as the closest STB-A (CIPT 140010059) and most distant (STB-K) M. canettii strains (Supplementary Table 1). Comparative alignments and genome annotation were performed based on BLAST searches and analysis of gene synteny, using Artemis and Artemis comparison tool66, as well as a custom Multiple Annotation of Genomes and Differential Analysis (MAGDA) software previously used for annotation of M. canettii and Helicobacter pylori genomes4, 67. Comparisons with orthologues from M. canettii STB-D, -E, -G, -H, -I, and -J in addition to STB-A and -K, and from M. marinum type strain M and M. kansasii genomes were additionally done using the Microscope platform v3.13.368. When applicable, annotations were transferred from those of M. tuberculosis or M. canettii orthologs in the TubercuList/Mycobrowser database, using BLAST matches of > 90% protein sequence identity, an alignable region of >80% of the shortest protein length in pairwise comparisons and visual inspection of the gene synteny. ACT comparison files were generated using MAUVE 2015-02-25 software to visualize the genome-wide distribution of SNP densities between the assembled L9 genome from Rwanda and M. tuberculosis H37Rv and M. canettii STB-A and STB-K genomes. Recombination between L8 and other MTBC lineages or M. canettii was assessed from a progressive MAUVE alignment of the PacBio assembled L8 genome and previously published closed genomes65 using ClonalFrameML34.

Accession codes

The complete genome sequence of the L8 strain from Rwanda was deposited in the NCBI repository under project PRJNA598991 with SRR10828835 and SRR10828834 accession codes for Illumina- and PacBio-derived genome sequences, respectively.

Author contributions

S.G., P.S., C.M., B.C.d.J., L.R. and J.C.S.N. designed the study. P.S., J.C.S.N., C.L., M.M., C.M. and S.G. analyzed data and wrote the manuscript, with comments from all authors. A.J. and C.M. performed the assembly of sequences. M.M. annotated the L9 genome. C.L., F.M., D.B. and A.J. performed SNP analyses and phylogenetic reconstruction. M.M. and P.S. conducted comparative analyses of complete mycobacterial genomes, with support from C.L. and O.T. J.C.S.N., E.B.N., I.M.H., J.B.M., W.M., K.F. and M.D. performed and/or analyzed data from mycobacterial isolation, growth assays, phenotypic characterization and/or molecular tests. S.D., C.G., J.C.S.N., E.B.N., E.A. and M.K.K. conducted targeted deep sequencing analyses. S.D., C.G. and W.S. L. Majlessi, F.S., C. Locht and C. Leclerc conducted and/or analyzed immune assays. J.T., A. Criscuolo and S.B. conducted MLST, recombination and/or phylogenetic analyses. L.F. conducted histopathological analyses. V.K., M.O. and C.P. created bioinformatics tools and analyzed data. M.F. isolated STB strains. T.S. and T.P.S. conducted core genome and NeighborNet analyses.

Acknowledgements

Calculations were partially performed at sciCORE (http://scicore.unibas.ch/) scientific computing core facility at University of Basel. This work was supported by EDCTP2 grant DRIA2014-326—DIAMA of the European Union, the Belgian General Directorate for Development Cooperation (PhD fellowship to JCSN), grant ANR-16-CE35-0009 from Agence Nationale de la Recherche, the Swiss National Science Foundation (grants 310030_188888, IZRJZ3_164171, IZLSZ3_170834 and CRSII5_177163), and the European Research Council (309540-EVODRTB).

The views and opinions of authors expressed herein do not necessarily state or reflect those of EDCTP. P.S., C.G. and S.D. declare the following competing interests: P.S. was a consultant of Genoscreen; C.G. and S.D. were employees of the same company. The other authors declare no competing interests. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Footnotes

  • ↵* Joint senior authors

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A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region
Jean Claude Semuto Ngabonziza, Chloé Loiseau, Michael Marceau, Agathe Jouet, Fabrizio Menardo, Oren Tzfadia, Esdras Belamo Niyigena, Wim Mulders, Kristina Fissette, Maren Diels, Cyril Gaudin, Stéphanie Duthoy, Willy Ssengooba, Emmanuel André, Michel K Kaswa, Yves Mucyo Habimana, Daniela Brites, Dissou Affolabi, Jean Baptiste Mazarati, Bouke Catherine de Jong, Leen Rigouts, Sebastien Gagneux, Conor Joseph Meehan, Philip Supply
bioRxiv 2020.01.20.912998; doi: https://doi.org/10.1101/2020.01.20.912998
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