Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of ‘ancient’ lineage 1 and prior to the Eurasian dispersal of ‘modern’ lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages – such as constitutive expression of nitrate reductase – as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host–pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

To explain the differences in morphology seen for L7 on these media we explored their contents. The base powders of Middlebrook 7H10 and Middlebrook 7H11 (both provided by BD) had the same composition except for the addition of 0.1% pancreatic digest of casein to Middlebrook 7H11 (Supplementary Table S4). Therefore, we propose that the added casein digest is the component in 7H11 that has a direct or indirect impact on the colony morphology of L7 strains leading to a raised crumpled structure on all tested 7H11 media types.

The impact of glycerol as a growth supplement
Independent of which Middlebrook medium (7H10 or 7H11 based) that was used in this study for comparing colony morphology of wild-type and complemented L7 strains, the colony size was consistently smaller for wild-type strains of L7 (mMA absent) as compared to L7 strains complemented with mmaA3 (mMA present). However, this difference could be reduced by adding glycerol to the growth medium, and especially when grown on 7H10. Fig. S4A shows the effect on morphology of BTBH-568 (L7) when a titration with glycerol at concentrations 0%, 0.5%, and 2.5% was performed on 7H10 agar. The colonies of all constructs (wild-type/mmaA3/EV control) increased significantly in size with increased glycerol concentration but complementation with mmaA3 had highest impact. These morphological effects were similar on both 7H10 and 7H11 (Supplementary Fig. S4A and S4B).
To investigate whether the effect of glycerol seen on solid medium could be observed also in liquid culture, we supplemented 7H9 liquid medium with 2% glycerol and performed TEM to explore whether it had an influence on L7 cell morphology. Indeed, the wild-type strain BTBH-568 (L7), which showed an abnormal phenotype when grown without glycerol (Fig. 3), displayed a cell morphology similar to reference strain H37Rv as well as BTBH-568 complemented with mmaA3, when grown with glycerol ( Supplementary Fig. S5).

The effect of glycerol and casein on L7 cell structure and colony morphology
Our experiments show that different media compositions (7H11 and 7H10 and supplementations thereof) influenced on the morphology, with glycerol and casein-digest being two media components that seemed to have a visual impact on the morphology (Supplementary Fig. S4A and S4B). An increased glycerol concentration in the agar medium compensated for the morphological artefact of the wild-type L7 strains, leading to larger colonies with eugonic features of the colonies. Interestingly, at cell-level the TEM results also indicated an effect when glycerol was added to the media ( Supplementary Fig. S5). The wild-type strain BTBH-568 of L7 showed a reverted cell morphology in the presence of glycerol. The reason for this may be that supplementation with glycerol as a carbon source influence the cell metabolism of the pathogen with altered cell-wall composition as a consequence, and/or because glycerol is taken up from the growth medium and directly incorporated into the cell envelope with structural effects. The same explanations can be proposed for the morphological difference when a pancreatic digest of casein was supplemented to 7H11 agar media.
Casein is a protein that has high proline content and a high degree of hydrophobic amino acids, and a digest of casein may provide the culture with peptides that have a direct or indirect effect on the cell and colony morphology of L7. It is also possible that glycerol can quench the effect seen by the caseindigest, through interaction with the same.
In conclusion, it can be proposed that the addition of glycerol or the absence of a casein-digest can restore the L7 morphology to an eugonic feature when grown on these media in vitro; however, these findings cannot clarify the possible role that mMA has in host-pathogen interactions and as a virulence factor in vivo.  Supplementary Fig. S1. Conserved Domain analysis of selected proteins from three gene clusters discussed in this study (Table 2 and Fig. 2A). Positions of the mutated amino acids conserved in L7 are highlighted by arrows. S1:A Rv3080c (pknK); S1:B Rv3089 (fadD13); S1:C Rv2931 (ppsA); S1:D Rv2940c (mas); S1:E Rv2946c (pks1); S1:F Rv2947c (pks15); S1:G Rv2952; S1:H Rv2962c; S1:I Rv0643c (mmaA3) glycerol. L7 strain BTBH-568 complemented with EV control and mmaA3, respectively, was used for this experiment in parallel to the reference strain Mycobacterium tuberculosis H 37 Rv. A Jeol 120 EX transmission electron microscope was used for analysis and images were acquired at 1,200x -4,000x magnification. Arrows highlight reduced cording phenotype of L7, suggesting an impaired cell envelope.