Fig 1.
General phenotypic characteristics of Mycobacterium strain FSD4b-SM.
A) Representative example of scant M. spongiae growth on simplified marine agar. B) Growth curve of M. spongiae in simplified marine broth. Each point is an average of measurements taken from three biological replicates (S2 Table). C) Ziehl-Neelsen stained M. spongiae cells. D) Electron micrograph of M. spongiae cells (x 33,000 magnification).
Fig 2.
Comparative genomics summary of Mycobacterium spongiae FSD4b-SM.
A) Pairwise average nucleotide identity (%ANI) between related Mycobacterium species. B) Maximum-likelihood phylogenetic tree (iqtree) with 1000 bootstrap iterations, inferred among 30 mycobacteria and based on amino acid sequence alignments from 107 conserved bacterial genes (bcgTree). M. smegmatis was used as an outgroup to root the phylogeny. Asterisk indicates node with >60% bootstrap node support. All other tree nodes had greater than 90% bootstrap support. MTBC encircled. Red branch length denotes M. spongiae FSD4b-SM placement. C) Upset plot showing shared coding sequences (CDS) at the 80% amino acid identity level between five related mycobacteria (Roary). D) Circos plot showing DNA sequence homology (Blastn) among M. spongiae and closely related species. Regions on the outermost ring represent the length of each individual genome, grey shading on the next innermost ring shows regions that are unique to M. spongiae among these comparator genomes. Links from the M. spongiae genome to other genomes show relative positions of regions encoding orthologues with >80% amino acid similarity. Link colours correspond to the colour of each genome on the outermost ring. E) Artemis Comparison Tool (ACT) plot showing comparative chromosome architecture and length between M. spongiae, M. marinum and M. tuberculosis. Links between the sequences denote regions with >75% nucleotide identity, with red links indicating the same DNA orientation, while blue links indicate inverse orientation with respect to each genome.
Fig 3.
Lipidomic analysis of M. spongiae.
A) MS/MS peak spot viewer of the M. spongiae extract in MS-DIAL showing retention time versus mass/charge ratio. Abundance (based on peak area) is shown as the colour of individual spots (blue = low, green = intermediate, orange = high abundance). Each lipid (sub)class is highlighted with a black circle. Note that not every spot within a circle belongs to the lipid (sub)class. B) Comparison of M. marinum and M. tuberculosis key membrane lipids with those predicted or detected from FSD4b-SM. The question mark above mycoketide for M. marinum indicates that the M. marinum genome contains a pks12 orthologue, but mycoketides have not been detected from M. marinum. AG: arabinogalactan; PG: peptidoglycan; PDIMs: phthiocerol dimycocerosates; PGLs: phenolic glycolipids; LOS: lipooligosaccharide.
Fig 4.
Construction of a recombinant M. bovis BCG with ESX-1FSD4b-SM and testing of its potential as an M. tuberculosis vaccine.
A) The FSD4b-SM ESX-1 locus was assembled from eight overlapping PCR products in a yeast-E.coli shuttle vector in Saccharomyces cerevisiae, followed by transfer and sub-cloning of the ESX-1 locus into a mycobacterial integrative vector in E. coli and subsequent transfer and integration into the M. bovis BCG chromosome. B) Western blot with anti-CFP-10 antibody showing detection of CFP-10FSD4b-SM from recombinant M. bovis BCG:ESX-1FSD4b-SM and M. tuberculosis H37Rv CFP-10, but not from empty vector containing M. bovis BCG:pYUB412. BCG:ESX-1FSD4b-SM 1 and 2 are two independent M. bovis BCG transformants. C) Establishment of a qPCR assay for detection of M. spongiae in mouse lung tissue. D) Mouse lung bacterial burden following intranasal inoculation of C57BL6 wild type mice with live M. spongiae. Shown are mean and SD at days 1, 7 and 56 post-infection (PI). Three mice were sacrificed at each time point and qPCR reactions were performed in technical triplicates. E) Vaccination trial using M. tuberculosis H37Rv infectious aerosol challenge. Mice were vaccinated intranasally with M. spongiae FSD4b-SM or either intranasally or subcutaneously with M. bovis BCG (BCG vax), or recombinant M. bovis BCG expressing ESX-1 from M. spongiae (rBCG::ESX-1FSD) and lungs were assayed for M. tuberculosis cells 4 weeks post challenge. Three mice were sacrificed in each group and M. tuberculosis counts were performed in duplicate. IN, intranasal; SC, subcutaneous.