Genomic analysis of Mycobacterium brumae sustains its nonpathogenic and immunogenic phenotype

Abstract

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384^T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.

Keywords: diversity; immunogenic; non-pathogenic; nontuberculous mycobacteria; therapeutic.

Figure 1

Safety profile of Mycobacterium brumae. Comparison of the pathogenicity and toxicity between M. brumae and M. bovis BCG Connaught. Summary of the results obtained in different in vitro studies infecting macrophages (Noguera-Ortega et al., 2016b) and bladder cancer cell lines (Noguera-Ortega et al., 2016a), and in vivo studies in different animal models: intrahemacoelical infection of Galleria mellonella (Bach-Griera et al., 2020), intravesical instillations in orthotopic murine model of bladder cancer (Noguera-Ortega et al., 2016a,b,c), and intravenous infection in SCID mice (Bach-Griera et al., 2020). Blue rising arrow (high cell immune infiltration).

Figure 2

Circular representation of the Mycobacterium brumae chromosome. The outermost ring shows the chromosome length in megabases, followed by the forward (blue) and reverse genes (yellow). SNPs in M. brumae strains (CR269, CR142, and CR103) with respect to the ATCC 51384^T reference genome is indicated in the next three rings, respectively. Moving inward, the next ring shows the GC content, with values over the mean filled in red and values below the mean in green. The last ring shows the GC skew (G − C)/(G + C) using a 20-kb window.

Figure 3

Gene annotation comparison of COG distributions shared in Mycobacterium brumae and in Mycobacterium tuberculosis H37Rv. COG refers to Clusters of Orthologous Groups. The vertical axis shows the percentage of genes in each category. The different categories are represented on the horizontal axis and the legend indicates the correspondence with each COG category. The corresponding strain for each bar is indicated in stripe pattern for M. tuberculosis H37Rv and in plain pattern for M. brumae. Fisher test p values for each category indicated as following: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; and ^****p < 0.0001.

Figure 4

Phylogenetic relationship of Mycobacterium brumae within the Mycobacterium genus. Maximum likelihood phylogenetic tree of type strains from the Mycobacterium genus based on an alignment with 177 core genes, highlighting the position of M. brumae ATCC 51384^T type strain. GTR model was used with bootstrap confidence of 1,000 replicates. Branch lengths are proportional to nucleotide substitutions and the topology is rooted with Hoyosella subflava. The phylogenetic tree was annotated with the pathogenicity, the ESX-1 presence or absence, and the growth rate (see Methods).

Figure 5

Genes presence or absence analysis. Venn diagram showing the comparison of the presence/absence and abundance of M. tuberculosis virulence-associated genes (A), antigens recognized by T cells (B), and antigens recognized by B cells (C) in the M. bovis BCG Connaught and M. brumae genomes.

Figure 6

Immunogenic capacity of Mycobacterium brumae compared to Mycobacterium bovis BCG Connaught. Summary of the results obtained in studies treating with both mycobacteria J774 macrophages cell line (Noguera-Ortega et al., 2016b), bladder cancer cell lines (Noguera-Ortega et al., 2016a), human peripheral mononuclear cells (Noguera-Ortega et al., 2016b), and murine bone-marrow macrophages (unpublished); and after treating intravesically with each mycobacteria bladder cancer tumor-bearing mice (Noguera-Ortega et al., 2016a,b, 2018). Blue rising arrow indicates higher than M. bovis BCG Connaught, red falling arrow indicates lower than M. bovis BCG Connaught and the equals symbol indicates similar to M. bovis BCG Connaught.