Despite M. simiae being one of the most relevant NTM due to its emergence as a human pathogen, the genomic features and genetic potential of this species remain poorly described. In collaboration with the State Laboratory of Public Health of Guanajuat, located in central Mexico, we had access to a clinical isolate of M. simiae, termed MsiGto. The sample was isolated from a 90 years old female patient from Leon, the largest and most industrialized city of the State of Guanajuato, Mexico, in 2011 (Table 2). This sample was selected for genomic sequencing due to the emerging clinical importance of strains from the M. simiae complex worldwide, combined with a lack of representative genomic sequences from Mexico to this date. The available genome sequences could allow comparative analyses in order to increase our understanding of this opportunistic bacterium.
MsiGto was isolated from a sputum specimen. Briefly, 2 mL sample were transferred to a sterile tube and decontaminated by adding an equal volume of 4% NaOH with phenol red. The mix was immediately vortexed and incubated at 37  C for 15 min. The liquefied sample was centrifuged at 3000 g at 4  C for 15 min, the supernatant was discarded and pH was adjusted between the 6.5 and 7.2 range using 1 N HCL. Finally, 0.2 mL of sample were inoculated into Lowenstein-Jenssen slants (Difco) and incubated at 37  C for three weeks.
Biomass was collected and suspended in phosphate buffered saline solution, pH 7.4, and inactivated by heating at 80  C for 45 min. After centrifugation, genomic DNA was extracted from the pellet using the FastDNA SPIN Kit for Soil (MP Biomedicals) according to manufacturer's instructions. Extracted gDNA was assessed for quantity and quality using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).
The MsiGto genome was sequenced on a HiSeq 2000 platform with a 100 bp paired-end cycle according to standard Illumina protocols, at Macrogen facilities. Quality of the generated sequencing reads (13,676,836 reads, total read length: 1,381,360,436 bp) was checked with FastQC. Reads were filtered before assembly, such that both sequences in paired-end reads exhibited more than 90% bases of quality greater than or equal to Q20. Post-filtering Q20% was 97.69 and Q30% was 88.6 at the base level. The filtered reads were assembled using SOAP de novo aligner, yielding 50 contigs. Scaffolding was performed with SSPACE Standard and resulted in 12 scaffolds, generating a genome size of 6.7 Mb with a coverage of 216X (Table 3).
Gene prediction and functional annotation were performed using the Rapid Annotation using Subsystem Technology platform. A prophage region prediction was completed using PHAST. CRISPRs were searched using the CRISPR finder, and antibiotic resistance genes were investigated using Resistance Gene Identifier from the Comprehensive Antibiotic Resistance Database. Genome-to-genome comparisons were performed using multiple approaches, including conservation analysis of protein families across genomes with the Protein Family Sorter from the PATRIC Platform, and comparing functionally related clusters using the function-based comparison of the RAST server. MsiGto and related genomes were also aligned using a genome-wide BLAST comparison, and visualized through the Artemis Comparative Tool for manual inspection.
Genomic assembly yielded a total length of 6,684,413 bp fragmented in 50 contigs, the largest M. simiae genome reported to date. The MsiGto genome consists of a unique chromosome, as no plasmid DNA was found. The GC content of the genome was 66.08%, consistent with other reported M. simiae strains. Gene prediction analysis documented 3 rRNAs, 49 tRNAs and 6409 coding sequences. Out of these, 4462 genes (69.62%) were assigned to putative functions, and 3669 genes (approximately 62.57%) were assigned to clusters of orthologous groups functional categories. Sequence searches using RGI evidenced resistance genes to amynoglycosides, ethambutol and beta lactams, which is a concern as to date standard antibiotic regimes for this species include the use of ethambutol, the aminoglycoside amikacin, as well as the macrolides azithromycin and clarithromycin. The genome properties and statistics are summarized in Tables 4 and 5. Gene distribution among the COG functional categories is shown in Table 6. A circular map of MsiGto chromosome is provided in Fig. 2.
We report here the third genome sequence of M. simiae. MsiGto was isolated from a clinical sample obtained from an elderly woman, and is the largest M. simiae genome reported to date with an approximate 745 Kbp additional to the next largest genome (strain MO323). As pathogenic mycobacteria tend to undergo genome decay, the large genomic size of MsiGto speaks out of an organism capable of thriving in both environmental conditions and in the human host. Indeed, by comparison, the genome of M. tuberculosis, an obligate pathogen, is significantly smaller with 4.41 Mb. Meanwhile, with 6406 genes, the genome size of MsiGto is similar to that of the opportunistic pathogen Pseudomona aeruginosa (5570 predicted ORFs), which thrives in a variety of environments including soil, water, as well as the multiple human tissues it infects. Analysis of the P. aeruginosa genome evidenced its large size arose from genetic expansion to enhance functional diversity rather than from gene duplication. Interestingly, investigating protein families in MsiGto, in comparison with MO323 and DSM 44165, evidenced a larger proportion of proteins uniquely found in MsiGto (1.17 times more than DSM 44165 and 4.7 times more than MO323), suggestive of a relatively increased versatility in accordance with the multiple niches it can thrive in (Fig. 3).
The genome of MsiGto is rich in virulence factors, genes conferring infective mechanisms, and host immune response evasion systems. Some of these proteins are common to other pathogenic mycobacteria, and shared by the two previously sequenced M. simiae genomes, such as ESAT-6, known to modulate host immune responses by affecting human T-cell responses. Interestingly, all M. simiae strains including MsiGto have three of the four antigen 85 complex genes (fbpA, fbpC, and fbpD) responsible for cell wall synthesis. The enzyme products of these genes are responsible for the conversion of trehalose monomycolate (TMM) into the Cord Factor trehalose dimycolate (TDM), which is considered one of the most important virulence factors of mycobacteria. Other genera belonging to the Mycobacteriaceae family that include both pathogenic and environmental species, such as Rhodococcus, Corynebacterium and Nocardia, also produce TDM.
Mammalian Cell Entry proteins are cell surface exposed proteins that play a crucial role in M. tuberculosis virulence by permitting the bacteria to enter mammalian cells and survive inside the macrophage, modulating the immune response. Mce clusters consist of 4 homologous operons in M. tuberculosis (mce1, mce2, mce3, mce4) with a similar arrangement: two genes encoding integral membrane proteins followed by six mce genes (A, B, C D, E and F). Mce proteins are also involved in lipid metabolism, acting as transporters and allowing cholesterol degradation to free carbon and energy for use by M. tuberculosis. In the genome of MsiGto we found that the cluster mce3 is overrepresented. While the DSM 44165 and MO323 genomes present none and a single copy of mce3, respectively, two complete copies of the mce3 cluster were found in MsiGto. As the transition from an environmental organism to a pathogen has been associated with the acquisition of Mce genes in actinobacteria, and Mce3 proteins are expressed by M. tuberculosis during the infection phase, it is tempting to speculate the increased number of Mce3 copies found in MsiGto provided the strain with human infection potential.
The presence of multiple copies of these potential lipid transporters in mycobacterial genomes is consistent with the finding that pathogenic mycobacteria switch from carbohydrates to lipids as their main carbon and energy source inside cells. The evolution of this locus, through duplication and divergence, has almost certainly contributed to virulence in mycobacteria, and even in other distantly related actinobacteria, such as Streptomyces. Given that the ability to acquire cholesterol from the host is crucial to maintain a chronic infection, we postulate that cells having a large mce copy number, such as that found in MsiGto, may potentially evolve pathogenicity relatively faster when compared with other environmental mycobacteria.
A total of 493 protein families were found exclusively present in MsiGto when compared to the other two available M. simiae genomes (MO323 and DSM 44165). Consistent with findings in the Pseudomonas aeruginosa genome, it seems that MsiGto has undergone genome expansion. Within the gene pool unique to MsiGto we found the arginine/ornithine antiporter gene arcD, which is involved in the persistence of the zoonotic pathogen Streptococcus suis in host cells. Additional unique genes found in the MsiGto genome participate in aromatic amino acid metabolic pathways. For instance, 2-oxo-hepta-3-ene-1,7-dioic acid hydratase (hpcG gene, EC 4.2.1.80) participates in the degradation of tyrosine and the 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase I alpha participates in the synthesis of chorismate (aroG gene, EC 2.5.1.54). The latter is an interesting observation as tyrosine is a key nutrient source during infectious growth within macrophages of some pathogenic fungus. At this stage, however, it would be risky to rule out the involvement of these specific genes in important environmental functions that allow MsiGto to survive outside the host, or during both lifestyles.