Insights From the Genome Sequence of Mycobacterium paragordonae, a Potential Novel Live Vaccine for Preventing Mycobacterial Infections: The Putative Role of Type VII Secretion Systems for an Intracellular Lifestyle Within Free-Living Environmental Predators

Abstract

Mycobacterium paragordonae (Mpg) is a temperature-sensitive Mycobacterium species that can grow at permissive temperatures but fails to grow above 37°C. Due to this unique growth trait, Mpg has recently been proposed as a novel live vaccine candidate for the prevention of mycobacterial infections. Furthermore, the increasing frequency of the isolation of Mpg from water supply systems led us to hypothesize that the free-living amoeba system is the natural reservoir of Mpg. In this study, we report the complete 6.7-Mb genome sequence of Mpg and show that this genome comprises four different plasmids with lengths of 305 kb (pMpg-1), 144 kb (pMpg-2), 26 kb (pMpg-3), and 17 kb (pMpg-4). The first two plasmids, pMpg-1 and -2, encode distinct Type VII secretion systems (T7SS), ESX-P5 and ESX-2, respectively. Genome-based phylogeny indicated that Mpg is the closest relative to M. gordonae, which has a 7.7-Mb genome; phylogenetic analysis revealed an average of 86.68% nucleotide identity between these two species. The most important feature of Mpg genome is the acquisition of massive genes related to T7SS, which may have had effect on adaptation to their intracellular lifestyle within free-living environmental predators, such as amoeba. Comparisons of the resistance to bacterial killing within amoeba indicated that Mpg exhibited stronger resistance to amoeba killing compared to M. gordonae and M. marinum, further supporting our genome-based findings indicating the special adaptation of Mpg to free-living amoeba. We also determined that, among the strains studied, there were more shared CDS between M. tuberculosis and Mpg. In addition, the presence of diverse T7SSs in the Mpg genome, including an intact ESX-1, may suggest the feasibility of Mpg as a novel tuberculosis vaccine. Our data highlight a significant role of lateral gene transfer in the evolution of mycobacteria for niche diversification and for increasing the intracellular survival capacity.

Keywords: M. gordonae; Mycobacterium paragordonae; Type VII secretion systems; genome sequence; lateral gene transfer.

FIGURE 1

Circular representation of the Mpg genome and plasmids. Whole-genome sequencing of Mpg revealed that Mpg harbors (A) a single circular chromosome (6,730,319 bp) and (B) four plasmids (pMpg-1, 305,730 bp; pMpg-2, 144,093 bp; pMpg-3, 26,922 bp; pMpg-4, 17,187 bp). From inside to outside, track 1 (black peaked line) indicates the GC content, and track 2 (red peaked line) indicates the GC skewness of the Mpg genome and plasmids. Track 3 and 4 represent the predicted ORFs in the reverse and forward orientation, respectively. In the case of the Mpg genome, track 5 indicates the locations of tRNAs. Tracks 6 through 8 represent the sequence identities compared with M. tuberculosis H37Rv^T, M. gordonae DSM 44160^T, and M. marinum M, respectively.

FIGURE 2

Phylogenetic tree based on the whole-genome sequences of Mpg and other mycobacterial strains. The tree was constructed using the neighbor-joining method using the MAUVE Genome Alignment software and visualized using the TreeViewX program. The bar indicates the number of substitutions per nucleotide position.

FIGURE 3

Venn diagrams showing orthologous CDS among Mpg and other mycobacterial species as determined by BLASTCLUST analysis (0.8 of length coverage threshold). (A) Comparison among the Mpg, M. gordonae DSM 44160^T, M. marinum M, and M. tuberculosis H37Rv^T strains. (B) Comparison among the Mpg, M. tuberculosis H37Rv^T, and M. indicus pranii strains.

FIGURE 4

Comparison of the genetic organization of the ESX loci in various mycobacterial genomes, including Mpg. Comparison of the genetic organization of the ESX loci identified in the genomes of M. tuberculosis, BCG, M. marinum, M. kansasii, M. gordonae, M. avium, and Mpg. (A) ESX-1 locus. (B) ESX-2 locus. (C) ESX-3 locus. (D) ESX-4 locus. (E) ESX-5 locus. The sequence similarities with M. tuberculosis are indicated for each ORF. The red dashed box indicates the deleted genes. The red lined box in panel (B) indicates the separation of the genes involved in the ESX-2 locus, and their location is also indicated above the box.

FIGURE 5

Comparison of the genetic organization of the ESX loci in various mycobacterial plasmids, including Mpg. (A) The ESX-P5 locus from pMpg-1 was compared with other loci in the mycobacterial plasmids pRAW (M. marinum E11), pMAH135 (M. avium subsp. hominissuis TH135), pMK12478 (M. kansasii ATCC 12478), and pMyong1 (M. yongonense). (B) The ESX-2-like locus in pMpg-2 was compared with the ESX-2 locus of M. tuberculosis. In panel (A), the sequence similarities are indicated for each ORF, which were calculated against genes from pRAW. Genes which were not matched with those of pRAW and additional insertions were represented as white arrows.

FIGURE 6

CFU enumeration of Mpg, M. gordonae and M. marinum from infected A. castellanii. CFU counts of Mpg, M. gordonae and M. marinum after infection of A. castellanii at a M.O.I. of 10 for 7 days. Statistical analyses (Student t-test) were performed among the CFUs of Mpg, M. gordonae and M. marinum at each infection time-point (^*P < 0.05 and ^∗∗ P < 0.01). Asterisks above each bar of M. gordonae and M. marinum indicate the statistical significance between M. paragordonae and those two strains at each time point.