Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria

Abstract

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Keywords: ESX-1; Mycobacterium tuberculosis; evolution; mycobacterial model; phylotype; virulence.

Fig 1

Growth measurement over time at an optical density of 600 nm for M. decipiens (A), M. lacus (B), M. riyadhense (C), M. shinjukuense (D), and M. kansasii (E). Growth was measured at 32°C, 35°C, and 37°C for each strain. Results from two independent experiments in classic culture conditions. Picture of M. decipiens growth at 35°C and 37°C in 7H9 ADC liquid cultures (F) and on LJ tubes (G) were taken after 10 days of incubation. Comparable quantities of bacteria were used for both temperatures.

Fig 2

Genomic comparisons of ESX loci of M. decipiens with Mtb and M. kansasii. Comparisons of ESX-1 (A), ESX-2 (B), ESX-3 (C), ESX-4 (D), and ESX-5 (E) were performed using the Artemis Comparison Tool ACT, and the “MicroScope” database from the Genoscope (https://mage.genoscope.cns.fr/microscope/mage/viewer.php?).

Fig 3

Western blot-based evaluation of the presence of ESAT-6 and CFP-10 in whole-cell lysate (left panel) and supernatant (right panel) fractions of in vitro cultures of M. decipiens (M. dec.), M. lacus (M. lac.), M. riyadhense (M. riy.), M. shinjukuense (M. shi.), and Mtb H37Rv that were grown to an OD_600nm of 0.6–0.8 using anti-ESAT-6, anti-CFP-10, as well as anti-Ag85B and anti-SigA control antibodies. An amount of 50 µg of proteins was migrated on SDS-PAGE for 30 min at 200 V and transferred onto a nitrocellulose membrane for 7 min at 110 V (A). Western blotting of whole-cell lysate (left panel) and supernatant (right panel) fractions from M. lacus (M. lac.), M. riyadhense (M. riy.), M. shinjukuense (M. shi.), and M. decipiens (M. dec.) cultures that were grown to an optical density higher than 1 (B). Genomic position of the orthologous genes of the espACD cluster that is present in the genome of M. decipiens, compared to the genomic locations of orthologous espACD clusters in selected other mycobacterial species that possess an espACD cluster in their genomes (C). Note that the orthologous flanking genes of the espACD cluster identified in the various species refer to the Mtb H37Rv gene nomenclature and relative genomic localization. Image adapted from reference (16) with permission and complemented with new information on the M. decipiens espACD cluster. Genes depicted as white arrows correspond to M. decipiens specific genes.

Fig 4

Infection of THP-1-derived macrophages determined by CFU counting at 0, 3, 5, and 7 days after infection. Results represent between three and six independent experiments at 35°C and 37°C for M. decipiens (A), M. lacus (B), M. riyadhense (C), M. shinjukuense (D), M. kansasii (E), M. bovis BCG Pasteur strain (F), and Mtb H37Rv (G). Data are represented as means and SEM of three to six biological experiments, each experiments containing three technical replicates. Statistical differences in CFU were determined by a two-way ANOVA test with Dunnett’s multiple comparisons test (*P < 0.033; **P < 0.002; ***P < 0.001; ns, non-significant).

Fig 5

Evaluation of in vivo growth capacity of M. decipiens, M. lacus, M. riyadhense, M. shinjukuense, M. kansasii, M. bovis BCG Pasteur, and Mtb H37Rv in two murine models. CFU counts in control mice at day 1 post-infection and titration of aerosolized culture of both experiments are indicated (A and E). Results from C3HeB/FeJ infection and ratios of different time points vs day 1 counts are indicated (B). Results from C3HeB/FeJ infection based on CFU counts at different time points. Results are shown at a log₁₀ scale for lungs (C) and spleens (D). Same types of results are presented for the C57BL/6J infection with ratios of CFUs D1/CFUs titration (F), CFU counts in lungs (G) and spleens (H). Each experiment was performed once, and the represented values correspond to the number of mice at each time point. "ND": not detected.