A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates

Abstract

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120–150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species

“…The variability of virulence factors within MKC species is supported by earlier data, comparing M. kansasii against other species 6,17,18 suggesting an MKC species-specific virulome. To address this, we defined 287 M. kansasii genes orthologs to M. tuberculosis H37Rv potential virulence factors and screened for their presence in the genomes collection.…”

“…Still, with respect to the species or strains virulome characteristics, even though each MKC species generally keeps a specific set of VFs, some strains are deviating from their species virulome gene set, by losing some VFs. This poses that distinct strains of MKC species can present a broad virulence spectrum, as already observed for M. kansasii 6 . Interestingly, KAN-43 is one of the two strains that belonged to phage type 2 and if the absence of the espACD operon has influence on the strain´s virulence is currently under investigation.…”

“…H37Rv that encode for potential virulence factors found by Mussi et al 6 . We applied a cutoff of 85% identity and coverage.…”

“…Conversely, an association of pathogenicity with genetic recombination has been observed, mediated by distributive conjugal transfer, also with possible correlation to MKC speciation events 7,8 . Regarding virulence, whereas M. kansasii contains a type-VII secretion system ESX-1 locus with all corresponding orthologs of the M. tuberculosis genes, some MKC species do not have an intact conservation of this locus 7,9. Furthermore, nearly three hundred shared orthologs between M. kansasii and M. tuberculosis are predicted to encode potential virulence factors 6 (VFs), but the conservation of these VFs in another MKC species has not been explored.…”

“…Even though some questions about the MKC pathogenicity, virulence, drug resistance, genetic diversity, and epidemiology have been addressed by analyzing genomic data 1,2,[6][7][8][9][10][12][13][14] , several aspects about the MKC species genomic characteristics and epidemiology remains unexplored. For instance, i) in addition to plasmids, the prevalence and characteristics of mobile genetic elements in MKC genomes, such as prophages; ii) the possible plasmids or prophages contribution to virulence or drug resistance; iii) the MKC pangenome structure and diversity; iv) the MKC viral defense mechanisms repertoire; v) the potential M. kansasii lineages and their association with certain geographical areas or niches; and vi) transmission mechanisms of the different MKC species.…”