Comparative Whole-Genomic Analysis of an Ancient L2 Lineage Mycobacterium tuberculosis Reveals a Novel Phylogenetic Clade and Common Genetic Determinants of Hypervirulent Strains

Abstract

Background: Development of improved therapeutics against tuberculosis (TB) is hindered by an inadequate understanding of the relationship between disease severity and genetic diversity of its causative agent, Mycobacterium tuberculosis. We previously isolated a hypervirulent M. tuberculosis strain H112 from an HIV-negative patient with an aggressive disease progression from pulmonary TB to tuberculous meningitis—the most severe manifestation of tuberculosis. Human macrophage challenge experiment demonstrated that the strain H112 exhibited significantly better intracellular survivability and induced lower level of TNF-α than the reference virulent strain H37Rv and other 123 clinical isolates.Aim: The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent M. tuberculosis strains of the same phylogenetic clade isolated in other global regions.Methods: A low-virulent M. tuberculosis strain H54 which belonged to the same phylogenetic lineage (L2) as strain H112 was selected from a collection of 115 clinical isolates. Both H112 and H54 were whole-genome-sequenced using PacBio sequencing technology. A comparative genomics approach was adopted to identify mutations present in strain H112 but absent in strain H54. Subsequently, an extensive phylogenetic analysis was conducted by including all publically available M. tuberculosis genomes. Single-nucleotide-polymorphisms (SNPs) and structural variations (SVs) common to hypervirulent strains in the global collection of genomes were considered as potential genetic determinants of hypervirulence.Results:Sequencing data revealed that both H112 and H54 were identified as members of the same sub-lineage L2.2.1. After excluding the lineage-related mutations shared between H112 and H54, we analyzed the phylogenetic relatedness of H112 with global collection of M. tuberculosis genomes (n = 4,338), and identified a novel phylogenetic clade in which four hypervirulent strains isolated from geographically diverse regions were clustered together. All hypervirulent strains in the clade shared 12 SNPs and 5 SVs with H112, including those affecting key virulence-associated loci, notably, a deleterious SNP (rv0178 p. D150E) within mce1 operon and an intergenic deletion (854259_ 854261delCC) in close-proximity to phoP.Conclusion: The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent M. tuberculosis. The causative role of these mutations in mycobacterial virulence should be validated in future study.

Revised nomenclature and SNP barcode for Mycobacterium tuberculosis lineage 2

“…Mtb L2 likely originated in a region around southern East Asia or Southeast Asia [3][4][5], expanded in China and spread to the rest of the world, particularly in Central Asia, Eastern Europe and East Africa [2,3,6]. L2 strains have been associated with higher virulence [7,8], increased transmissibility [9][10][11] and high prevalence of multidrug resistance [12,13]. However, global genomic diversity and population structure of Mtb L2 remain poorly characterized.…”

Construction of lux-based promoter-reporter platforms in Mycobacterium bovis BCG for screening of drug repurposing small-molecule compounds as new anti-tuberculosis drugs

“…Notably, the development of anti-virulence drugs requires an in-depth understanding of the roles that diverse virulence factors have in disease processes. Previous studies from our team and other research groups suggested that phoP is essential for the intracellular growth of the M. tuberculosis complex inside macrophages [10,11]. Disruption of phoP could impair intramacrophage and facilitate the host's immune system to eradicate the bacteria.…”

“…It was shown that disruption of phoP in M. tuberculosis caused impaired multiplication within macrophages, suggesting that this gene possibly plays an essential role in the intracellular growth of M. tuberculosis [10]. In addition, our previous study identified a common mutation in the promoter region of phoP, which could confer aggressive intramacrophage growth of hypervirulent M. tuberculosis strains [11]. Therefore, we hypothesized that suppression of phoP expression might be able to impair intramacrophage survival of M. tuberculosis, facilitating the host's immune system to eradicate the bacteria.…”

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes ofMycobacterium tuberculosis

“…In addition, the MIRU-profiler was also tested on four circulating strains from Hong Kong, for which we performed complete genome-sequencing and described in our recent publications ( Leung et al, 2017 ; Rajwani et al, 2018 ). The genomes of these four strains were sequenced on the PacBio RS II platform with average read-length >10 Kb and de-novo assembled into a single contig.…”