AUTHOR=Rajwani Rahim , Yam Wing Cheong , Zhang Ying , Kang Yu , Wong Barry Kin Chung , Leung Kenneth Siu Sing , Tam Kingsley King Gee , Tulu Ketema Tafess , Zhu Li , Siu Gilman Kit Hang 

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=7

YEAR=2018

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2017.00539

DOI=10.3389/fcimb.2017.00539

ISSN=2235-2988

ABSTRACT=<p><bold>Background:</bold> 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, <italic>Mycobacterium tuberculosis</italic>. We previously isolated a hypervirulent <italic>M. tuberculosis</italic> 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 <italic>H37Rv</italic> and other 123 clinical isolates.</p><p><bold>Aim:</bold> The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent <italic>M. tuberculosis</italic> strains of the same phylogenetic clade isolated in other global regions.</p><p><bold>Methods:</bold> A low-virulent <italic>M. tuberculosis</italic> 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 <italic>M. tuberculosis</italic> 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.</p><p><bold>Results:</bold>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 <italic>M. tuberculosis</italic> genomes (<italic>n</italic> = 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 (<italic>rv0178</italic> p. D150E) within <italic>mce1</italic> operon and an intergenic deletion (854259_ 854261delCC) in close-proximity to <italic>phoP</italic>.</p><p><bold>Conclusion:</bold> The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent <italic>M. tuberculosis</italic>. The causative role of these mutations in mycobacterial virulence should be validated in future study.</p>