Defining the molecular virulence mechanism of Mycobacterium bovis

The Mycobacterium tuberculosis complex (MTBC) contains the causative agents of tuberculosis (TB) in mammals. The archetypal members of the MTBC, Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis), cause human tuberculosis and bovine tuberculosis, respectively. Although M. tuberculosis and M. bovis share over 99.9% genome identity, they show distinct host adaptation for humans and animals; hence, while the molecular basis of host adaptation is encoded in their genomes, the mechanistic basis of host tropism is still unclear. Exploration of the in vitro phenotypic consequences of known genetic difference between M. bovis and M. tuberculosis offers one route to explore genotype-phenotype links that may play a role in host adaptation. Thus, my PhD project mainly studied two such DNA regions, TbD1 and RD900. The TbD1 (‘Mycobacterium tuberculosis deletion 1 region’) locus encompasses the mmpS6 and mmpL6 genes and this region is absent in M. tuberculosis ‘modern’ lineages (Lineages 2, 3, 4). The function of TbD1 has previously been investigated in M. tuberculosis, where conflicting data has emerged on the role of TbD1 in sensitivity to oxidative stress, while the underlying mechanistic basis of such a phenotype is unclear. The TbD1 project aimed to shed further light on the role of the TbD1 locus by exploring its function in M. bovis. M. bovis AF2122/97 and BCG Denmark TbD1 knockout (¿TbD1) strain were generated and conducted comparative transcriptomics to define global gene expression profiles of M. bovis wild type (WT) and the ¿TbD1 strains under in vitro culture conditions (rolling and standing cultures). This analysis revealed differential induction of a hypoxia-driven copper response in WT and ¿TbD1 strains. In vitro phenotypic assays demonstrated that the deletion of TbD1 sensitized M. bovis to H2O2 and hypoxia-specific copper toxicity. The Region of Difference 900 (RD900) locus contains two pknH genes (pknH1 and pknH2) flanking the tbd2 gene. Gene pknH1 and pknH2 both encodes a eukaryotic-like serine/threonine protein kinase and tbd2 encodes a potential ABC transporter. The RD900 is deleted in M. tuberculosis H37Rv and thus only one pknH gene is left. Mycobacterium bovis AF2122/97 has an intact RD900 locus with two pknH genes however none of the PknH proteins has the proline-rich region as compared to PknHTB from M. tuberculosis H37Rv. To understand the function of RD900 locus in M. bovis and identify different pathways that regulated by PknH1, PknH2 or PknHTB, I collaborated with Carlos Martin’s group at the University of Zaragoza, Spain, and conducted WGS and RNA-seq on their M. bovis AF2122/97 WT and WT::pknHTB strains. Analysis found that the knock-in of the pknHTB in M. bovis AF2122/97 altered a range of cellular processes, mainly in intermediate metabolism and respiration, cell wall and cell processes, lipid metabolism and these alterations may contribute to the attenuated in vivo virulence of the M. bovis AF2122/97 WT::pknHTB mutants observed in their mouse infection model. On the other hand, I knocked out the entire RD900 locus on M. bovis AF2122/97 to make a ‘clean’ background and complemented ¿RD900 mutants with pknH1, pknH2 or pknHTB separately for comparative transcriptomic studies. Although RNA-seq could not assign different pathways that each PknH initiates, the results suggested that in M. bovis AF2122/97 the PknHs phosphorylate a dormancy survival regulator (DosR) under the trigger of standing-induced hypoxia, and the full initiation of DosR regulon genes in response to hypoxia needs the RD900 locus. To summarize, this PhD project provides new information on the functions of the TbD1 and RD900 loci in stress responses in M. bovis and implied their important roles in host adaptation.