Determination of functional impacts of differentially expressed PhoP gene on mycobacterial virulence in causing tuberculous meningitis

Abstract

Tuberculous meningitis is a life-threatening extrapulmonary tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) in the central nervous system. Such a serious clinical manifestation of tuberculosis is associated with an increased level of virulence in some strains of the causative agent. The situation is further aggravated by the lack of effective vaccines and drugs against tuberculosis. Improved understanding of the pathogenesis of tuberculosis and genetic determinants responsible for increased virulence in M. tuberculosis strains is highly needed to alleviate the problems. Recently, a set of genetic mutations have been identified in the genome of the hypervirulent M. tuberculosis strains isolated from tuberculous meningitis patients. These mutations were found to be associated with differentially elevated expression of the phoP gene in the hypervirulent M. tuberculosis strains but were absent in the low virulent M. tuberculosis strains of the same phylogenetic lineage. However, the causative role of these genetic factors in the development of tuberculous meningitis is still unknown. This thesis explains the findings of the study that help to bridge the existing research gap. This study has two objectives. 1) To construct a transformant Mycobacterium marinum (M. marinum) strain with enhanced expression of phoP gene of M. tuberculosis. 2) To elucidate the causative role of the enhanced expression of the phoP gene in mycobacterial virulence in causing tuberculous meningitis using zebrafish-M. marinum infection model. In the first part of the study, the phoP gene and blue fluorescent protein (BFP) gene regions were amplified from the genome of M. tuberculosis H37Rv and the plasmid pME-loxP-mTagBFP2-stop-pA-loxP, respectively, and cloned into Escherichia coli-mycobacteria shuttle vector (pVV16). The recombinant pVV16-phoP gene-BFP gene plasmid was transferred into the M. marinum strain M/E11 using electroporation. To label the control M. marinum strain M/E11 with blue color, we also constructed a recombinant pVV16-BFP gene plasmid and transferred it into the mycobacterium using the same procedures. PCR and Sanger sequencing analyses and blue fluorescent transformants recovered from culture confirmed the presence of the target genes in the transformants. qPCR analysis of the transcription level of the phoP gene in the transformant transformed with pVV16-phoP gene-BFP gene plasmid indicated that the expression of the gene was increased about 3-fold relative to that of the control strain transformed with pVV16-BFP gene plasmid. In the second part of the study, wild-type and absolute (ednrbab140;mitfab692) (transparent) lines of zebrafish were injected with the transformant (M. marinum strain M/E11) and control strain (M. marinum strain M/E11). The effects of the infection were examined in the brain of the zebrafish using bacteriological, histopathological, and immunohistochemistry analyses. Fluorescence microscopy was used for the detection and monitoring of blue fluorescent granuloma-like signals or aggregates in the body and brain region of the M. marinum E11 infected transparent zebrafish. Among the control strain-infected wild-type zebrafish, 6.25% (4/64) developed infection in their brains, whereas from those wild-type zebrafish infected with transformant, 28.13% (18/64) had brain infection (P=0.0018) with a higher bacillary load. Among the zebrafish that developed brain infection, 4 (3 from transformant-infected and one from control strain-infected) of them were from the M. marinum strain M-infected group. Notably, granuloma-like lesions were only observed in the brain of transformant-infected zebrafish. A lower survival rate (36.8%) was also observed in the transformant M. marinum E11 infected zebrafish relative to that of the control M. marinum E11 infected zebrafish (68.4%). Importantly, 47.37% (9/19) and 6.25% (1/16) of the wild-type zebrafish that died after being infected with the transformant and control strain, respectively, had brain infections. Among these transformant-infected zebrafish that developed brain infections, 77.8% (7/9) died within a week. In transparent live zebrafish, granuloma-like signals were also detected in the brain of 16.7% (2/12) and 41.7% (5/12) of the zebrafish injected with control and transformant M. marinum E11, respectively. Moreover, qPCR analysis showed a lower level of tumor necrosis factor-alpha (TNF-α) in the brain of transformant-infected zebrafish than that of the control-infected zebrafish (P=0.2186). Taken together, our results indicated that the transformant M. marinum is more neurotropic and virulent than the control strain, reflecting the fact that differentially elevated expression of the phoP gene may enhance the virulence of mycobacteria in causing infection in the central nervous system. To our knowledge, this study is the first to validate the functional impacts of differentially expressed phoP gene on mycobacterial virulence in the development of tuberculous meningitis inside the host. Thus, the findings may provide a knowledge base for the development of novel and effective vaccines and drugs against tuberculosis particularly tuberculous meningitis.