The unique pattern of mannose-capped lipoarabinomannan expression in Mycobacterium tuberculosis with different drug resistant profiles following isoniazid stress

Tuberculosis (TB) is a global health problem caused by Mycobacterium tuberculosis (MTB) infection. The main problem of TB treatment is the emergence of drug resistance, which can occur by inappropriate of antibiotic used. Isoniazid (INH) is the first-line anti-TB drug that inhibits mycolic acid synthesis, an important part of the mycobacterial cell wall. Mannose-capped lipoarabinomannan (ManLAM) is an essential cell wall part that plays a role as an immunomodulator and acts as a virulence factor. In this study, MTB clinical isolates with different drug resistant profiles were used to determine the expression of ManLAM related genes including pimB, mptA, mptC, dprE1, dprE2 and embC by qRT-PCR. Stress-related genes including hspX, tgs1, and sigE were determined by multiplex real-time PCR with probe assay. Sanger sequencing of ManLAM related genes and genes associated with drug resistance (inhA, katG, and rpoB) were analyzed. In response to INH, the expression pattern of ManLAM related genes was different among four strains. Interestingly, MDR-TB markedly up-regulated ManLAM related genes greater than others. Stress-related genes hspX and tgs1 were significantly upregulated in MDR response to INH, whereas sigE was significantly upregulated in MDR response to RIF and INH-R. DprE1 is crucial for MTB and it is a valuable target for anti-TB drugs. RIF-R and MDR isolates show C[->]T mutation at nucleotide position 459 of the dprE1 gene leading to the same amino acid at codon 153. Codon usage analysis for DprE1 showed that RIF-R and MDR preferred ACT codon over drug sensitive strains. This work provides the expression pattern of ManLAM related genes and stress responder genes, which are key factors in the interaction between MTB and host. Moreover, ManLAM is a possible factor that plays an important role in the adaptive mechanism and the drug resistance mechanism of mycobacteria. Author summaryThe adaptive mechanism of mycobacteria in response to stressors is an important strategy to promote their virulence and pathogenesis. This study determined the effect of antibiotic stress on Mycobacterium tuberculosis (MTB) focusing on mannose-capped lipoarabinomannan (ManLAM), which is one of the virulence factors that modulate host immune response. Multiplex real-time PCR with probe assay targeting stress responder genes and qRT-PCR targeting ManLAM related genes were performed. Isoniazid acts as a stressor to induce stress response in mycobacteria, as shown in the up-regulation of stress-related genes including hspX, tgs1, and sigE. The expression pattern of ManLAM related genes in drug resistant and drug sensitive-MTB in response to INH was different, causing a unique pattern. ManLAM related genes respond to isoniazid mostly in drug resistant strains and are present at high expression levels in INH-R and MDR. The results suggest that ManLAM is one factor involved in the adaptive mechanism of MTB response to antibiotic stress and probably associated with the emergence of MTB drug resistance. This work provides new insights into the adaptive mechanism of mycobacterial response to isoniazid that will improve understanding of how mycobacteria develop drug resistance.