Rv1273c-Rv1272c For Copper Transport And RpoC Mutations In Rifampicin-resistant Mycobacterium Tuberculosis

Tuberculosis is still a major global health problem and drug-resistant tuberculosis remains a continuing threat.Therefore,studies on M.tuberculosis virulence will contribute to drug development and disease control.For the irreplaceable roles copper plays in physiology,copper uptake is required for mycobacterial viability.However,copper uptake machinery used by M.tuberculosis remains poorly explored.ABC transporters are responsible for various substance across the cell membrane.Several lines of evidence indicate that ABC transporter Rv1273c-Rv1272c is closely related with mycobacterial virulence,whose function remains unclear.Our previous work identified the homologies of Rv1273c-Rv1272c,Ms5008-Ms5009,respectively,in M.smegmatis mc~2155.We also found that Ms5008-Ms5009 deletion mutant grew faster in vitro than its parental strain,which is associated with copper depletion in culture media.Therefore,we further studied on Rv1273c-Rv1272c for copper transport and its role in mycobacterial pathogenesis in Chapter II.In Chapter Ⅱ,results from EB accumulation assay and in vitro growth indicate that both Rv1273c-Rv1272c and Ms5008-Ms5009 are importers belonging to ABC transporter superfamily and involved in mycobacterial growth.Intracellular metal content showed lowered copper concentration in mutant strain than wild type,suggesting that Ms5008-Ms5009 is involved in copper uptake.Transcriptional profiles reviewed downregulated expression of ribosomal protein-coding genes and dysregulation of genes in oxidative phosphorylation pathway.Results from macrophage infection model suggest that copper uptake contributes to reduced phagocytosis and enhanced survival.It has been reported that laboratory-generated rifampicin-resistant mutants showed reduced fitness whereas some clinical isolates who carry mutations in rpoA or rpoC showed no fitness defect.Whether mutations in rpoA or rpoC have compensatory effects for fitness defect remains unknown.Therefore,studies in Chapter III aim at explore this potential effects of mutations in rpoA or rpoC by investigate their roles in transcriptional regulation in rifampicin-resistant clinical isolates.In Chapter Ⅲ,we enrolled 9 rifampicin-resistant clinical isolates for further study.We determined their transcriptional profiles when cultured with or without rifampicin using RNA-Seq and screened for dysregulated genes associated with mutations in rpoC.In drug-free conditions,mutations in rpoC are related with the upregulation of ribosomal protein-coding genes,dysregulation of growth-related essential genes and balancing the expression of arginine and glutamate synthesis associated genes.Toward drug stimulation,mutations in rpoC are related with the upregulation of the inhibited oxidative phosphorylation machinery,stabilizing the expression of essential genes toward rifampicin and balancing sulfur-containing amino acids metabolim associated genes’expression.