Molecular Mechanism Of Vancomycin Resistance And Defective Biofilm Formation Mediated By Mycobacterium Tuberculosis Toxin Rv2872

Tuberculosis?TB?is caused by Mycobacterium tuberculosis infection,is highly contagious and high mortality rate of chronic infectious diseases.Causing a serious damage to public health.The emergence of extensively drug resistance?XDR?of M.tuberculosis,multidrug-resistant strains?MDR?,even totally drug resistanct?TDR?MTB make tuberculosis epidemic even worse.We are urgent to explore new methods and new antibiotics for the treatment of tuberculosis.TA system was firstly discovered to stabilize plasmid.The labile antitoxin can keep the toxin in check.Once activated,toxins can kill the cell,preventing the plasmid from loss.Among the 79 TA systems identified in M.tuberculosis H37 Rv,VapBC TA systems are very widespread in M.tuberculosis genome.The PIN-domain TA systems are now called VapBC TAs?virulence associated proteins?,where VapB is the inhibitor of VapC as well as the PIN-domain ribonuclease toxin.The roles of VapBC toxin antitoxin system in M.tuberculosis still remain unknown,which drawed our attention.We provided evidence that the expression of some toxins were up-regulated after vancomycin treatment,we found that MS_Rv2872 was more resistant to vancomycin than MS_Vec.The results suggested that Rv2872 may regulate some vancomycin related genes.Previous global transcriptome data showed that over 100 M.tuberculosis genes were differentially regulated upon exposure to vancomycin,RT-PCR was used to determine the transcriptional levels of vancomycin related genes in MS_Rv2872 and MS_Vec.In comparison with control strains,two genes were up-regulated?transcriptional regulatory protein MSMEG_Rv0051 and transcriptionalregulatory protein ClgR MSMEG₂694?,three genes were down-regulated?ribosome binding protein MSMEG₀424,L-lysine-epsilon aminotransferase MSMEG₁764,and sulfate adenylyltransferase subunit MSMEG₄979?.Previous studies founded that MSMEG₀424,MSMEG₁764 and MSMEG₄979 showed different degrees of sensitivity against vancomycin.To determine whether MSMEG_Rv0051 and MSMEG₂694 were involved in the vancomycin response.The recombinant M.smegmatis strains expressing MSMEG_Rv0051 and MSMEG₂694 genes were constructed.We found that overexpressing of this two genes decrease the susceptibility of host bacteria to vancomycin.We also found that MSMEG₂916 can down-regulate the transcription of MSMEG₀051.MSMEG₂916-MSMEG₀051 might form a regulatory cascade to modulate the transcription of genes responsive to vancomycin treatment.The purified Rv2872 toxin protein can digest MSMEG₂916,as regulator of vancomycin responsive to MSMEG₀051.The gene Rv2872 also caused other significant phenotypic changes,such as changes in cell wall structure,alteration of amino acid content and the composition of the bacterial biofilm.The specific cleavage of single-stranded RNA of biofilm development gene MSMEG₆092 might cause the MS_Rv2872 biofilm difference.The Rv2782 play a crucial role in M.tuberculosis pathogenesis,genetics and as well as physiology progress.Its regulation cascade proved the similar function as TAs.The toxin Rv2872 was firstly reported to be a sequence-specific endonuclease involved in vancomycin stress responses and biofilm development.The Rv2872 gene is involved in intrinsic of vancomycin resistance and considered as potential vancomycin adjuvant target.