Function Of Protein Rv2624c Broad Spectrum Of Mycobacterium Tuberculosis Stress

Mycobacterium tuberculosis (Mtb) is a pathogenic bacteria species in the genus Mycobacterium and the causative agent of most cases of tuberculosis (TB). TB is the second leading cause of death in the world from a bacterial infectious disease. The World Health Organizaiton (WHO) estimates that approximately one-third of the world’s population is infected with Mtb and2million individuals die of TB. Only about10%of tuberculosis infections lead to active pulmonary disease. In the remaining90%of tuberculosis cases, initial infection is contained by the host’s immune system and active disease dose not develop. This situation is called Latent TB Infection (LTBI). The stable LTBI state is achieved by the ability of Mtb to attenuate and evade host mycobactericidal responses. Inadequate immunity leads to mycobacterial multiplication and clinical disease. It’s dangerous when dormant Mtb turn reactivation. Unfortunately, the mechanisms that regulate these processes remain unclear. Recent researches show that Universal Stress Protein(USP) superfamily is associated with the latency. We study functions of these proteins in order to provide valuable insight into tuberculosis dormancy and uncover new chances for the development of antituberculosis therapies.The USP superfamily is an acient and well-conserved protein family represented in diverse organisms from Archae and Eubacteria to yeast, fungi and plants. USP can be reduced in response to a wide variety of growth-arrested conditions, for examples, heat shock, DNA damage, NO, exhaustion of any one of number of nutrients, presence of a variety of toxic agents including heavy metals, hypoxia. Researchs about Rv2623which is one of the Mtb USP, suggest that i) Rv2623regulates growth of Mtb in vivo and vitro, ii)Rv2623is required for the Mtb entry into the chronic phase of infection in the host, iii)Rv2623binds ATP, iv)the growth-regulatory is related to its ATP-binding activity. The results indicate that USP and Mtb dormancy are relevant.Our studies show that Rv2624c which is another USP of Mtb binds ATP while it isn’t an ATPase when it is alone in vitro. We engineered mutations within D17E and G109A conserved amio acids to disrupt ATP binding. ATP quantificaition analysis of nucleotides bound from E.coli-purified Rv2624cD17E,Rv2624cG109and Rv2624cD17EG109revealed that the mutant proteins are deficient in ATP-binding. The wild type and D17E, G109A, D17EG109A mutant proteins were overexpressed in M.smegmatis and tested by anti-Rv2624c rabbit polyclonal antibody. The D17E and D17EG109A overexpressed at different levels compared to wile type Rv2624c. We examined the effect of overexpression of Rv2624c and mutants on the rapidly growing M.smegmatis MC2155. The results showed that the growth kinetics of mutants are comparable to that of wildtype. We also determined M.smegmatis which overexpressed Rv2624c exhibited a increase survival in macrophages THP1. We utilized RNA-seq to explore the transcriptome of M.smegmatis which overexpressed Rv2624c in order to explain above phenotype. Two profiles of M.smegmatis which overexpressed Rv2624c and control were generated after a physical ribosome RNA removal step. We systematically described the transcriptome and analyzed the functions for the differentiated expressed genes between the two strains, and finally chose arginine/proline metabolism pathway and tryptophan metabolism pathway which predicted to be related to the survival in macrophages.10upregulated genes selected from two pathways were validated by RT-qPCR.7homologous genes of above10genes can be found in M.bovis and also were validated upregulated by RT-qPCR in M.bovis which overexpressed Rv2624c. So we predicted the fuction of Rv2624c is related to arginine/proline metabolism pathway and tryptophan metabolism pathway. Our studies indicate that Rv2624c is very impotant in survival in macrophage for mycobacterium and provide clues for USP studies.