Characterization Of Mammalian Cell Entry Gene Family Of Mycobacterium Tuberculosis Rv0590A

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality of humankind throughout the world. The HIV/AIDS pandemic, deterioration in public health systems in some developing and resources limited countries, and the occurrence of multi-and extensive-drug resistant forms of tuberculosis exacerbated this public health exigency. With the development of medical treatments, the biological characteristics of M. tuberculosis, which is the pathogen of TB, have been further researched. Then treatment medicine has been developed, such as streptomycin, rifampin, isoniazid, ect. This has made great achievements in controlling the spread of TB. However, the abuse of chemical drugs, the prevalence of drug-resistant bacteria, HIV coinfection and accelerating global transportation exacerbated the TB predicament. New TB drug targets and new anti-TB drugs are urgently needed to combat TB.As an intracellular pathogen, M. tuberculosis mainly residing in mononuclear phagocytes. Survival and multiplication in host macrophages and inhibit acidification or mature of phagosome is one important ability of M. tuberculosis. The genome of M. tuberculosis contains four mce operons (mcel-4). Each mce operon contains 8-13 genes, with a similar arrangement within each other. The recombinant E. coli harboring this mce gene enabled this non-pathogenic bacterium to invade and survive inside macrophages, which are closely involved in the invasion and prolonged existence of M. tuberculosis in host macrophages. As a putative conservation protein, Rv0590A is peculiar to mce2 and it is absent in other mce operons, may regulate the expression of mce2 with Mce2R. Furthermore, mce2B and Rv0590A is an interrupted coding sequences (ICDSs), which are one of the evolution evidence between M. tuberculosis and M. bovis with other 18 ICDSs. In this study, Structure and function of the Rv0590A gene encoding protein were predicted through bioinformatics tools, in order to offer related reference for research of the protein. Through TMHMM Serverv 2.0, Jpred, Vector NTI 9, Clustal X, SignalP, swiss model and other bioinformatics resources, the basic properties of Rv0590A were analyzed, including protein primary structure (hydrophilic/hydrophobic, transmembrane and signal peptide, etc.); secondary structure and three-dimensional structures. The results showed that Rv0590A posses no signal peptide and no transmembrane region; two a-helices and twoβ-sheets form the secondary structure. Based on its secondary structure, three-dimensional structure was established by homology. In using of software from http://string.embl.de/, the net of the interaction of proteins was predicted. The results showed that proteins interaceted with Rv0590A mainly encoded by mce2 operon and Rv0586. This is useful for understanding the predicted function of coregulation in the expression of mce2 operon. Comparative genomics analysis of distribution and homology of Rv0590A in the genus Mycobacterium showed that M. tuberculosis H37Rv Rv0590A is highly conservative in Mycobacterium.Rv0590A gene in M. tuberculosis H37Rv was cloned into the prokaryotic vector pET28(+). And the fusion protein was expressed and its activity was assayed. The nucleotide sequence of Rv0590A gene in M. tuberculosis H37Rv was obtained from the GenBank database, and two pairs of primers was designed. M. tuberculosis H37Rv genome was used as a template, two Rv0590A genes were amplified by PCR, respectively. The PCR products were ligated to the pMD19-T Simple Vector, and then subcloned into vector pET28a(+) and pcDNA3.1(+), respectively. The recombinant plasmid pET28/Rv0590A and pcDNA3.1/Rv0590A were identified through colony PCR, plasmid restriction enzyme digestion and sequencing. The recombinant plasmid pET28/Rv0590A was transformed into Escherichia coli BL21(DE3). The fusion protein obtained under the optimial expression culture conditions was purified by Ni+ affinity chromatography, then through western bloting assay verified the fusion expression of the Rv0590A. The impact of Rv0590A overexpression on the host bacteria, including the growth, fatty acid synthesis and oxidative stress tolerance, was explored. The recombinant plasmid pcDNA3.1/Rv0590A was transformed into U937, the expression of Rv0590A on transcription level, and the expression of the cytokine in U937 after transfection by Rv0590A was detected by RT-PCR. In summary, this study demonstrated the physiological function of Rv0590A, and we hope that these studies may provide new clues for revealing the mechanism of Mce family and new insights into the mycobacteria pathogenesis.