The Effect Of Mycobacterium Tuberculosis Exopolyphatase Rv1026 On The Colony Morphology And Biofilm Of Mycobacterium Smegmatis

Tuberculosis is one of the infectious diseases seriously threatening human health with global significance. About 1/3 of the global population was infected by Mycobacterium tuberculosis. With the progress of human society and the development of medical treatments, the biological characteristics of M. tuberculosis, the pathogen of tuberculosis, has been further researched.Then treatment medicine has been developed, for instance streptomycin, isoniazid, rifampin, and so on. This has made great achievements in curbing the spread of tuberculosis. However, the abuse of chemical drugs, HIV coinfection, accelerating global transportation and the prevalence of drug-resistant bacteria exacerbated the tuberculosis predicament. New tuberculosis drug targets and new anti-tuberculosis drugs are urgently needed to combat the rampant tuberculosis.Inorganic polyphosphate (Poly P) is a polymer consisting of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds, which is abundantly found in all organisms and nature. PolyP plays an important role in many aspect including:expression and regulation of genes, role in DNA uptake, motility of microorganism, function in stress response, the virulence of pathogens, as well as the proliferation of mammary cancer cells, blood coagulation, cell calcification and the modulation of mitochondrial activity. Enzymes involving the metabolism of polyP include endopolyphosphatase, exopolyphosphatase, glucokinase, NAD kinase, Poly P:AMP phosphotransferase. The enzymes synthesizing and hydrolyzing polyP are polyphosphate kinase and exopolyphosphatase, respectively.the study on the M. tuberculosis PPK protein are completed to some extent. The downregulation of ppkl of M. tuberculosis is associated with drug-induced inhibition of translation, suggesting a role of polyP metabolism in the response to translation inhibition. Kamakshi Sureka et al reported that ppkl is involved in survival of the bacterium under conditions of hypoxia, detergent stress and oxidative stress, because ppkl is required for the transcription of the two-component signal transduction system mprAB,which in turn regulates the expression of sigE, a stress-regulatedσ-factor regulating the transcription of the stringent response regulator rel. Down regulation of ppkl of M. tuberculosis is associated with a loss in the ability of the bacterium to survive in macrophages supporting the view that ppkl plays an important role in the intracellular life of the bacterium. In addition, The PPK2 catalyse the conversion of GDP to GTP using polyP as phosphate donor, sustain the nucleotide pools in mycobacteria. A proper balance of nucleotide triphosphates is important for many physiological processes. For example, one of the major cell-wall components of mycobacteria, lipoarabinomannan, is synthesized from mannose-1-phosphate and GTP. Downregulation of ppk2 impairs survival of M. tuberculosis in macrophages suggests that polyP and PPK2 play an important role in the physiology of the bacteria residing within macrophages.However, the other enzyme involving in polyP metabolism is PPX, of which intracellular physiological function remains to be revealed. Therefore, the study on the function and the feature of the protein contributes to the understanding mechanism underlying M. tuberculosis intracellular survival and immune escape.Phylogenetic analysis indicate that the homologues of both PPX1 (Rv0496) and PPX2 (Rv1026) might be active as exopolyphosphatases. Using GenTHREADER program at PSIPRED server to search the structure related with Rv0496 and Rv1026, we found the PPX/GPPA of Aquifex aeolicus and Rv1026 and Rv0496 share most structural similarity. A structure-based sequence comparison based on the crystal structure of PPX/GPPA from A. aeolicus was performed using PPX protein sequences from M. tuberculosis and others bacterium studyed. The protein sequences from M. tuberculosis contained the putative catalytic Glu and Arg residues corresponding to Glul21 and Arg93 from A. aeolicus, and the pyrophosphoric acid-binding site corresponding to Asp22, Arg267, Glu144 and Glu211, and the glycine-rich phosphate-bingding loop (P-loop Glyl43-Serl46). All information about the Rv0496 and Rv1026 genes from M. tuberculosis is available from a transposon mutant screen study. Both genes were implied in M. tuberculosis pathogenicity, as Rv0496 was identified as a novel T-cell antigen and Rvl026 was shown to be induced when M. tuberculosis infects macrophages. While, whether the PPX1 and PPX2 homologues encoded by Rv0496 and Rv1026 in M. tuberculosis are indeed active as exopolyphosphatases, the experimental valdation of their enzymatic activity remains need to be studied. In present study, the nucleotide sequence of ppx gene in M. tuberculosis H37Rv was obtained from the GenBank database, and a pair of primers was designed based on these sequence. Then, M. tuberculosis H37Rv genome was used as a template, ppx gene was amplified by PCR. The PCR product was ligated to the pMD19-T Simple Vector, and then subcloned into expression vector pMV261. The recombinant plasmid pMV261-ppx was identified through colony PCR, plasmid restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Mycobacterium smegmatis. We culture recombinant M. smegamtis at 37℃to induce it express Rv1026, and found altered colony morphology, defective sliding motility and biofilm formation of recombinant M. smegmatis, and fourthemore, these alternation are sensitive to nonionic denaturant agent Tween80. Due to the associativity of cell wall fatty acid with colonial morphology and biofilm formation of M. smegmatis, we abstracted the cell wall fatty acid of recombinant and the control M. smegamtis, and use gas chromatographic to analyze the fatty acid constituent. We found that there are distinct peak between recombinant and the control strains. In summary, in this study we demonstrated the enzymatic activity of M. tuberculosis ppx, and that the reduction of polyP can cause the alternation of colonial morphology and reduction of biofilm formation, and these changes are sensitive to nonionic denaturant agent Tween80.