Study Of Mycobacterium Tuberculosis Signal Transduction System And Screening,Identification And Preliminary Application Of Novel Antigen

Tuberculosis (TB) remains a major threat of global public health, and it is a chronic infectious disease caused by single etiological agent, Mycobacterium tuberculosis (M.tb). The death caused by TB is second only to the acquired immunodeficiency syndrome (AIDS) among infectious diseases. The burden of disease caused by TB is still very heavy and one third of global population was infected by M.tb, most of which are latent infection. However, the'dormant bacteria'will resuscitate and develop into active TB when the host immune system is compromised due to various reasons, especially in human immunodeficiency virus (HIV) positive patients. According to report from World Health Organization (WHO) in2011, globally there were8.5-9.2million active cases and0.9-1.2million of deaths among human immunodeficiency virus (HIV)-negative cases of TB. Incomplete therapy due to long therapy time, unreasonable use of drug and continuous evolution of bacteria corporately lead to the initiation and spread of multidrug-resistant TB(MDR-TB) and extensively drug-resistant TB(EDR-TB), which make the anti-tubercular agent in difficulty. Mycobacterium bovis BCG, the only vaccine currently used in TB prevention, can provide efficient protection for infants and children but unsatisfactory and variable protection for adult. TB prevention is a global arduous task, especially in depressed area and developing countries.China is still one of the22High-burden countries of TB. There were about1.3million active cases in China in2011, which is14%of global cases. More and more MDR-TB has been found and it might lead the epidemic condition in China in the coming years. The increasing number of TB/HIV co-infection patient requires us to reinforce the prevention and cure, especially in central and west regions, and the vast rural areas in China. At the same time, development of novel drugs, vaccines and diagnostic techniques are urgently needed.Diagnosis is the prerequisite to treatment. Currently, TB diagnostic methods largely base on imaging, microbiology, immunology, molecular biology with vary sensitivity and specificity. Serodiagnosis is gaining more and more attention due to time-saving, simplicity of operating, easy to sampling and extremely versatile. Many rapid TB test kits based on immunogold filtration assay strip are marketing. However, the performance evaluation sponsored by WHO/TDR in2008suggested that these kits were not recommended for clinical use mainly due to low sensitivity and specificity. The sensitivity and specificity of serodiagnosis largely hinge on the choice of diagnostic markers and test methods. Electrochemical immunosensor, combining the high specificity of immunoassay and high sensitivity of sensing technology, is widely used in detection of trace immunogenic substance. Here, cellular extracts of M.tb was separated by two-dimensional gel electrophoresis and electro-transferred completely to a PVDF membrane, followed by hybridization with TB patient sera. According to LC-MS/MS analysis, the strongest hybridization spot correspond to the transcriptional factor Rv2175c encoded by gene rv2175c. Recombinant Rv2175c protein was produced from E.coli and antibodies were generated in rabbit using the recombinant antigens. Rv2175c specific IgGs are too low in both health and TB patient serum and indistinguishable between them based on ELISA. But electrochemical immunosensor can detect a stronger signal from TB group than from health serum. This indicates that antibodies against protein Rv2175c or other proteins that can interact with Rv2175c exist in TB group. Thees results were thoroughly discussed, which will provide precious experience for our further work.In addition, during dissemination and infection, M.tb has to handle harsh extracellular and cellular situations it encounters. A robust and precise signal transduction system is imperative for handling the complex, volatile, harsh niche and persistent in the host. Signal transduction system of M.tb consists mainly of12two-component systems (TCSs),11eukaryotic-like Ser/Thr protein kinases(STPKs) systems,1protein tyrosine kinase(PTK) system and11extracytoplasmic function sigma factor (ECF-σ) systems. PtkA is the only tyrosine protein kinase present in M.tb and both autophosphorylation and substrate phoshorylation are occur on tyrosine residue. Tyrosine encoding codons are UAU or UAC, which are low GC content. Comparing to other amino acid, tyrosine is relatively low in M.tb with high GC content (65%). However, PtkA has been reserved during the long term evolution. So, it must undertake some important roles in M.tb. But until now, the substrate PtpA is the only thing we know about PtkA.To explore more physiological function of PtkA, protein expression profile between wild type and ptkA deletion mutant were compared in both M.tb H37Rv strain and M.bovis BCG by two-dimensional gel electrophoresis. The results indicate that the deletion of ptkA in M.tb H37Rv cause a remarkable change in protein expression profile, but no obvious change in M.bovis BCG LC-MS/MS analysis found that the3prominently down-expressed proteins in H37Rv ΔptkA strain are hypoxic response protein1, heat shock protein hspX (alpha-crystallin) and universal stress protein Rv2623. All these proteins involve in various stress response, immunogenicity and belong to DosR regulon, which is the main switch controlling the active or dormancy state in M.tb. This indicates PtkA might involve in stress response, immunogenicity and persistant directly or indirectly. And the sequence and location of ptkA and its neighboring genes are some between M.tb H37Rv strain and M.bovis BCG strain, but the effect of ptkA deletion are different. This hints that PtkA might regulate some global regulator in M. tb.Protein kinases mainly play their role by phosphorylating their substrates. PtpA is the only substrate has been identified for PtkA. In macrophage, PtpA block the fusion between phagosome and lysosome and inhibit phagosome acidification by binding to the subunit H of H+-ATPase and dephosphorylating human class C Vacuolar Protein Sorting VPS33in BHOPS (homotypic vacuole fusion and vacuole protein sorting) complex. This is a very important strategy for M.tb to persistant in the host. But what signal can activate PtkA and then to phosphorylate PtpA? Is PtkA itself been regulated by other protein? Does PtkA play some other roles in M.tb? To identify more cognate substrates, cellular extracts of M.tb was incubated with recombinant PtkA. Be interesting, PtkA phosphorylation level was increased when incobutae with M.tb cellular extracts compared to PtkA alone. This suggests that PtkA by itself can serve as a substrate for endogenic M.tb protein kinases. Combinating the existing information, we hypothesize that PtkA might be phosphorylated by one or more of the STPKs. Then, PknB, PknD, PknG and PknH were expressed and purified as GST or His-tag recombinant protein. The purified kinases were incubated with PtkA by using the γ-[32P] ATP as phosphate group donor. And we found that all these four kinases can phosphorylate PtkA with different intensity, in which PknD is the strongest one and followed by PknG. PknD phosphorylate PtkA in time and dose dependent manner further confirm that PtkA is a specific substrate of PknD. LC-MS/MS analysis reveal that PknD phosporylate PtkA on T119and another motif, which is close to the autophosphorylation site Y262. This reminds us PknD might modify the autophosphorylation activity by phosphorylating this motif. At last, STPKs might regulate PtpA through PtkA and affect the pathogen-host interaction and persistant of M.tb indirectly. This is another new example of convergence of different signal transduction systems in M.tb, which further reveal that M.tb signal transduction systems are very complicated and themselves can be regulated in multi-levels.