Studies On The Invasion Mechanism Of Virulence Mce1a Gene Of Mycobacterium Tuberculosis And The Rapid Classification And Detection Of Microbial 16S RDNAs

In recent years, the morbidity and mortality of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remain high. In the world, infected persons by Mtb are almost one third of the global population, and in every year, new sufferers and death patients for TB are about nine million and two million, respectively. In China, present patients with TB (mainly pulmonary TB) are about five million (80% in the countryside) and remain second most in the world. The numbers of onset or death TB patients constantly list first most in the A & B infectious diseases reported by Ministry of Health. Nowadays, the prophylactic effect of traditional BCG vaccine is not much satisfied, and incidents caused by drug-resistant Mtb are progressively increased. However, detailed pathogenesis of Mtb is still unclear, which becomes the great obstacle to develop new vaccines and more efficient and sensitive anti-TB drugs. In this study targetting mcela gene associated with Mtb invasion, firstly, we explored expression profiles of mcela. under states of dormant survival or active propagation of Mtb, cloned the full-length and truncated mcela genes, and then expressed and purified target proteins in E.coli, respectively. Secondly, two pieces of wcela-specific small interfering RNAs (siRNAs) were designed, in vitro transcribed, and determined their inhibitive activity and efficiency through cultured HEK 293-T cells. Based upon above work, mechanism of McelA protein involved in bacterial invasion was explored in cultured murine macrophages RAW 264.7 and human colonic carcinoma CT 26.CL25 epithelial cells, through T-Mcel A protein stimulations, interference experiments by specific siRNA, and the in vitro invasion assay by recombinant McelA-expressing E.coli. In addition, on the bases of multiple sequence alignments and phylogenetic tree analyses of various microbial 16S rDNAs, we designed and synthesized bacterial universal and specific primers, and developed the corresponding rapid classification and detection methods by real-time fluorescence quantitative PCR (FQ-PCR).1. Expression profiles of mcela gene under states of dormancy or propagation of MtbAs a kind of obligate aerobic intracellular bacterium, Mtb can survive inactively in the host for long period, and then resuscitate and propagate to cause disease under certain conditions. In this study, to investigate the expression profile of mcela in non-replicating Mtb (below 25℃) which were close to intracellular circumstances of latent bacilli, we detected mRNA transcripts of this gene in two standard strains (virulent H37Rv and attenuated H37Ra) and eight clinically isolated strains (four strains are sensitive, two are rifampicin resistant, and two are isoniazide resistant) through RT-PCR, all of which were stored at 4 ℃ for three months. We also detected expressions of mcela in the same ten MTB strains cultured at 37 ℃ from two weeks till five weeks. Results showed that mcela expressed stably at a similar level in different Mtb strains during actively growing stages, and that transcription of this gene was not detected in non-replicating bacilli, which hintedthat expression of wee la might be related with the invasion and latency abilities of Mtb. 2. Mechanism of Mcel A protein involved in bacterial invasionFirstly, the full-length and truncated mcel a genes were cloned and expressed in E.coli DH5a. After three pairs of PCR primers including Exo-primer, In-primer, and In-primer 2, were designed and synthesized, the full-length (wee la) and truncated wee la (T- ntce la) genes were amplified by nest-PCR, and inserted into the prokaryotic expression vectors pROEX-HTb with 6xHis tag and pBV220 induced through heating. Target proteins were induced to express in corresponding recombinant E.coli by IPTG or heating to 42 °C, and purified by the Ni-NTA column or molecular sieve. Then, bacterial lysates and purified products were subjected to SDS-PAGE, and samples from recombinant E.coli (pROEX-mcela) were analyzed by Western blot with the anti-6xHis MAb. Results demonstrated as follows, (i) The recombinant E.coli contained pROEX-mcela or pBV-Tmcela, expressed an about 45 or 27 kDa protein in accordance with the predicted molecular weight (MW), respectively. The intact Mcel A protein containing N terminal signal peptide expressed in non-soluble form, and might locate stably on the cell wall or membrane. The truncated T-Mcel A protein containing no signal peptide expressed mainly in the inclusion body form, and partly in soluble form (about one third), which reflected that N terminal signal sequence influences the location of Mcel A. (ii) After purifications by Ni-NTA column or molecular sieve, both target proteins with correct MW were acquired. However, Mcel A was of low yield because of its membrane location, and T-Mcel A was of relatively high yield (about 20% of whole bacterial proteins).Secondly, wee la-specific siRNAs were designed, transcribed, and subjected to the interference experiments in HEK 293-T cells. For the convenience of detection, wee la was cloned to the upstream of enhanced green fluorescence protein (EGFP) gene to construct a eukaryotic pEGFPNl-mcela plasmid which expresses McelA-EGFP fusion protein. After designing and screening of siRNA templates through online softwares "siRNA Target Finder" and "BLAST", wee la-specific si312 and si410, and the positive and negative control siEGFP and silRR were synthesized through the T7 in vitro transcription kit. We co-transfected pEGFPNl-mcela and different siRNAs (2.0 ul, 1.5 nmol/ul) in HEK 293-T cells with lipofectamine, and determined the inhibitive activity of specific siRNAs through fluorescence microscopy, semi-quantitative RT-PCR, and Western blot. Then, different siRNAs were co-transfected with various dosage gradients, and their silencing effects and efficiencies were detected by fluorescence microscopy, flow cytometry, FQ-PCR, and Western blot. Results showed as follows, (i) It is rapid and convenient to synthesize siRNAs with 21 bp in length through the in vitro transcription method by T7 RNA polymerase. The siRNAs can be efficiently delivered to target cells by transfection with lipofectamine. HEK 293-T cells express Mcel A at high level and can be chose as the idealcell line in related studies, (ii) In transcription level, si410 and si312 can inhibit mcel a mRNA by 90% and 88%, respectively, which approach to the inhibitive effeciency of siEGFP (92%). In translation level, si410 and si312 silence McelA expression by 51% and 49%, respectively, which are lower than the inhibitive effeciency of siEGFP (70%). (iii) As Western blot exhibited, two close intact bands with 85 kDa in MW present target proteins, from which we supposed that part of McelA-EGFP fusion protein might be of glycosylation modification while expressed in HEK293-T cells, (iv) Various siRNAs targeting the same gene get to the equivalent inhibitive effeciency at different time point, which might be related with different cutting effeciency of corresponding RISC complex.Thirdly, the mechanism of McelA protein in inducing bacterial invasion was explored. On one hand, since we have acquired purified T-McelA protein and efficient specific siRNAs, it was studied that T-McelA's effects upon the expressions of TLR-2 and related signal molecules in macrophages from the obverse (stimulating by T-Mcel A protein with various concentrations) and reverse (silencing of McelA expression by si410) angles, respectively. On the other hand, the recombinant Mcel A-expressing E.coli was took for the in vitro invasion assay in cultured RAW 264.7 and CT 26.CL25 cells. Results showed as follows, (i) The expression of TLR-2 was enhanced in RAW 264.7 cells stimulated by T-McelA protein, following which expressions of downstream signal molecules including IRAK-1 and TRAF-6 were up-regulated too, with IRAK-l's expression changed more significantly. As known, activations of TLR-2, IRAK-1, and TRAF-6 lead to the activation of NF-kB, one of nuclear factors, which plays a central regulation role in the cellular inflammation response. Therefore, the activation function of McelA protein upon TLR-2 and even IRAK-1 and TRAF-6 in RAW 264.7 cells, might be one of important factors in the cellular inflammation response induced by Mtb. (ii) The recombinant E.coli was conferred abilities of invasion and survival in RAW 264.7 cells for at least 48 h, accompanied with the suffered RAW 264.7 and CT 26.CL25 cells exhibiting some pathological changes. Especially in RAW 264.7 cells, the recombinant bacilli caused the suffered cells becoming larger, and showing several bigger nuclei and significant membrane protrusions, which mean the cytoskeletal reorganization. So, it is concluded that the Mcel A-expressing E.coli has certain invasion ability and can escape the immune killing of macrophages, and that the mechanism of McelA protein in inducing bacterial invasion might be associated with cytoskeletal reorganizations of target cells.To summarize all the results, it can be inferred that McelA protein might induce the invasion, the immune escape, and the progression to dormancy or pathogenesis of Mtb, through activating TLR-2 and its downstream inflammatory signal molecules including IRAK-1, and participating in regulations of host inflammation response. Moreover, according to recent reports about invasion mechanisms of Listeria, Salmonella, Shigella, Yersinia, and Mycobacterium avium complex (MAC), we are like to raise the possibleinvasion mechanism of Mtb as follows, (i) The invasion mechanism of Mtb may be the same to or like that of MAC, the zipper-trigger mechanism, (ii) The regulation function of McelA in host inflammation responses might be one important initial factor causing the zipper-trigger related cytoskeletal reorganization.3. Rapid classification and detection of microbial 16S rDNAs by FQ-PCRFounded on the multiple sequence alignments and phylogenetic tree analyses of 16S (18~28S) rDNAs of 58 strains of bacteria, Chlamydia, mycoplasma, and fungi, the bacterial universal and specific FQ-PCR primers were designed in the high homologous and specific regions, respectively. Total DNAs were extracted and purified from over 20 standard or clinically isolated strains including P. aeruginosa, S. aerus, S. citreus, S. typhi, S. paratyphi, S. Jlexneri, B. proteus, S. epidermis, S. hominis, S. pneumoniae, E. coli, B. anthracis, Mtb, U. urealyticum, C. albicans, and C. krusei. Target fragments of bacterial 16S rDNAs were amplified by PCR with universal and specific primers, respectively, and used to construct recombinant pMD18-T plasmids as the standards of FQ-PCR. The 20 ul of reaction systems were developed with TaKaRa SYBR? Premix Ex Taq? and used for rapid detections of dilution' gradients of various DNA samples by FQ-PCR. Results of phylogenetic tree analyses showed that 16S (18-28S) rDNA can be chose as the ideal target gene for rapid classifications and detections of bacteria, Chlamydia, mycoplasma, and fungi. Findings in FQ-PCR demonstrated that rapid detections of various bacterial 16S rDNAs can be performed in about 2 h with universal and specific primers, and that the method is of good specificity and sensitivity (about 2~5 pg/ul of total bacterial DNA, or 2xl03 + 3*102 copies of 16S rDNA gene).