Biological Characteristics And Functions Of CagT And CagM In Helicobacter Pylori

Helicobacter pylori, a microaerophilic, Gram-negative bacterium that efficiently colonizes in the stomach of about half of the world's population, is one of the most common infectious agents and can be considered as a prototype for microbial persistence. Infections occur in early childhood and persist for decades in the absence of targeted antimicrobial therapy. Studies of H. pylori have revealed not only its ability to coexist for many decades within a single host, but also that the species has coexisted with its host for much of human history. Using sequences from a large data set of bacterial strains that, as in humans, genetic diversity in H. pylori decreases with geographic distance from east Africa, the cradle of modern humans before 58 000 years. While many H. pylori infected individuals are clinically asymptomatic, most will exhibit some degree of gastritis. Approximately 10% of the infected subjects will develop more severe gastric pathologies like peptic ulcer disease and atrophic gastritis. Gastric cancer develops in approximately 1% of infected individuals, a correlation that prompted the World Health Organization to declare H. pylori the first bacterial class I carcinogen in 1994.The cytotoxin-associated gene pathogenicity island (cag PAI) of H. pylori is a 40-kb genomic insert region that is comprised of 28 to 30 genes. A portion of these genes, encode a type IV secretion system (T4SS), are responsible for the injection of the only known effector molecule, CagA, into host cell. Then, the host cell is tyrosine-phosphorylated and subsequently induces proliferation, differentiation and cell elongation. CagA has been associated with rearrangements of the actin cytoskeleton resulting in phenotypic changes called the scattering or "hummingbird" phenotype. Another cellular response caused by the cag secretion system is the production and secretion of interleukin-8 (IL-8) by epithelial cells, which is independent of the CagA protein in some H. pylori strains. CagT (HP0532) is one of the cag PAI proteins; its mutant strain can block the translocation of CagA into host cell, induction of the "hummingbird" phenotype and IL-8 secretion. For the important role of cag PAI, many researchers at home and abroad carried out studies on it. CagT is an essential gene in cag PAI; it plays an important role in host pathological damage caused by H. pylori. The CagT was found at the base of the rigid needle structure of cag PAI, covered by CagY.Both proteins construct the framework of cag PAI. CagM is one of the absolutely essential components for CagA delivery and IL-8 secretion. However, there is little known about both of the mechanism of action in the pathogenesis of cag PAI. My research mainly discussed the biological characteristics and functions of CagT and CagM in H. pylori,1. H. pylori CagT, CagM and CagA protein:Expression, purification and their immunoreactivityThe genome of H. pylori 26695 was purified with Bacteria DNA Kit. The cagM and cagT genes without the signal peptide sequences, and a segment of cagA gene were amplified by PCR with genomic DNA from H. pylori 26695 strain.The three amplified products were cloned by T/A method. Then, the target fragments were produced with corresponding restriction enzymes. The cagMfragment was linked to pQE30 vector, and then transferred into the host bacteria E. coli M15. As the same, the cagT fragment was linked to pET-28a(+) vector and the cagA fragment was linked to pET-30a(+) vecter, then both of the recombinant'expression plasmids were transferred into the host bacteria E. coli BL21 (DE3).CagM, CagT and CagA proteins were expressed with lmmol/L IPTG inducing, and all of three proteins could be expressed in both soluble form and inclusion body. Inclusion bodies of CagM and CagA were washed with inclusion body eluent, and the washed inclusion bodies and soluble CagT were purified by affinity chromatography. Antibody against CagM, CagT and CagA proteins were acquired by immunization of rabbits and the immunoreactivity of the proteins were evaluated through respective antibodies. ELISA and Western blotting assays proved that three recombinant proteins have perfect immunoreactivity.2. Construction and identification of the H. pylori cagT,cagM and cagA isogenic knockout mutantsThe Cam gene conserved in pHel2 plasmid was linked to pBluescript SK(-) plasmid after the two plasmid were dealt with SacⅡand XbaⅠ. The upstream and downstream fragments of cagM, cagT and cagA were amplified by PCR with genomic DNA from H. pylori 26695 strain. The amplified products were cloned into pMD19-T vectors by T/A method, respectively. The upstream fragments of three genes were dealt with SacⅡand Xbal, then introduced into pBluescript SK(-) which contained Cam, and the downstream fragments of three genes were dealt with Apal and SalⅠ, and introduced into pBluescript SK(-) which was inserted the Cam gene and the respective upstream fragment, so that to construct suicide plasmid of the cagM, cagT and cagA knockout strains. The constructed isogenic knockout plasmids were introduced into H. pylori 26695 strain by electroporation. The transformants were grown on skirrow's blood agar plates supplemented with 25μg/ml of chloramphenicol. The resulting chloramphenicol-resistant colonies were selected and evaluated by PCR and Western blotting to confirm the replacement of the wild-type with the mutant strains. The mutants were designated asΔcagT,ΔcagM, and AcagA.The successful construction of these mutant strains could provide wonderful tools for researching the biological effect of CagT and CagM in the translocation of CagA into host cell.3. The influence of CagT and CagM on the translocation and phosphorylation of CagA and the IL-8 secretion of host cells.H. pylori strains 26695,11637,ΔcagA,ΔcagT and AcagM were grew on liquid cultures. AGS gastric epithelial cells were infected with these H. pylori strains at a multiplicity of infection of 200 per cell when the bacteria concentration up to OD 0.5-1.0.The host response was monitored using three different read-out systems:(i) induction of a scattering phenotype, (ii) translocation and phosphorylation of CagA, and (iii) IL-8 secretion. Infection of AGS cells with H. pylori wild-type strains 26695 and 11637 induced IL-8 secretion and translocation and phosphorylation of CagA. Cells infected with these wild-type strains were significantly elongated and had a spindle-like morphology in the infected cells that has been referred to as the scattering or hummingbird phenotype. In agreement with these results, we found that all cag PAI gene knockout strains (ΔcagT and AcagM) of the contact-dependent type IV transporter complex were defective for IL-8 secretion, translocation and phosphorylation of CagA, and induction of the hummingbird phenotype, demonstrating the essential function of CagT and CagM in each of these host responses and translocation of CagA. As the control, cagA isogenic mutant strain was defective for translocation and phosphorylation of CagA, and induction of the hummingbird phenotype, but sufficient for IL-8 secretion.4. Biological characteristics and several functions of CagT and CagM in H. pyloriThis section mainly researched the biological characteristics and several biological functions of CagT and CagM. Firstly, we detected whether the molecular modification of CagT and CagM occur and molecular size charged in wild-type H. pylori which contain the intact cag PAI. H. pylori wild-type strains were broke by sonication. CagT and CagM did not modify in wild-type strains by Western blotting detection. The H. pylori were fractionated though ultracentrifugation and detergents, then, the location of CagT and CagM in strain were identified. Combined bioinformatics prediction with our result, we demonstrated that both CagT and CagM located to the inner and outer membrane in bacteria. For CagM, CagT and CagA can locate to the inner membrane, we detected the interaction between CagA and CagT/CagM. The coimmunoprecipitation outcome indicated that CagA could interact with CagT; however, it could not interact with CagM. Moreover, we also investigated whether CagM and CagT secret into extracellular supernatants. Interestingly, the two proteins did not secrete into extracellular supernatant. Their function could be depended on the anchoring to cytomembrane. Finally, Western blotting provided that cagM isogenic knockout mutant did not influence the expression and stability of CagT.Overall, our results suggest that CagT may serve as a unique multifunctional component of the T4SS that may be involved in CagA secretion at the bacterial membrane. It is one of key structural proteins which are involved in cag PAL It is also a chaperonin that interacts with CagA at the inner membrane and help the translocation of CagA into host cells. CagM is one of key structural proteins and is essential for translocation and phosphorylation of CagA and induction of the hummingbird phenotype.