Pathogenic Bacteria Cytotoxin And Infection Relataed High Level Ca2+Induce Cell Apoptosis

Part1:Apoptosis-inducing effect and its relative mechanism of Vibrio vulnificus cytolysin to HUVEC, SGC-7901and SMMC-7721Cells in vitroBackground and Objective Vibrio vulnificus is a gram-negative halophilic bacterium originating from warm coastal waters. It causes fatal septicemia and serious necrotizing wound infection in those who ingest raw contaminated seafood or have open wounds exposed to contaminated seawater. V. vulnificus is known to be a life-threatening pathogen for its high lethality rate of70%. Vibrio vulnificus cytolysin (VVC) is one of the most important causative agents of V. vulnificus. It is known to be a pore-forming toxin which shows hemolytic activity for mammalian erythrocytes. Besides the hemolytic activity, VVC had also proved to be cytotoxic to a variety of mammalian cell lines such as mast cell, endothelial cell, and macrophage. And apoptosis induced by VVC in endothelial cells has also been demonstrated. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, we constructed a prokaryotic expression system of vvhA gene to express large amount of recombinant VVC (rVVC). Subsequently, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901and SMMC-7721cells were investigated.Methods The entire vvhA gene that encoding VVC from V.vulnificus strain GTC333was amplified by PCR and sequenced after T-A cloning. E.coli BL21DE3PET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then constructed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The hemolytic and cytotoxic activity of the purified cytolysin was confirmed by rabbit erythrocyte hemolysis test and MTT test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+in the supernatants of rVVC-treated HUVEC, SGC-7901and SMMC-7721cultures, respectively. Morphologic change of rVVC-treated cells was observed under transmission electron microscope. The effect of rVVC inducing apoptosis of HUVEC, SGC-7901and SMMC-7721was detected by flow cytometry. The activation of caspase-3,-8,-9in rVVC-treated cells was measured by Caspase-3,-8,-9Fluorometric Assay Kits. In the further experiment, rVVC was labeled with FITC and the location of FITC-labeling rVVC in HUVEC, SGC-7901and SMMC-7721cells was observed by laser confocal microscopy.Results The cloned vvhA gene had96.09%and98.26%similarities of nucleotide and amino acid sequences compared to the corresponding sequences in GenBank. rVVC acted as an agent to induce the death of HUVEC, SGC-7901and SMMC-7721cells in a dosage-dependent manner from1～100μg/mL. rVVC, with a dosage of1μg/mL, could dissolve rabbit erythrocytes (P<0.01).10μg/mL rVVC was able to promote the increases of K+content (P<0.01) but no change of LDH content could be found in the cell supernatants. Typical apoptotic morphological characters were observed in the HUVEC, SGC-7901and SMMC-7721cells treated with10μg/mL rVVC for4h. The VVC-induced apoptosis was further confirmed by flow cytometry to be time-and dosage-dependent, which was associated with the activation of caspase-9and-3, but not caspase-8. It was also detected that FITC-labeling rVVC attached itself initially to the surface of all the target cells and entered the cytoplasma subsequently in the10～240min duration of co-incubation. When FITC-labeling rVVC treated cells for30min, most of the rVVC was found to be intracellular location.Conclusion rVVC has cytolytic activity. VVC exerts apoptotic action on HUVEC, SGC-7901and SMMC-7721cells vai caspase-9/3dependent pathway, which may be the major pathogenic mechanism of VVC. The cytolysin is able to quickly enter the cytoplasma of target cells after a brief superficial attachment, rather than act locally at the cell membrane. VVC not only acts as a hemolysin but also has an ability to induce apoptosis in human vascular endothelial cells and tumor cells. Part2:Changes of cytosolic free calcium and induction of apoptosis by Leptospira interrogans infectionBackground and Objective Leptospirosis is a world-wide amphixenosis caused by spirochetes belonging to the genus Leptospira. Infection with pathogenic leptospira species can cause acute febrile and systemic illness. The basic clinical symptom is hemorrhage and jaundice. Despite the recent advances in Leptospira-host cell interaction, the knowledge of the physiopathology of leptospirosis remains very limited. Alteration of intracellular Ca2+concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. It was reported that the elevation of Ca2+level was induced by many kinds of bacteria after they adhered to host cell, which triggered rapid and precise signals and spatially assemble host molecules for internalization in a specific intracellular compartment. Ca2+has been implicated as a second messenger during macrophage response to infectious agents, and bacterial pathogens have developed strategies to divert Ca2+-dependent responses, including phagocytosis or superoxide production in macrophages. Many bacterial pathogens have developed specific strategies to survive challenge with professional phagocytes, including the induction of macrophage apoptosis, inhibition of phagocytosis, escape from the phagocytic vacuole, or diversion of intracellular routing. In order to determine wether Leptospira can induce the elevation of Ca2+level after adhenrence to cells and understand the potential mechanism of the Ca2+elevation and its relationship with cell apoptosis, the changes in [Ca2+]i induced by leptospira infection in J774A.1and THP-1cells were observed by using Laser Scanning Confocal Microscope after cytosolic free calcium was fluorescent stained with Fluo-4/AM. Various Ca2+blockers were employed to analysis the source of elevated Ca2+and the relationship of high level Ca2+and apoptosis induced by Leptospira.Methods Changes in [Ca2+]i induced by leptospira infection in J774A.1and THP-1cells were observed before or after by using Laser Scanning Confocal Microscope after cytosolic free calcium was fluorescent stained with Fluo-4/AM. To determine the source of elevated Ca2+, the Cells were pretreated with extracellular Ca2+chelator EGTA, intracellular Ca2+chelator BAPTA/AM, L-type Ca2+channel blocker verapamil, T-type Ca2+channel blocker mibefradil, receptor-operator Ca2+channel blocker SKF96365and phospholipase C inhibitor U73122, and then the Ca2+fluorescence intensity after infecting with leptospira were detected again. The cell apoptosis induced by leptospira before or after Ca2+blockage were detected by flow cytometry. Through bioinformatics analysis, PLC related genes in leptospira were predicted, and the prokaryotic expression systems were constructed. The PLC activity of the recombinant proteins were determined by Malachite Green Phosphatase Assay Kit and HitHunterTM Inositol(1,4,5)-Triphosphate Assay Kit. The gene (LB361) which its product confirmed to have PLC activity was knock-out and its revertant was constructed based on the LB361mutant. Changes in [Ca2+]i induced by leptospira LB361mutant and LB361com revertant were also determined.Results The resting fluorescence intensity of intracellular free Ca2+levels in normal J774A.1and THP-1cells were99.3±3.7%and105.3±7.7%, respectively. After cells were infected with L. interrogans strain lai, the calcium fluorescence intensities were observed to be significant elevated, and the concentration reached a maximum of436.9±14.4%(J774A.1) and486.8±20.1%(THP-1) after infected for60min. Chelation of extracellular Ca2+with EGTA (extracellular Ca2+chelator) and suspension of cells in Ca2+free buffer, preloading cultures with intracellular Ca2+chelator BAPTA/AM, both of them lead to a less elevated peak of Ca2+compared with chelator untreated cells. Receptor-operator Ca2+channel blocker SKF96365inhibited the elevation of Ca2+level notably. While the voltage-sensitive L-type Ca2+channel inhibitor Verapamil and T-type Ca2+channel inhibitor mibefradil presented less effectiveness on inhibiting Ca2+elevation. The PLC inhibitor U73122and Neomycin also presented to be of great efficiency in inhibiting the elevation of Ca2+. These inhibitors are not toxic to the host cells at the concentration used. Apoptosis were induced in J774A.1and THP-1cells after infected with L. interrogans strain lai, and blockage of cytosolic free Ca2+can inhibit leptospira induced apoptosis. Three PLC related genes (LA0543,LA2250,LB361) were predicted through bioinformatics analysis, and the product of LB361were confirmed to has PI-PLC activity. LB361- mutant can cause slight elevation of Ca2+level, which is far low from the peak value of Ca2+elevation induced by wild strain.Conclusion L. interrogans strain lai infection induced a significant elevation of Ca2+level in J774A.1and THP-1cells, which was suggested to be the combination effect of both intracellular calcium store depletion and Ca2+influx from extracellular environment. Ca2+influx from extracellular environment was mainly mediated by receptor-operator Ca2+channel. Leptospira infection first induce the rise of [Ca2+]i through receptor-operator Ca2+channel, and subsequently activated PLC to induce calcium release from intracellular calcium store. Leptospira infection induces cell apoptosis in J774A.1and THP-1cells, which may be related with its induction of Ca2+elevation. The product of LB361gene was confirmed to be a PI-PLC, which can hydrolyze PIP2to generate IP3, IP3binds and opens endoplasmic IP3gated calcium channel, resulting in the release of bound calcium into the cytosol. LB361gene hence is a virulence factor of Leptospira, playing an impotant role in Leptospira infection induced Ca2+elevation and cell apoptosis, virulence factor...