Establishment Of Susceptible Cell Models And Host-bacterial Pathogen Interactions Of Edwardsiella Tarda

Edwardsiella tarda is an important gram-negative bacterial pathogen of fish.However, due to the lack of adequate cell models, the invasive features of E. tarda tofish cells and the pathogenesis of host cell death have not been thoroughlyinvestigated. In this study, three fish cell lines were used for establishment ofsusceptible cell models, and for further investigation of the interactions between E.tarda and its cellular hosts. E. tarda invaded and replicated in all cell lines, andsusceptible cell models were established. Epithelioma papulosum cyprini (EPC) cellswere more sensitive to E. tarda infection than the flounder gill cell line FG-9307andzebrafish embryonic cell line ZF4, with higher levels of intracellular bacteria in EPCcells. E. tarda strains from different orgins showed distinct adherence and cytotoxicityto the FG-9307cells. The invasion and intracellular replication of E. tarda inFG-9307cells were studied at the ultrastructural level, and increased formation ofstructures resembling cellular protrusions were found in the proximity of the site ofbacterial entry. Concurrently observed was the membrane absence at the point ofcontact in some cases, implicating an entry process through a―trigger mechanism‖.The bacteria were firstly internalized in a membrane-bound vacuole. Large amountsof replicated bacteria were found free in the cytosol after extended incubation ingentamicin resistance assay, indicating that the bacteria managed to escape from thevacuole and gained access to the cytosol of host cells. Extensive electron-lucent areaswith destroyed organelles in the cytoplasm and impaired plasma membrane withescaping bacteria were also observed following extended incubation. Infection ratesincreasd as the incubation time extended after gentamicin treatment, suggesting thatthe large number of intracellular replicated bacteria were at least partly on account ofsecondary infection. Moreover, Apoptosis was observed in EPC cells upon infection,characterized by the occurrence of apoptotic bodies, DNA ladder, increased Annexin V binding and the activation of caspase-3, whereas E. tarda infected FG-9307cellsand ZF4cells were negative for the above features. Both intrinsic and extrinsicpathways were activated in EPC cells upon E. tarda infection. And E. tarda infectionin FG-9307cells failed to protect the staurosporine-induced apoptosis. Furthermore,mutants with in-frame deletion of two genes involved in ATP-binding cassettesystems, respectively, were constructed to investigate their roles in antibioticresistance and virulence. The complemented strains0613+and0907+were alsoconstructed, and it was found that the complemented genes in0613+and0907+wereboth overexpressed, as determined by qRT-PCR. The Δ0613mutant showed decreasedresistance to several aminoglycosides and tetracycline antibiotics, but no significantchange in invasion abilities and virulence.0613+partly restored the wild-typeantibiotic resistance, and was attenuated in respect to internalization in EPC cells,mortalities in zebrafish embryos and LD50in adult zebrafish.0907+also showedattenuated virulence in zebrafish. In conclusion, the present study revealed that E.tarda interacts with fish cells in different manners, and divergent pathways wereactivated in these cellular hosts to mediate cell death. The study also indicated that theABC transporter0613of E. tarda could be a new multidrug resistance pump, which,together with0907, can severely attenuate virulence when overexpressed. Theseresults provided new information on the interactions between E. tarda and fish cells.