The type III secretion system (T3SS) of Edwardsiella piscicida plays a crucial role in its pathogenesis. Our previous study indicated that the T3SS effector protein EseJ inhibits the bacterium’s adhesion to epithelioma papillosum cyprini (EPC) cells, while the mechanism of the inhibition remains elusive. In this study, we revealed that EseJ negatively regulates the fimA gene, as demonstrated by comparative transcription analysis of ΔeseJ and wild-type (WT) strains. As well, the dramatically increased production of FimA was detected in the absence of EseJ compared to that by the WT strain. The adherence of the ΔeseJ strain decreased far below that of the WT strain in the absence of FimA, demonstrating that FimA plays a pivotal role in the hyperadhesion of the ΔeseJ strain. Adherence analysis with a strain with truncated eseJ demonstrated that the C-terminal region of EseJ (Gly1191 to Ile1359) is necessary to inhibit the transcription of the type 1 fimbrial operon. Binding between the EseJ fragment from amino acid residues 1191 to 1359 and the DNA fragment upstream of fimA was not detected, indicating that EseJ might indirectly regulate the type 1 fimbrial operon. Our study reveals that EseJ controls E. piscicida adherence to EPC cells by negatively regulating the type 1 fimbrial operon.

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild-type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining and cleaved caspase-3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase-8, caspase-9, and caspase-3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre-treatment of macrophages with caspase-8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase-8, caspase-9, and caspase-3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae. K E Y W O R D Sapoptosis, Edwardsiella piscicida, type 1 fimbriae, type III protein EseJ