Secreted in a Type III Secretion System-Dependent Manner, EsaH and EscE Are the Cochaperones of the T3SS Needle Protein EsaG of Edwardsiella piscicida.

The intracellular EscE protein tightly controls the secretion of the type III secretion system (T3SS) middle and late substrates in Edwardsiella piscicida. However, the regulation of secretion by EscE is incompletely understood. In this work, we reveal that EscE interacts with EsaH and EsaG. The crystal structures of the EscE-EsaH complex and EscE-EsaG-EsaH complex were resolved at resolutions of 1.4 Å and 1.8 Å, respectively. EscE and EsaH form a hydrophobic groove to engulf the C-terminal region of EsaG (56 to 73 amino acids [aa]), serving as the cochaperones of T3SS needle protein EsaG in E. piscicida. V61, K62, M64, and M65 of EsaG play a pivotal role in maintaining the conformation of the ternary complex of EscE-EsaG-EsaH, thereby maintaining the stability of EsaG. An in vivo experiment revealed that EscE and EsaH stabilize each other, and both of them stabilize EsaG. Meanwhile, either EscE or EsaH can be secreted through the T3SS. The secondary structure of EsaH lacks the fourth and fifth α helices presented in its homologs PscG, YscG, and AscG. Insertion of the α4 and α5 helices of PscG or swapping the N-terminal 25 aa of PscG with those of EsaH starkly decreases the protein level of the chimeric EsaH, resulting in instability of EsaG and deactivation of the T3SS. To the best of our knowledge, these data represent the first reported structure of the T3SS needle complex of pathogens from Enterobacteriaceae and the first evidence for the secretion of T3SS needle chaperones. IMPORTANCE Edwardsiella piscicida causes severe hemorrhagic septicemia in fish. Inactivation of the type III secretion system (T3SS) increases its 50% lethal dose (LD50) by ~10 times. The secretion of T3SS middle and late substrates in E. piscicida is tightly controlled by the intracellular steady-state protein level of EscE, but the mechanism is incompletely understood. In this study, EscE was found to interact with and stabilize EsaH in E. piscicida. The EscE-EsaH complex is structurally analogous to T3SS needle chaperones. Further study revealed that EscE and EsaH form a hydrophobic groove to engulf the C-terminal region of EsaG, serving as the cochaperones stabilizing the T3SS needle protein EsaG. Interestingly, both EscE and EsaH are secreted. Our study reveals that the EscE-EsaH complex controls T3SS protein secretion by stabilizing EsaG, whose secretion in turn leads to the secretion of the middle and late T3SS substrates.

Identification and Characterization of EvpQ, a Novel T6SS Effector Encoded on a Mobile Genetic Element in Edwardsiella piscicida.

In this study, a hypothetical protein (ORF02740) secreted by Edwardsiella piscicida was identified. We renamed the ORF02740 protein as EvpQ, which is encoded by a mobile genetic element (MGE) in E. piscicida genome. The evpQ gene is spaced by 513 genes from type VI secretion system (T6SS) gene cluster. Low GC content, three tRNA, and three transposase genes nearby evpQ define this MGE that evpQ localizes as a genomic island. Sequence analysis reveals that EvpQ shares a conserved domain of C70 family cysteine protease and shares 23.91% identity with T3SS effector AvrRpt2 of phytopathogenic Erwinia amylovora. Instead, EvpQ of E. piscicida is proved to be secreted at a T6SS-dependent manner, and it can be translocated into host cells. EvpQ is thereof a novel T6SS effector. Significantly decreased competitive index of ΔevpQ strain in blue gourami fish (0.53 ± 0.27 in head kidney and 0.44 ± 0.19 in spleen) indicates that EvpQ contributes to the pathogenesis of E. piscicida. At 8-, 18-, and 24-h post-subculture into DMEM, the transcription of evpQ was found to be negatively regulated by Fur and positively regulated by EsrC, and the steady-state protein levels of EvpQ are negatively controlled by RpoS. Our study lays a foundation for further understanding the pathogenic role of T6SS in edwardsiellosis.

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae, which stimulate the cleavage of caspase-8.

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.

The Edwardsiella piscicida Type III Effector EseJ Suppresses Expression of Type 1 Fimbriae, Leading to Decreased Bacterial Adherence to Host Cells.

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.