Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZ Ep , was characterized in E. piscicida. hutZ Ep is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZ Ep is probably not related to hemin utilization. To explore the biological role of HutZ Ep , a markerless hutZ Ep in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZ Ep did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZ Ep deletion, which indicated that HutZ Ep was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZ Ep significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZ Ep was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZ Ep is essential for E. piscicida biofilm formation and contributes to host infection.

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Edwardsiella piscicida is an important fish pathogen that causes substantial economic losses. In order to understand its pathogenic mechanism, additional new virulence factors need to be identified. The bacterial thioredoxin system is a major disulfide reductase system, but its function is largely unknown in E. piscicida. In this study, we investigated the roles of the thioredoxin system in E. piscicida (named TrxBEp, TrxAEp, and TrxCEp, respectively) by constructing a correspondingly markerless in-frame mutant strain: ΔtrxB, ΔtrxA, and ΔtrxC, respectively. We found that (i) TrxBEp is confirmed as an intracellular protein, which is different from the prediction made by the Protter illustration; (ii) compared to the wild-type strain, ΔtrxB exhibits resistance against H2O2 stress but high sensitivity to thiol-specific diamide stress, while ΔtrxA and ΔtrxC are moderately sensitive to both H2O2 and diamide conditions; (iii) the deletions of trxBEp, trxAEp, and trxCEp damage E. piscicida’s flagella formation and motility, and trxBEp plays a decisive role; (iv) deletions of trxBEp, trxAEp, and trxCEp substantially abate bacterial resistance against host serum, especially trxBEp deletion; (v) trxAEp and trxCEp, but not trxBEp, are involved in bacterial survival and replication in phagocytes; (vi) the thioredoxin system participates in bacterial dissemination in host immune tissues. These findings indicate that the thioredoxin system of E. piscicida plays an important role in stress resistance and virulence, which provides insight into the pathogenic mechanism of E. piscicida.