HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

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.