Filgrastim-txid

Edwardsiella piscicidais 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, HutZEp, was characterized inE. piscicida.hutZEpis encoded with two other genes,hutWandhutX, in an operon that is similar to the haem utilization operonhutWXZidentified inV. cholerae. However, protein activity analysis showed that HutZEpis probably not related to hemin utilization. To explore the biological role of HutZEp, a markerlesshutZEpin-frame mutant strain, TX01ΔhutZ, was constructed. Deletion ofhutZEpdid 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 byhutZEpdeletion, which indicated that HutZEpwas probably a novel factor promoting biofilm formation inE. piscicida. Compared to the wild-type TX01, TX01ΔhutZexhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation ofhutZEpsignificantly impaired the ability ofE. piscicidato invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZwas defective in blocking host macrophage activation. The expression ofhutZEpwas 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 HutZEpis essential forE. piscicidabiofilm formation and contributes to host infection.

Universal stress proteins (Usps) exist ubiquitously in bacteria and other organisms. Usps play an important role in adaptation of bacteria to a variety of environmental stresses. There is increasing evidence that Usps facilitate pathogens to adapt host environment and are involved in pathogenicity. Edwardsiella piscicida (formerly included in E. tarda) is a severe fish pathogen and infects various important economic fish including tilapia (Oreochromis niloticus). In E. piscicida, a number of systems and factors that are involved in stress resistance and pathogenesis were identified. However, the function of Usps in E. piscicida is totally unknown. In this study, we examined the expressions of 13 usp genes in E. piscicida and found that most of these usp genes were up-regulated expression under high temperature, oxidative stress, acid stress, and host serum stress. Particularly, among these usp genes, usp13, exhibited dramatically high expression level upon several stress conditions. To investigate the biological role of usp13, a markerless usp13 in-frame mutant strain, TX01Δusp13, was constructed. Compared to the wild type TX01, TX01Δusp13 exhibited markedly compromised tolerance to high temperature, hydrogen peroxide, and low pH. Deletion of usp13 significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis showed that the inactivation of usp13 significantly impaired the ability of E. piscicida to invade into host cell and infect host tissue. Introduction of a trans-expressed usp13 gene restored the lost virulence of TX01Δusp13. In support of these results, host immune response induced by TX01 and TX01Δusp13 was examined, and the results showed reactive oxygen species (ROS) levels in TX01Δusp13-infected macrophages were significantly higher than those in TX01-infected cells. The expression level of several cytokines (IL-6, IL-8, IL-10, TNF-α, and CC2) in TX01Δusp13-infected fish was significantly higher than that in TX01-infected fish. These results suggested that the deletion of usp13 attenuated the ability of bacteria to overcome the host immune response to pathogen infection. Taken together, our study indicated Usp13 of E. piscicida was not only important participant in adversity resistance, but also was essential for E. piscicida pathogenicity and contributed to block host immune response to pathogen infection.