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

Abstract

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.

Edwardsiella piscicida YefM-YoeB: A Type II Toxin-Antitoxin System That Is Related to Antibiotic Resistance, Biofilm Formation, Serum Survival, and Host Infection

“…TX01, TX01Δ yoeB , TX01Δ yefM-yoeB , and TX01Δ yefM-yoeB C were cultured in LB medium to exponential phase and diluted to 10 5 CFU/mL. Crystal violet staining of biofilm assay was performed as previously ( Shi et al, 2019 ).…”

“…The observation of biofilms by confocal laser scanning microscopy (CLSM) was performed as described by Chan et al (2018) and Shi et al (2019) . Briefly, TX01, TX01Δ yoeB , TX01Δ yefM-yoeB , and TX01Δ yefM-yoeB C were grown in LB medium on glass-bottom dishes for 24 h at 28°C.…”

“…The biofilms were observed using a Leica TCS-SP2-AOBS-UV confocal laser scanning microscope equipped with an argon ion laser. The experimental procedures were performed three times ( Shi et al, 2019 ).…”

“…The speculative promoter of yefM-yoeB (the 345 bp of upstream of the yefM-yoeB operon), P345, was cloned with the primer pair YefM-YoeBP-F/YefM-YoeBP-R and inserted into the SwaI site of pSC11, a promoter probe plasmid ( Hu et al, 2009a ), which resulted in pSCP345. pSCP345 was introduced into E. coli DH5α by transformation and cultured on LB agar plates adding 20 μg/mL X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside, Solarbio, Beijing, China) ( Shi et al, 2019 ). To construct the overexpression plasmid pTYefM and pTYefM-YoeB, ORFs of yefM and yefM-yoeB were amplified with the primer pairs YefMF4/YefMR4 and YefMF4/YoeBR4, respectively.…”

“…The observation of biofilms by confocal laser scanning microscopy (CLSM) was performed as described by Chan et al (2018) and Shi et al (2019). Briefly, TX01, TX01 yoeB, TX01 yefM-yoeB, and TX01 yefM-yoeBC were grown in LB medium on glass-bottom dishes for 24 h at 28 • C. The dishes were rinsed to remove non-adherent bacteria and then stained with a LIVE/DEAD BacLight bacterial viability kit L-13152 (Invitrogen-Molecular Probes, Carlsbad, CA, United States) for observation of biofilms.…”