Effect of the Flagellar Gene fliL on the Virulence of Pseudomonas plecoglossicida to Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Abstract

Pseudomonas plecoglossicida is the pathogen of visceral white spot disease in marine fish, which usually occurs at 16–19 °C and has resulted in heavy economic losses. Our previous RNA sequencing revealed that the expression of the fliL gene in P. plecoglossicida was significantly up-regulated during infection of the host. In order to study the influence of the fliL gene on the virulence of P. plecoglossicida, the fliL gene of the NZBD9 strain was knocked out by the homologous recombination method, the fliL gene-deleted strain (ΔfliL strain) constructed, and complemented the fliL gene to the ΔfliL strain to obtain the C-ΔfliL strain. The growth curves of the NZBD9 strain, ΔfliL strain, and C-ΔfliL strain did not show significant differences. Compared with the NZBD9 strain, the motility, adhesion, and biofilm formation ability were tendered in the ΔfliL strain (p < 0.05); the complement of the fliL gene enhanced these abilities to the level of the NZBD9 strain. The results of artificial infection experiments showed that the LD50 of NZBD9 strain, ΔfliL strain, and C-ΔfliL strain in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) were 5.0 × 103 CFU/fish, 6.3 × 104 CFU/fish, and 1.3 × 103 CFU/fish, respectively. RNA sequencing was performed on wild-type strains and ΔfliL strains. A total of 126 differentially expressed genes (DEGs) were screened (p < 0.05), of which 114 were downregulated and 12 were upcontrolled, among which several genes related to the six-type secretion system and transport activity were significantly downregulated. The DEGs were aligned to the GO and KEGG databases and enriched to 44 GO pathways and 39 KEGG pathways, respectively. The active pathways of ABC transporters were significantly enriched in both databases. These results indicate that the fliL gene is related to the movement, biofilm formation, and adhesion ability of P. plecoglossicida, and may reduce virulence by affecting substance transport and bacterial secretion.

Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

“…The wild-type strain of P. plecoglossicida (NZBD9) used in this study was previously isolated from large yellow croaker with visceral white spot disease in our laboratory ( 20 ). The Δ fliL strain was obtained by homologous recombination after knocking out fliL gene of wild strain, and then replacing fliL gene to obtain C-Δ fliL strain ( 21 ). Bacteria were cultured in Luria-Bertani (LB) broth containing ampicillin (100 µg/mL) or tetracycline (10 µg/mL) at 18 °C and 220 rpm.…”