sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum interact with immune genes of rainbow trout

Outer membrane vesicles (OMVs) released by gram-negative bacteria during host-pathogen interactions harbor cargos, such as DNA, RNA, toxins, and virulence factors. We hypothesized that sRNAs carried within OMVs of Flavobacterium psychrophilum interact with host immune genes and affect their expression. OMVs were isolated from F. psychrophilum and visualized using transmission electron microscopy (TEM). RNA-Seq datasets generated from whole-cell F. psychrophilum and their OMVs indicated enrichment of specific sRNAs in the OMVs compared to the parent cell. Fluorescent in situ hybridization (FISH) and confocal microscopy confirmed the expression of a randomly chosen sRNA. Integrated RNA-Seq analyses of host transcriptome and bacterial sRNAs on day 5 post-infection of F. psychrophilum -resistant and -susceptible rainbow trout genetic lines revealed 516 protein-coding, 595 lncRNA, and 116 bacterial sRNA differentially expressed (DE) transcripts. Integrated and network analyses of these DE transcripts revealed immune genes targeted by bacterial sRNAs. On the top of these genes, an isoform encoding anaphase-promoting complex subunit 13 (ANAPC13_1) was highly upregulated and exhibited interaction and reciprocal expression with 21 DE sRNAs enriched in OMVs and/or located in pathogenicity islands (PAIs). In vitro treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cell autolysis accompanied by dynamic changes in expression of host genes when profiled 24h following treatment. The OMV-enriched sRNAs, soFE013584 and soFE002123, showed high interactions with the protection of telomeres 1 gene (POT1); essential for chromosome stability and cellular viability. Modulation of the host gene expression following OMV-treatment, which favors elements from the phagocytic, endocytic, and antigen presentation pathways in addition to HSP70, HSP90, and cochaperone proteins, provided evidence for a potential role of OMVs in boosting the host immune response. In conclusion, our work identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China

Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist.

Comparative Genomic Analyses of Flavobacterium psychrophilum Isolates Reveals New Putative Genetic Determinants of Virulence Traits

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly provided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence.

Skin bacteria of rainbow trout antagonistic to the fish pathogen Flavobacterium psychrophilum

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.