Characterization and expression profiling of fadd gene in response to exogenous Aeromonas hydrophila or Edwardsiella tarda challenge in the hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂).

In mammals, fas-associated protein with death domain (FADD) is involved in the process of cell apoptosis and plays a key role in innate immune signaling. Nevertheless, its detailed molecular mechanisms underlying apoptosis and immune responses to exogenous bacterial infections in teleosts remain largely unknown. In this study, a group of 60 hybrid yellow catfish (with the body weight of 25 ± 0.5 g) were used in subsequent experiments, we examined the expression profiling of fadd gene through comparative genomics and comparative immunological methods. Our results showed that fadd in the hybrid yellow catfish (hycfadd) exhibited similar gene and spatial structures to those in other vertebrates, and formed an independent clade in phylogeny. An expression pattern analysis revealed that hycfadd widely transcribed in various tissues, with the highest transcription level in the liver. Furthermore, expression profiling of hycfadd when intraperitoneally infected with 50 µL of exogenous Aeromonas hydrophila (2.0 × 107 CFU/mL) or Edwardsiella tarda (2.0 × 107 CFU/mL) within 48 h were significantly up-regulated in the kidney, spleen, liver and intestine. Important genes in the toll like receptor (tlr) 1-tlr2- myeloid differentiation primary response 88 (MyD88)-fadd-caspase (casp) 8 cascades of TLR signaling pathway in liver were significantly up-regulated after the A. hydrophila stimulation, suggesting that apoptosis through the TLR signaling pathway may have been triggered and activated, which were further verified in the liver, kidney, spleen, intestine and gill by a TUNEL assay. Overall, this study provides solid evidence for the bacterial induction of fadd-related apoptosis in teleosts.

Toll-like Receptor 3 in the Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂): Protein Structure, Evolution and Immune Response to Exogenous Aeromonas hydrophila and Poly (I:C) Stimuli.

As a major mediator of cellular response to viral infection in mammals, Toll-like receptor 3 (TLR3) was proved to respond to double-stranded RNA (dsRNA). However, the molecular mechanism by which TLR3 functions in the viral infection response in teleosts remains to be investigated. In this study, the Toll-like receptor 3 gene of the hybrid yellow catfish was identified and characterized by comparative genomics. Furthermore, multiple sequence alignment, genomic synteny and phylogenetic analysis suggested that the homologous TLR3 genes were unique to teleosts. Gene structure analysis showed that five exons and four introns were common components of TLR3s in the 12 examined species, and interestingly the third exon in teleosts was the same length of 194 bp. Genomic synteny analysis indicated that TLR3s were highly conserved in various teleosts, with similar organizations of gene arrangement. De novo predictions showed that TLR3s were horseshoe-shaped in multiple taxa except for avian (with a round-shaped structure). Phylogenetic topology showed that the evolution of TLR3 was consistent with the evolution of the studied species. Selection analysis showed that the evolution rates of TLR3 proteins were usually higher than those of TLR3-TIR domains, indicating that the latter were more conserved. Tissue distribution analysis showed that TLR3s were widely distributed in the 12 tested tissues, with the highest transcriptions in liver and intestine. In addition, the transcription levels of TLR3 were significantly increased in immune-related tissues after infection of exogenous Aeromonas hydrophila and poly (I:C). Molecular docking showed that TLR3 in teleosts forms a complex with poly (I:C). In summary, our present results suggest that TLR3 is a pattern recognition receptor (PRR) gene in the immune response to pathogen infections in hybrid yellow catfish.

Characterization of TLR1 and expression profiling of TLR signaling pathway related genes in response to Aeromonas hydrophila challenge in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂).

Toll-like receptor 1 (TLR1) mediates the innate immune response to a variety of microbes through recognizing cell wall components (such as bacterial lipoproteins) in mammals. However, the detailed molecular mechanism of TLR1 involved in pathogen immunity in the representative hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂) has not been well studied. In the present study, we identified the TLR1 gene from the hybrid yellow catfish, and further comparative synteny data from multiple species confirmed that the TLR1 gene is highly conserved in teleosts. Phylogenetic analysis revealed distinguishable TLR1s in diverse taxa, suggesting consistence in evolution of the TLR1 proteins with various species. Structural prediction indicated that the three-dimensional structures of TLR1 proteins are relatively conserved among different taxa. Positive selection analysis showed that purifying selection dominated the evolutionary process of TLR1s and TLR1-TIR domain in both vertebrates and invertebrates. Expression pattern analysis based on the tissue distribution showed that TLR1 mainly transcribed in the gonad, gallbladder and kidney, and the mRNA levels of TLR1 in kidney were remarkably up-regulated after Aeromonas hydrophila stimulation, indicating that TLR1 participates in the inflammatory responses to exogenous pathogen infection in hybrid yellow catfish. Homologous sequence alignment and chromosomal location indicated that the TLR signaling pathway is very conserved in the hybrid yellow catfish. The expression patterns of TLR signaling pathway related genes (TLR1- TLR2 - MyD88 - FADD - Caspase 8) were consistent after pathogen stimulation, revealing that the TLR signaling pathway is triggered and activated after A. hydrophila infection. Our findings will lay a solid foundation for better understanding the immune roles of TLR1 in teleosts, as well as provide basic data for developing strategies to control disease outbreak in hybrid yellow catfish.