Hung-Yueh Yeh

Channel catfish, Ictalurus punctatus, cysteine proteinases: cloning, characterisation and expression of cathepsin H and L.

Abstract The antigen recognition by the host immune system is a complex biochemical process that requires a battery of enzymes. Cathepsins are one of the enzyme superfamilies involved in antigen degradation. We observed the up-regulation of catfish cathepsin H and L transcripts during the early stage of Edwardsiella ictaluri infection in preliminary studies, and speculated that cathepsin H and L may play roles in infection. We identified, sequenced and characterized the complete channel catfish cathepsin H and L cDNAs, which comprised 1415 and 1639 nucleotides, respectively. The open reading frames of cathepsin H appeared to encode a protein of 326-amino acid residues, which that of cathepsin L encoded a protein of 336 amino acids. The degree of conservation of the channel catfish cathepsin H and L amino acid sequences in comparison to other species ranged from 61% to 77%, and 67% to 85%, respectively. The catalytic triad and substrate binding sites are conserved in cathepsin H and L amino acid sequences. The cathepsin L transcript was expressed in all tissues examined, while the cathepsin H was expressed in restricted tissues. These results provide important information for further exploring the roles of channel catfish cathepsins in antigen processing.

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop‐mediated isothermal amplification method

Aims:  To evaluate the loop‐mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus.

Complete Structure, Genomic Organization, and Expression of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Matrix Metalloproteinase-9 Gene

In this study, the channel catfish (CC) matrix metalloproteinase-9 (MMP-9) gene was cloned, sequenced, and characterized at both the cDNA and the genomic DNA levels. The complete sequence of the CC MMP-9 cDNA consisted of 2,551 nucleotides, including one open reading frame and 5′- and 3′-end untranslated regions. The open reading frame potentially encoded a 686-amino-acid peptide with a calculated molecular mass (without glycosylation) of approximately 77.4 kDa, which included a signal peptide and potentially heavy O-glycosylation sites. CC MMP-9 did not have the tripeptide Arg-Gly-Asp motif. The degree of conservation of the CC MMP-9 amino acid sequence to human and mouse counterparts was 55%, while to those of other fish species was 67–74%. The full-length CC MMP-9 genomic DNA comprised 5,663 nucleotides, much shorter than human or mouse counterparts. The exon-intron structure followed the splice acceptor/donor consensus rule, and the sequence contained 13 exons. The MMP-9 transcript was constitutively expressed in restrictive CC tissues. This result should provide fundamental information for further exploration of the role of MMP-9 in fish pathophysiology.

Over-expression, purification and immune responses to Aeromonas hydrophila AL09-73 flagellar proteins.

Aeromonas hydrophila is ubiquitous in aquatic environments worldwide and causes many diseases in fish as well as human. Recent outbreaks of aeromonad diseases in channel catfish prompted us to investigate catfish immune responses during infection of A. hydrophila. In this communication, we report to amplify, over-express, purify and characterize 19 A. hydrophila flagellar proteins. All recombinant proteins were confirmed by nucleotide sequencing of expression plasmids, SDS-PAGE analysis and His tag Western blot of induced proteins. Our preliminary result also showed that the purified recombinant FlgK protein reacted strongly to sera from experimentally infected catfish, suggesting that this protein has potential for a novel target for vaccine development. It is also anticipated that these recombinant proteins will provide us with very useful tools to investigate host immune response to this microorganism.

Sequence analysis, characterization and mRNA distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) chemokine (C-X-C motif) receptor 4 (CXCR4) cDNA

Chemokine receptor CXCR4, a member of the G protein-coupled receptor superfamily, binds selectively CXCL12. This protein plays many important roles in immunological as well as pathophysiological functions. In this study, we identified and characterized the channel catfish CXCR4 transcript. The full-length nucleic acid sequence of channel catfish CXCR4 cDNA comprised of 1994 nucleotides, including an open reading frame, which appears to encode a putative peptide of 357 amino acid residues with a calculated molecular mass of 40.1kDa. By comparison with the human counterpart, the channel catfish CXCR4 peptide can be divided into domains, including seven transmembrane domains, four cytoplasmic domains, and four extracellular domains. The CXCR4 transcript was detected in spleen, anterior kidney, liver, intestine, skin and gill of all catfish examined in this study. Because four CXCL of channel catfish have been identified, the result provides valuable information for further exploring the channel catfish chemokine signalling pathways and their roles in immune responses to infection.

Identification, phylogenetic relationships, characterization and gene expression patterns of six different annexins of channel catfish (Ictalurus punctatus Rafinesque, 1818)

Annexins are Ca(2+)-dependent phospholipid-binding proteins. They are ubiquitous in living organisms and are involved in many cellular processes. In the course of studying Edwardsiella ictaluri pathogenesis in channel catfish, we identified that six annexin expressed sequence tags (A1, A2, A4, A5, A6 and A11) were up-regulated at the early stage of infection. In this study, we cloned and characterized these transcripts. The full-length nucleic acid sequences of channel catfish annexins ranges from 1231 (annexin A1) to 2476 (annexin A6). Each transcript has one open reading, which appears to encode peptides ranges from 317 to 662 amino acid residues with the calculated molecular masses from 35.0 (annexin A5) to 74.5kDa (annexin A6). Phylogenetic and sequence analyses demonstrate that each channel catfish annexin had a diversified amino terminus, and had four structurally conserved 70-amino acid repeats. In addition, several important features for annexin functions were conserved in channel catfish. For expression profile, channel catfish annexin A1, A4 and A6 transcripts were detected in spleen, anterior kidney, liver, intestine, skin and gill of fish examined. However, annexin A2, A5 and A11 cDNAs were variously detected in tissues of fish sampled. This result provides important information for further elucidating channel catfish annexin functions in vivo.

Sequence analysis, characterization and tissue distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) myeloperoxidase cDNA.

Myeloperoxidase (EC 1.11.1.7), a heme-containing lysosomal glycoprotein, is found predominantly in azurophilic granules of neutrophils. This enzyme upon activation catalyzes hydrogen peroxide in the presence of various halide ions to form hypohalous acids. Subsequently, these reagents are able to kill the invading microorganisms. In this study, we report the identification, characterization and expression analysis of the channel catfish myeloperoxidase transcript. The full-length nucleotide sequence of channel catfish myeloperoxidase cDNA had 3157 nucleotides, including an open reading frame, which appears to encode a putative peptide of 771 amino acid residues with a calculated molecular mass of 87.14 kDa. By comparison with the human counterpart, the channel catfish myeloperoxidase peptide can be divided into domains and has conservative features, including peroxidase catalytic sites, covalent linkage sites for the heme group and all cysteine residues. The channel catfish myeloperoxidase transcript was detected by RT-PCR in anterior kidneys, where the major leukocyte population is neutrophil precursors. Reagent development and the role of this enzyme in Edwardsiella ictaluri infection are under investigation.

Channel catfish (Ictalurus punctatus Rafinesque, 1818) tetraspanin membrane protein family: identification, characterization and expression analysis of CD63 cDNA.

CD63, known as lysosome associated membrane protein 3 (LAMP-3), is a member of the tetraspanin integral membrane protein family. This protein plays many important roles in immuno-physiological functions. In this communication, we report the identification, characterization, and expression analysis of the channel catfish CD63 transcript. The complete nucleic acid sequence of channel catfish CD63 cDNA was comprised of 1159 nucleotides, including an open reading frame, which appears to encode a putative peptide of 237-amino-acid residues. Like other tetraspanin proteins, the channel catfish CD63 peptide can be divided into domains, including four transmembrane domains, three intracellular domains, and one of each small and large extracellular loops. The channel catfish CD63 peptide shares 52-55% identity among fish counterparts, but only 43-46% identity among mammalian counterparts. The characteristic Cys-Cys-Gly motif and four Cys residues in the large extracellular loop were conserved. The channel catfish CD63 transcript was detected by RT-PCR in spleen, anterior kidney, liver, intestine, skin and gill. This result provides important information for further elucidating CD63 functions in channel catfish.

Channel catfish, Ictalurus punctatus Rafinesque 1818, tetraspanin membrane protein family: characterization and expression analysis of CD81 cDNA.

CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1) in human, is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune and other physiological functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. The full-length of channel catfish CD81 cDNA comprised of 1130 nucleotides, including an open reading frame which appears to encode a putative peptide of 234 amino acid residues. By comparison with the human counterpart, the channel catfish CD81 peptide could be divided into domains, including four transmembrane domains, three intracellular domains, and one of each small and large extracellular loops. The degree of conservation of the channel catfish CD81 amino acid sequence to that of mammalian counterparts ranged from 65% to 67%. The large extracellular domain shows the least conservation between fish and mammals. However, the characteristic Cys(159)-Cys(160)-Gly(161) motif and Cys(176/188) in this domain were conserved. The channel catfish CD81 transcript was detected by RT-PCR in spleen, head kidney, liver, intestine, skin and gill. This result provides important information for further elucidating CD81 functions in channel catfish.

Pathogenicity of Streptococcus ictaluri to Channel Catfish

The pathogenicity of a Streptococcus ictaluri isolate in channel catfish Ictalurus punctatus at the fry (0.5 g), fingerling (15 g), and juvenile (55 g) stages was determined by experimental bath immersion and injection experiments. Channel catfish were exposed in 1-L immersion baths containing 10(8), 10(9), 10(10), 10(11) or 10(12) colony-forming units (cfu) of S. ictaluri. Fish were also injected intraperitoneally with 0.1 mL of bacterial solution for final doses of 10(4), 10(5), 10(6), 10(7), or 10(8) cfu of S. ictaluri per fish. Streptococcus ictaluri caused mortality in fry, fingerling, and juvenile channel catfish within 21 d postinfection. When mortalities were calculated based on size and challenge route, the cumulative percent mortalities were 11% for fry and 0% for fingerlings by the bath immersion route and 14% for fingerlings and 6% for juveniles by the injection route. Isolation of S. ictaluri from moribund and dead catfish was confirmed by the newly established BIOLOG profile (MicroLog3 system). The results indicate that channel catfish were only susceptible to high concentrations of S. ictaluri and that juvenile channel catfish were less susceptible, possibly explaining why little mortality has been attributed to S. ictaluri infection in catfish aquaculture.