Eric Peatman

JAM-A regulates permeability and inflammation in the intestine in vivo

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus.

The mucosal surfaces of fish (gill, skin, gastrointestinal tract) are important sites of bacterial exposure and host defense mechanisms. In mammalian systems, the intestinal epithelium is well characterized as both a selectively permeable barrier regulated by junctional proteins and as a primary site of infection for a number of enteric pathogens including viruses, bacteria, and parasites. The causative bacterium of enteric septicemia of catfish, Edwardsiella ictaluri, is believed to gain entry through the intestinal epithelium, with previous research using a rat intestinal epithelial cell line (IEC-6) indicating actin polymerization and receptor-mediated endocytosis as potential mechanisms of uptake. Here, we utilized high-throughput RNA-seq to characterize the role of the intestinal epithelial barrier following E. ictaluri challenge. A total of 197.6 million reads were obtained and assembled into 176,481 contigs with an average length of 893.7 bp and N50 of 1676 bp. The assembled contigs contained 14,457 known unigenes, including 2719 genes not previously identified in other catfish transcriptome studies. Comparison of digital gene expression between challenged and control samples revealed 1633 differentially expressed genes at 3 h, 24 h, and 3 day following exposure. Gene pathway analysis of the differentially expressed gene set indicated the centrality of actin cytoskeletal polymerization/remodelling and junctional regulation in pathogen entry and subsequent inflammatory responses. The expression patterns of fifteen differentially expressed genes related to intestinal epithelial barrier dysfunction were validated by quantitative real-time RT-PCR (average correlation coeff. 0.92, p < 0.001). Our results set a foundation for future studies comparing mechanisms of pathogen entry and mucosal immunity across several important catfish pathogens including E. ictaluri, Edwardsiellatarda, Flavobacterium columnare, and virulent atypical Aeromonas hydrophila. Understanding of molecular mechanisms of pathogen entry during infection will provide insight into strategies for selection of resistant catfish brood stocks against various diseases.

Generation of genome-scale gene-associated SNPs in catfish for the construction of a high-density SNP array

BackgroundSingle nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing.ResultsTranscriptome sequencing was conducted for channel catfish and blue catfish using Illumina next generation sequencing technology. Approximately 220 million reads (15.6 Gb) for channel catfish and 280 million reads (19.6 Gb) for blue catfish were obtained by sequencing gene transcripts derived from various tissues of multiple individuals from a diverse genetic background. A total of over 35 billion base pairs of expressed short read sequences were generated. Over two million putative SNPs were identified from channel catfish and almost 2.5 million putative SNPs were identified from blue catfish. Of these putative SNPs, a set of filtered SNPs were identified including 342,104 intra-specific SNPs for channel catfish, 366,269 intra-specific SNPs for blue catfish, and 420,727 inter-specific SNPs between channel catfish and blue catfish. These filtered SNPs are distributed within 16,562 unique genes in channel catfish and 17,423 unique genes in blue catfish.ConclusionsFor aquaculture species, transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. When multiple individuals with different genetic backgrounds are used, RNA-Seq is very effective for the identification of SNPs. The SNPs identified in this report will provide a much needed resource for genetic studies in catfish and will contribute to the development of a high-density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for genome association studies and whole genome-based selection in catfish.

Transcriptomic signatures of attachment, NF-κB suppression and IFN stimulation in the catfish gill following columnaris bacterial infection.

Outbreaks of columnaris disease (Flavobacterium columnare) are common in wild and cultured freshwater fish worldwide. Disease occurrences, particularly those caused by virulent genomovar II isolates, in aquaculture species such as channel catfish can be devastating. In contrast to other important aquaculture pathogens, little is known about host immune responses to columnaris. Adhesion of F. columnare to gill tissue has been correlated in some previous studies to virulence and host susceptibility. Here, therefore, we conducted the first transcriptomic profiling of host responses to columnaris following an experimental challenge. We utilized Illumina-based RNA-seq expression profiling to examine transcript profiles at three timepoints (4h, 24h, and 48h) in catfish gill after bath immersion infection. Enrichment and pathway analyses of the differentially expressed genes revealed several central signatures following infection. These included the dramatic upregulation of a rhamnose-binding lectin, with putative roles in bacterial attachment and aggregation, suppression of NF-κB signalling via IκBs, BCL-3, TAX1BP1, and olfactomedin 4, and strong induction of IFN-inducible responses including iNOS2b, IFI44, and VHSV genes. Fifteen differentially expressed genes with varying expression profiles by RNA-seq, were validated by QPCR (correlation coefficients 0.85-0.94, p-value <0.001). Our results highlight several putative immune pathways and individual candidate genes deserving of further investigation in the context of development of therapeutic regimens and laying the foundation for selection of resistant catfish lines against columnaris.

Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity

Chemokines are a superfamily of cytokines responsible for regulating cell migration under both inflammatory and physiological conditions. CC chemokines are the largest subfamily of chemokines, with 28 members in humans. A subject of intense study in mammalian species, the known functional roles of CC chemokines ligands in both developmental and disease conditions continue to expand. They are also an important family for the study of gene copy number variation and tandem duplication in mammalian species. However, little is known regarding the evolutionary origin and status of these ligands in primitive vertebrates such as teleost fish. In this paper, we review the evolution of the teleost fish CC chemokine gene family, noting evidence of widespread tandem gene duplications and examining the implications of this phenomenon on immune diversity. Through extensive phylogenetic analysis of the CC chemokine sets of four teleost species, zebrafish, catfish, rainbow trout, and Atlantic salmon, we identified seven large groups of CC chemokines. It appeared that several major groups of CC chemokines are highly related including the CCL19/21/25 group, the CCL20 group, CCL27/28 group, and the fish-specific group. In the three remaining groups that contained the largest number of members, the CCL17/22 group, the MIP group, and the MCP group, similarities among species members were obscured by rapid, tandem duplications that may contribute to immune diversity.

Efficient assembly and annotation of the transcriptome of catfish by RNA-Seq analysis of a doubled haploid homozygote.

BackgroundUpon the completion of whole genome sequencing, thorough genome annotation that associates genome sequences with biological meanings is essential. Genome annotation depends on the availability of transcript information as well as orthology information. In teleost fish, genome annotation is seriously hindered by genome duplication. Because of gene duplications, one cannot establish orthologies simply by homology comparisons. Rather intense phylogenetic analysis or structural analysis of orthologies is required for the identification of genes. To conduct phylogenetic analysis and orthology analysis, full-length transcripts are essential. Generation of large numbers of full-length transcripts using traditional transcript sequencing is very difficult and extremely costly.ResultsIn this work, we took advantage of a doubled haploid catfish, which has two sets of identical chromosomes and in theory there should be no allelic variations. As such, transcript sequences generated from next-generation sequencing can be favorably assembled into full-length transcripts. Deep sequencing of the doubled haploid channel catfish transcriptome was performed using Illumina HiSeq 2000 platform, yielding over 300 million high-quality trimmed reads totaling 27 Gbp. Assembly of these reads generated 370,798 non-redundant transcript-derived contigs. Functional annotation of the assembly allowed identification of 25,144 unique protein-encoding genes. A total of 2,659 unique genes were identified as putative duplicated genes in the catfish genome because the assembly of the corresponding transcripts harbored PSVs or MSVs (in the form of pseudo-SNPs in the assembly). Of the 25,144 contigs with unique protein hits, around 20,000 contigs matched 50% length of reference proteins, and over 14,000 transcripts were identified as full-length with complete open reading frames. The characterization of consensus sequences surrounding start codon and the stop codon confirmed the correct assembly of the full-length transcripts.ConclusionsThe large set of transcripts assembled in this study is the most comprehensive set of genome resources ever developed from catfish, which will provide the much needed resources for functional genome research in catfish, serving as a reference transcriptome for genome annotation, analysis of gene duplication, gene family structures, and digital gene expression analysis. The putative set of duplicated genes provide a starting point for genome scale analysis of gene duplication in the catfish genome, and should be a valuable resource for comparative genome analysis, genome evolution, and genome function studies.

Quality assessment parameters for EST-derived SNPs from catfish

BackgroundSNPs are abundant, codominantly inherited, and sequence-tagged markers. They are highly adaptable to large-scale automated genotyping, and therefore, are most suitable for association studies and applicable to comparative genome analysis. However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries. Such genome resources are not yet available for many species including catfish. A large resource of ESTs is to become available in catfish allowing identification of large number of SNPs, but reliability of EST-derived SNPs are relatively low because of sequencing errors. This project was designed to answer some of the questions relevant to quality assessment of EST-derived SNPs.Resultswo factors were found to be most significant for validation of EST-derived SNPs: the contig size (number of sequences in the contig) and the minor allele sequence frequency. The larger the contigs were, the greater the validation rate although the validation rate was reasonably high when the contigs contain four or more EST sequences with the minor allele sequence being represented at least twice in the contigs. Sequence quality surrounding the SNP under test is also crucially important. PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.ConclusionStringent quality assessment measures should be used when working with EST-derived SNPs. In particular, contigs containing four or more ESTs should be used and the minor allele sequence should be represented at least twice. Genotyping primers should be designed from a single exon, completely avoiding introns. Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

BackgroundThrough the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energys Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.ResultsA total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.ConclusionsThis project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Nod-like subfamily of the nucleotide-binding domain and leucine-rich repeat containing family receptors and their expression in channel catfish.

The NLRs (nucleotide-binding domain and leucine-rich repeat containing family receptors) are a recently identified family of pattern recognition receptors in vertebrates. Several subfamilies of NLRs have been characterized in human, mouse, and zebrafish, but studies of NLRs in other species, especially teleost species, have been lacking. Here we report characterization of five NLRs from channel catfish: NOD1, NOD2, NLRC3, NLRC5, and NLRX1. Structural analysis indicated that the genes were organized in a similar fashion as in the mammals and in zebrafish. Phylogenetic analysis suggested that they were orthologous to the NOD-like subfamily of NLRs. All five NOD-like genes exist as a single copy gene in the catfish genome. Hybridization of gene-specific probes allowed mapping of three NLR genes to the catfish physical map, laying a foundation for genome characterization and for establishing orthologies with NLR genes from other species. These genes are widely expressed in various tissues and leukocyte cell lines. While the majority of the NLR genes appeared to be constitutively expressed, NOD1 was induced after infection with a bacterial pathogen, Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), suggesting its involvement in immunity against the intracellular pathogen.

Toll-like receptor 3 and TICAM genes in catfish: species-specific expression profiles following infection with Edwardsiella ictaluri.

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize specific pathogen-associated molecular patterns and use conserved signaling pathways to activate proinflammatory cytokines and type-1 interferons to fight infection. TLR3 in mammals is best known for its recognition of dsRNA as ligand and its MyD88-independent signaling. TLR3, upon recognition of dsRNA, recruits and binds its adaptor protein TIR domain-containing adapter molecule (TICAM) 1. Here we report the genomic sequences and structures of TLR3 and a TICAM adaptor from channel catfish (Ictalurus punctatus). Whereas a partial TLR3 cDNA sequence has been reported from channel catfish, and complete TLR3 genes are known from other teleost fish species, a complete TICAM sequence has not been previously reported from a nonmammalian species. Analysis of catfish TLR3 and TICAM expression after infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), suggested a conserved TLR3-TICAM receptor–adaptor relation in catfish. Comparison of TLR3 and TICAM expression profiles in channel catfish with those from the closely related blue catfish species (Ictalurus furcatus), which exhibits strong resistance to ESC, revealed a striking pattern of species-specific expression. A dramatic downregulation of TLR3 and TICAM gene expression was observed in blue catfish head kidney and spleen, which we speculate may be the result of maturation and migration of different cell types to and from the lymphoid tissues following infection.