Genome-wide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection

Fish & Shellfish Immunology 49 (2016) 154e162

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Genome-wide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection Lin Song a, b, Chao Li a, Yangjie Xie c, Shikai Liu b, Jiaren Zhang b, Jun Yao b, Chen Jiang b, Yun Li b, Zhanjiang Liu b, * a

Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109, China Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit, School of Fisheries, Aquaculture and Aquatic Sciences, Program of Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849, USA c Fisheries College, Jimei University, Xiamen, 361021, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2015 Received in revised form 12 November 2015 Accepted 10 December 2015 Available online 13 December 2015

Heat shock proteins 70/110 (Hsp70/110) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during infection. In this study, we identified 16 Hsp70/110 geness in channel catfish (Ictalurus punctatus) through in silico analysis using RNA-Seq and genome databases. Among them 12 members of Hsp70 (Hspa) family and 4 members of Hsp110 (Hsph) family were identified. Phylogenetic and syntenic analyses provided strong evidence in supporting the orthologies of these HSPs. In addition, we also determined the expression patterns of Hsp70/110 genes after Flavobacterium columnare and Edwardsiella ictaluri infections by meta-analyses, for the first time in channel catfish. Ten out of sixteen genes were significantly up/down-regulated after bacterial challenges. Specifically, nine genes were found significantly expressed in gill after F. columnare infection. Two genes were found significantly expressed in intestine after E. ictaluri infection. Pathogen-specific pattern and tissue-specific pattern were found in the two infections. The significantly regulated expressions of catfish Hsp70 genes after bacterial infections suggested their involvement in immune response in catfish. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Heat shock protein Hsp70 Catfish Genome Immunity Infection

1. Introduction Heat shock proteins (HSPs) consist of a large group of chaperones whose expression is induced by high temperature, hypoxia, infection and a number of other stresses or constitutively expressed in non-stressed cells as housekeeping proteins [1,2]. However, recent studies have revealed many immune responding abilities of HSPs in various hosts [3]. HSPs are classified based on their molecular weight and their functional domains to include Hsp110 (HSPH), Hsp90 (HSPC), Hsp70 (HSPA), Hsp60 (HSPD), Hsp40 (DNAJ), Hsp10 (HSPE), and small HSPs (HSPB) [4,5]. Among them, Hsp70 is one of the most conserved proteins in evolution. It is found in all organisms from archaebacteria and plants to humans. A high amino acid identity of approximately 50% is shared between the

* Corresponding author. 203 Swingle Hall, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.fsi.2015.12.009 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

prokaryotic Hsp70 protein DnaK and eukaryotic Hsp70 proteins [6]. Hsp70 is one of the most heat inducible and protective proteins [7]. Hsp70 proteins of all known species display highly conserved amino acid sequences and domain structures consisting of: (i) a conserved ATPase domain; (ii) a middle region with protease sensitive sites; (iii) a peptide binding domain; and (iv) a G/P-rich Cterminal region containing an EEVD-motif enabling the proteins to bind co-chaperones and other [8,9]. Furthermore, the members localized to specific cellular compartments have a localization signal in their N-terminus. For example, the localization of HSPA5 (also known as Bip or Grp78) is within the lumen of the ER and HSPA5 has a C-terminal retention signal sequence that inhibits its exit from the ER [10]. The conserved domain structure consolidates the chaperone function of the Hsp70 proteins and enables them to bind and release extended stretches of hydrophobic amino acids, exposed by incorrectly folded globular proteins in an ATPdependent manner. The C-terminal contains the least conserved sequences that may explain the non-redundant functions of Hsp70

L. Song et al. / Fish & Shellfish Immunology 49 (2016) 154e162

family members [11]. Another group of Hsps, Hsp110, have a high homology to Hsp70 members except for the existence of a longer linker domain between the N-terminal ATPase domain and the Cterminal peptide binding domain. Due to the high level of similarity in domain structure with Hsp70, the Hsp110 were often studied and discussed with Hsp70 (HSPA) as Hsp70/110 family [5]. Plenty of evidence have suggested the involvement of Hsp70/ 110s in immune response in vertebrates in the range of non-specific immune response to adaptive immune response. For non-specific immune, Vabulas et al. showed that HSP70 induced interleukin12 (IL-12) and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) promoters in macrophages [12]. Members of the Hsp70 cytosolic group are either constitutively expressed (Hspa8) or can be induced by a broad range of stress factors. The inducible Hsp70 has been recently characterized as a potent maturation stimulus for Dendritic Cells (DCs), which is an important professional antigen presenting cell (APC) that express Major Histocompatibility Complex (MHC) class II molecule on its surface and process the antigen into epitopes while migrating to the thymus to present the epitopes to the CD4þ T cell. This process is considered as the bridge of innate immune response and adaptive immune response [13,14]. For adaptive immune response, heat shock proteins participate as a danger signal in the host immune response. The acute immune response is organized and executed by innate immunity influenced by the neuroendocrine system. This response starts with sensing of danger signals by pattern-recognition receptors on the immune competent cells and endothelium. TLR4 is involved in signaling of both exogenous and endogenous danger signals. Hsp70s are one of these danger signals and act as the agonist of TLR4 [15]. One rare type of CD4þ T cells, which is called gd T cell, are believed to be triggered by alarm signals such as HSPs and it is only found abundant in gut mucosa. In human, the expression of HSPA12B was up-regulated in lipopolysaccharide (LPS)-induced inflammatory response in spinal cord, and mostly located in active microglia; this induced expression may be regulated by activation of MAPK-p38, ERK1/2 and SAPK/JNK signaling pathways. Overexpression of HSPA12B also protects against LPS-induced cardiac dysfunction and involves the preserved activation of the PI3K/Akt signaling pathway. HSPA12B is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses [16]. In teleost, Hsp70 genes have been found to be involved in bacterial kidney disease in coho salmon [17] and vibriosis infection in rainbow trout [18]. In olive flounder, one hsp70 gene was expressed in heat-shocked and virus treated FEC cells, and hsp40s were also found to be up-regulated in flounder embryonic cells (FEC) after viral infection. In tilapia, it was reported that tHsp70 alone and antigen-tHsp70 compound increased the proliferations of lymphocytes and macrophages, significantly increased the NO release and phagocytotic ability of macrophages, and enhanced the levels of immune-related genes in lymphocytes and macrophages. Hsp70 also assisted antigens to enhance the vaccine-induced protection against Streptococcus iniae [19]. In channel catfish, one member of Hsp70s was significantly induced in the anterior kidney (48, 72, and 96 hpi) and spleen (48 and 72 hpi) upon exposure to Edwardsiella ictaluri, which indicates the Hsp70s play a role in the early stages of the response to bacterial infection [20]. Channel catfish (Ictalurus punctatus) is the leading aquaculture species in the United States. It is also an important species for the study of the teleost immune system [21]. However, in recent years, the catfish industry has encountered huge losses due to diseases outbreaks. Columnaris disease is the most frequently occurring freshwater disease in fish including catfish [22]. It is responsible for significant economic losses in freshwater fish aquaculture worldwide. Compared with columnaris disease, enteric septicemia of catfish (ESC) is less frequent in occurrence, but is among the most

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severe diseases of catfish [23]. Economic losses to the catfish industry are in the tens of millions of dollars annually and the problem may become even more severe with the growth of the catfish industry. The catfish genomic resources have been well developed in recent years, particularly ESTs [24,25], transcriptome sequences generated by RNA-Seq [26,27] and draft whole genome sequence (unpublished data). These resources make it feasible to conduct systematic analysis of interested genes in channel catfish genome. On the basis of our previous work on catfish Hsp40 genes [28] and Hsp60, 90 and small Hsp genes [29], here we report the genome-wide identification of a full set of 16 Hsp70s and Hsp110s genes, their phylogenetic and syntenic analyses, and their expression in disease responses after bacterial infection with E. ictaluri and Flavobacterium columnare through meta-analysis of RNA-Seq datasets. Although regulated expression of HSPs after infection have been reported in several fish species, systematic analysis of their involvement in diseases has not been conducted. This work, to our best knowledge, represents the first systematic analysis of Hsp70/110s involvement after bacterial infection among all species. 2. Materials and methods 2.1. Database mining and sequence analyses In order to identify the full set of hsp70/110 genes in channel catfish, we collected all Hsp70s and Hsp110s proteins from teleost fishes (zebrafish, three-spined stickleback, medaka, tilapia and fugu) and other species (human, mouse, platypus, chicken, turtle and frog) (Supplementary Table 1). These sequences were retrieved from NCBI (http://www.ncbi.nlm.nih.gov) and Ensembl (http:// www.ensembl.org) databases and used as queries to search against channel catfish RNA-Seq datasets. The e-value was set at intermediately stringent level of e10 for collecting as many as potential hsp70/110-related sequences for further analysis. The retrieved sequences were then translated using ORF finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). Further, the predicted ORFs were verified by BLASTP against NCBI non-redundant (Nr) protein sequence database. The simple modular architecture research tool (SMART) [30] was used to predict the conserved domains based on sequence homology and further confirmed by conserved domain prediction from BLAST. The predicted catfish Hsp70/110 proteins and all other query sequences were utilized to search against catfish genome database using TBLASTN program. The retrieved genome scaffolds were then predicted by FGENESH in SoftBerry (http://linux1.softberry.com/berry.phtml? topic¼fgenesh&group¼programs&subgroup¼gfind). The amino acid similarity matrix of each Hsp70/110 from channel catfish and other fishes are generated by Clustal Omega software (http://www. ebi.ac.uk/Tools/msa/clustalo/). 2.2. Phylogenetic and conserved syntenic analyses All the amino acids of Hsp70/110s from channel catfish and other species were used to construct phylogenetic tree. Multiple protein sequences alignments were conducted using the Clustal W2 program [31] and MUSCLE 3.8 [32]. Three alignment methods: L-INS-i, E-INS-I and G-INS-i were applied from MAFFT 7.01 [33] and the best alignment with highest score was evaluated by program MUMSA [34]. JTTþIþG model (Jones-Taylor-Thornton (JTT) matrix incorporated a proportion of invariant sites (þI) and the gamma distribution for modeling rate heterogeneity (þG)) was selected as the best-fit model by ProtTest 3 program [35] according to the Bayesian information criterion. Maximum likelihood phylogenetic tree was constructed using MEGA5.2.2 [36] with bootstrap test of 1000 replicates. Final phylogenetic tree was separated into three

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different phylogenetic trees according to classification of subfamilies due to the large size. Conserved syntenic regions surrounding the relevant hsp70/110 genes were searched by examining the conserved co-localization of neighboring genes on scaffold of channel catfish and other species based on genome information from Ensembl (Release 77) and NCBI database. Neighbor genes of channel catfish Hsp70 genes were predicted by FGENESH [37] and BLASTP. 2.3. Meta-analysis of expression of Hsp70/110 genes after bacterial challenge

Nomenclature Guidelines (https://wiki.zfin.org/display/general/ ZFINþZebrafishþNomenclatureþGuidelines). Twelve heat shock protein 70 (hspa) genes were identified in the catfish genome including hsp70.2, hsp70.3, hspa8a.1, hspa8a.2, hspa8b, hsc70 hspa5, hspa9, hspa12a, hspa12b, hspa13 and hspa14. Four heat shock protein 110 (hsph) genes were identified in channel catfish including hspa4a, hspa4b, hspa4L and hyou1 (Table 1). Catfish Hsp70s share high amino acid identity (>80%) with each other within and among species however they share lower amino acid identity (<34%) with Hsp110s (Table 2). 3.2. Phylogenetic analysis of channel catfish Hsp70/110s

To determine the expression profiles of Hsp70/110 genes after bacterial infections, meta-analysis of RNA-Seq data was conducted with CLC Genomics Workbench (version 6.5.2; CLC bio, Aarhus, Denmark). The Illumina-based RNA-Seq reads were retrieved from two bacterial challenge experiments in catfish: intestine samples challenged with E. ictaluri at 0h, 3h, 24h and 3d (SRA accession number SRP009069) [38] as well as gill samples challenged with F. Columnare at 0h, 4h, 24h and 48h (SRA number SRP012586) [39]. For mapping of the trimmed high-quality RNA-Seq reads, both of the assembled transcripts of those two experiments were used as reference sequences respectively. Mapping parameters were set as  95% of the reads in perfect alignment and 2 mismatches. The total mapped reads number for each transcript was determined and normalized to analyze RPKM (Reads Per Kilobase of exon model per Million mapped reads). The proportions-based Kal's test was performed to identify the differentially expressed genes comparing each timepoint sample with control sample and fold changes were calculated. Transcripts with p-value  0.05, absolute fold change value  1.5, and total reads number 5 were included in the analyses as significantly differently expressed genes. 3. Results 3.1. Identification of Hsp70/110 genes in catfish A total of 16 Hsp70/110 genes were identified in channel catfish. All the information of domain structures and GenBank accession numbers are summarized in Table 1. Only one of these catfish hsp70 genes were identified before as ‘heat shock cognate 70 protein’ (NP_001187202.1) and now is named as hspa8a.2 after our phylogenetic and syntenic analysis [40]. Among all these genes, almost all sequences were identified in both transcriptome and genome databases with full-length hsp70/110 in both databases. These catfish hsp70/110 genes were named following Zebrafish

Table 1 Summary of 16 Hsp70/110 genes identified in the catfish genome. Name

Accession number

Domain

hsp70.2 hsp70.3 hspa8a.1 hspa8a.2 hspa8b hsc70 hspa4a hspa4b hspa4L hyou1 hspa5 hspa9 hspa12a hspa12b hspa13 hspa14

KT961621 JT412120.1 JT315838.1 NP_001187202.1 JT418846.1 JT408276.1 JT406739.1 JT407108.1 JT407194.1 JT411259.1 JT415554.1 JT408761.1 KT961622 JT411717.1 JT413763.1 JT406759.1

HSPA1-2_6-8-like_NBD HSPA1-2_6-8-like_NBD HSPA1-2_6-8-like_NBD HSPA1-2_6-8-like_NBD HSPA1-2_6-8-like_NBD HSPA1-2_6-8-like_NBD HSPA4_NBD HSPA4_NBD HSPA4L_NBD HYOU1_like_NBD HSPA5-like_NBD HSPA9-like_NBD HSPA12A-like_NBD HSPA12B-like_NBD HSPA13-like_NBD HSPA14-like_NBD

A total of 16 channel catfish Hsp70/110 genes have been phylogenetically analyzed. In a few cases where it was difficult to establish orthologies due to duplications and ambiguous names (for example, hspa8a.1, hspa8a.2 and hspa8b), syntenic analyses were conducted and their names were standardized followed by the zebrafish or human orthologues. The phylogenetic trees were then reconstructed after standardizing all the names. In the phylogenetic tree, all the members of catfish Hsp70/110 were well distributed into distinct clades and grouped with corresponding genes of zebrafish or other fishes, which were supported by strong bootstrap value (Fig. 1). 3.3. Syntenic analysis of channel catfish Hsp70/110s Though phylogenetic relationships provide support for the identities of most Hsp70 genes based on the similarity of gene sequences, it can't provide a strong and clear classification of the sequence share very similar sequence or the tandem repeats which occur specifically in the catfish. Hspa1, Hspa2, Hspa6 and Hspa8 genes have a common domain called HSP1_2_6_8Nucleoid Binding Domain. In catfish, there were six genes in this subfamily that were found through the genome. However, it was difficult to assign the names to each of them according to the clade of the phylogenetic tree due to some tandem repeat and gene duplications. Sytenic analyses were required to provide additional evidence for orthologies or otherwise the paralogies for these hsp70 genes. Positions of these catfish hsp70s genes and their neighbor genes were identified from the draft genome scaffolds. The genes and their neighborhoods were also identified from the zebrafish and human genome. As shown in Fig. 2, the neighbor genes of Hspa1 were analyzed in human, zebrafish and catfish chromosomes. The genes in same color showed a homology of each other. Three tandem repeat genes, HSPA1A, HSPA1B, HSPA1L, were found on human chromosome 6 within the MHC-complex loci (6p21), which was mapped to short arm of chromosome 6 from 29.7 M to 33.3 M. Four genes from zebrafish were related to HSPA group. Two of them, hsp70.2 and hsp70.3 as tandem repeats located on chromosome 3, were found orthologous to human HSPA genes. The other two genes, hspa-like and hsp70-like, were located on the chromosome 8 and were less related to human HSPA. Two copies of catfish hsp70 genes were found highly orthologous to zebrafish hsp70.2 and hsp70.3. Both of them were named after the zebrafish orthologues. One member of MHC-complex molecules was found on the same chromosome of catfish hsp70.2 and hsp70.3. The MHC genes was not found on the same group link of tilapia hsp70. This may due to each assembled group link of tilapia genome is not long enough. Even though the MHC complex loci or locus were found on the same chromosome of all these fish (except tilapia) hspa genes, the loci are relative far ahead of the hspa genes, which is different with the situation in human genome. Three hspa8-related genes were analyzed (Fig. 3). One copy of zebrafish hspa8 gene has the highest level of conservation in gene

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Table 2 Percentage of amino acid identity of each Hsp70/110 from channel catfish (Ictalurus punctatus) compared to each other and other fish Hsp70 and Hsp110. Ip represents channel catfish (I. punctatus). Dr_hsp70 is a Hsp70 from zebrafish (Danio rerio). Dr_hspa4a is a Hsp110 from zebrafish (D. rerio). Ol_hsp70.3 is a Hsp70 from medaka (O. latipes) and Ol_hspa4a is a Hsp70 from medaka (O. latipes). aa% Identity

Ictalurus punctatus hsp70.2

hsp70.3

hspa8a.l

hspa8a.2

hspa8b

hsc70

hspa4a

hspa4b

hspa4L

hvou1

hspa5

hspa9

hsp12a

hspa12b

hspa13

hspa14

Ip_hsp70.2 lp_hsp70.3 lp_hspa8a.1 Ip_hspa8a.2 lp_hspa8b Ip_hsc70 Ip_hspa4a Ip_hspa4b lp_hspa4L lp_hyou1 Ip_hspa5 Ip_hspa9 Ip_hsp12a Ip_hspa12b Ip_hspa13 Ip_hspa14 Dr_hsp70 Dr_hspa4a Ol_hsp70.3 Ol_hspa4a

100.00 93.16 89.38 88.91 91.02 87.98 31.31 31.31 29.23 29.77 64.23 51.36 21.02 23.33 39.75 33.80 89.27 30.03 87.95 30.51

100.00 89.19 82.68 85.78 84.56 31.55 31.88 29.77 29.83 63.83 51.13 20.59 23.33 40.00 33.80 92.16 30.26 88.66 31.07

100.00 95.37 95.17 93.63 33.67 33.67 32.26 32.03 68.93 55.93 21.87 23.21 40.95 33.20 90.54 33.06 88.61 33.47

100.00 93.03 87.98 29.39 29.39 27.32 27.68 62.52 48.64 20.68 22.99 39.95 31.79 84.24 28.27 81.95 28.75

100.00 90.56 31.46 30.98 29.21 28.59 64.86 50.56 21.11 23.44 40.20 33.00 86.72 30.02 84.93 30.50

100.00 30.62 29.98 29.03 30.59 63.80 50.64 21.11 23.44 39.20 33.20 85.65 29.19 84.93 29.82

100.00 79.43 64.03 29.19 32.95 27.86 19.24 17.12 30.43 28.87 32.20 85.22 31.92 81.58

100.00 63.97 28.11 32.28 28.36 18.84 18.18 30.18 28.87 32.20 78.58 31.76 76.25

100.00 26.50 30.46 26.20 20.44 18.16 29.41 26.19 30.58 64.73 29.80 61.74

100.00 28.16 25.51 17.56 17.02 23.72 25.55 30.30 28.28 30.65 27.55

100.00 47.88 20.77 22.27 39.09 33.33 63.08 31.95 63.77 32.12

100.00 21.29 20.04 35.15 31.69 50.08 27.69 51.22 28.69

100.00 60.97 17.01 17.97 21.23 19.64 21.66 18.27

100.00 18.78 19.64 23.56 17.34 24.22 16.29

100.00 31.06 40.25 30.43 40.75 30.43

100.00 34.21 27.84 34.21 28.66

contents and gene orders as compared with the human HSPA8. In addition most of the neighboring genes are duplicated genes and named as “gene a”, whereas the other zebrafish hspa8 gene neighbored by several “gene b”. Thus, we renamed the first hspa8 gene as hspa8a and the latter hspa8 gene as hspa8b. The counterpart catfish hspa8 genes were named according to zebrafish hspa8 genes. However, a tandem repeat was found in the catfish hspa8a gene, therefore, they were named as hspa8a.1 and hspa8a.2 respectively (Fig. 3). Another copy of hsp70 gene was found on the same chromosome of all fish hspa8 genes though in a distance. In zebrafish, this copy of hsp70 was named as hsc70, which means an alias of hspa8 by name. However, we couldn't find a strong evidence from both phylogenetic and syntenic analysis to support that this gene is an ortholog of hspa8, instead, according to the information from the database online (NCBI and Ensembl), we found this gene is more close to Hspa1a. However, we still named this gene as hsc70 following the zebrafish hsc70. 3.4. Hsp70/110 copy number variation among species The human Hsp70/110 family comprises 16 unique gene products that differ from each other by amino acid sequences, expression levels and sub-cellular localization. Twelve of them are belong to HSPA (HSP70) family, whereas the remaining four are classified in to HSPH (HSP110) family. Catfish have almost all the orthologues of HSP70/110 in human and zebrafish, with exception of several genes existing in humans, but absent from teleost fishes including Hspa2, Hspa6, and Hspa7. Hsph1 wasn't found in all teleosts under study except zebrafish (Table 3). It is interesting that a few of the hsp70/110 genes have more duplicated copies in teleost species than in mammal, bird and amphibian such as hspa8, hspa4. Compared to zebrafish, channel catfish has fewer copies for hspa1 and hapa12 (Table 3). 3.5. Expression analysis of Hsp70/110 genes in catfish after bacterial infection Using two bacterial challenged RNA-Seq datasets (intestine sample infected by E. ictaluri and gill sample infected by F. columnare), the involvement of Hsp70/110 genes after bacterial

infection was determined. As shown in Table 4 and Table 5, ten out of a total of sixteen Hsp70/110 genes were involved in disease defense responses. Specifically, 9 Hsp70/110 genes were found significantly regulated in the gill following F. columnare infection. Among them, eight genes were up-regulated and only one gene, hsc70, was down-regulated (Table 4). It is notable that all of these regulated Hsp70/110 genes were found the highest up-/downregulated fold change at 24h post challenge, except the hspa8b, whose most regulated fold change occurred at the 48h. This suggests a time pattern that how Hsp70/110s involve in the response of F. columnare infection. Two of the regulated Hsp70/110 genes, hspa8b and hyou1 are transiently up- or down-regulated. In contrast, hsp70.3, hspa4a, hspa4b, hspa5 and hspa9 were upregulated at at least two time points after infection, suggesting their up-regulated expression was more lasting. Similar expression patterns were observed for down-regulated gene, hsc70 (Table 4). Different from F. columnare infection, of the 16 catfish Hsp70/110 genes, only two genes were found to be significantly regulated in the intestine after E. ictaluri infection (Table 5). Hsp70.2 was found significantly down-regulated at 3 day post challenge while hspa4b was found significantly down-regulated at 24h post challenge. Within the remaining 14 genes, thirteen genes were not significantly regulated by E. ictaluri infection, while one, hspa12a, was not included for analysis because its expression reads were lower than the threshold (>5) set for the analysis (Table 5). However, expression of five Hsp70/110 genes, hsp70.3, hsc70, hspa4L, hyou1 and hspa14, appeared to be up- or down-regulated, although they were not statistically significant. 4. Discussion Heat shock proteins 70/110 (Hsp70/110) are a family of conserved ubiquitously expressed heat shock proteins in response to stressful conditions. Besides the chaperone functions, they are now known to be also involved in immune response during infection [41,13,14]. In spite of many studies have been done in mammalian species, systematic analysis of Hsp70/110 among teleost fish species has been lacking. In present study, systematic analysis of Hsp70/110s genes was conducted. A total of 16 Hsp70/ 110 genes were identified and annotated in channel catfish,

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Fig. 1. Phylogenetic tree of Hsp70 family. The phylogenetic tree was constructed by Mega5.2.2 using the Maximum Likelihood method based on the JTT matrix-based model of amino acid substitution as described in detail in Material and method section. The bootstrap consensus tree inferred from 1000 replicates is taken. Suffix “L” indicated “-like”, for instance, Hspa4L means Hspa4-like.

phylogenetic and syntenic analyses were conducted, and their expression profiles after bacterial infections were examined to determine their involvement in defense responses. Such genome resource information should be useful for genome analysis and annotation as well as for evolutionary studies in fish species, a group representing over 50% of all vertebrates. Although exhaustive searches were performed with all catfish genomic resources, the hsph1 was not found in the channel catfish genome. Hsph1 can't be found in all the fish genome except zebrafish genome database. It is not known at present if hsph1 was truly missing from the channel catfish genome or it has not been found yet. Other than hsph1, catfish harbored most of the hsp70/ 110. Although most Hsp70/110s are conserved through evolution, the teleost tended to have more duplications than mammals, likely as a consequence of whole genome duplication. Among various fish species, more paralogues of hspa8 and hspa4 were found than that in the genome of mammals, birds and amphibians. Besides, tandem

repeats of hspa8 were also found on the same scaffold of catfish genome. After syntenic analysis, we name them as hspa8a.1 and hspa8a.2 (Fig. 3). Due to Hspa1, Hspa2, Hspa6 and Hspa8 genes have a common domain called HSP1_2_6_8 Nucleoid Binding Domain, therefore the genes with this domain have little difference in the sequence to distinct each other. In catfish, there were six genes in this subfamily were found through the genome. After syntenic analyses they were named as hsp70.2, hsp70.3, hspa8a.1, hspa8a.2, hspa8b and hsc70 according to the nomenclature of zebrafish (Figs 2 and 3). Furthermore, we found another difference after syntenic analysis between human and catfish hsp70 gene. As we all known that MHC complex molecules bound the digested antigen and can be recognized by the T cell receptor (TCR) on the membrane of antigen presenting cell (APC). Hsp70s help MHC complex molecules select and bind to epitopes in this antigen-presenting step. In human genome three HSP70 genes (HSPA1A, HSPA1B and HSPA1L) have

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Fig. 2. Schematic presentation of the conserved synteny blocks neighboring homologs of human HSPA1A gene.

Fig. 3. Schematic presentation of the conserved synteny blocks neighboring homologs of human HSPA8 gene.

been mapped within the MHC loci in the short arm of chromosome 6 (6p21). A hypothesis was brought forward that the location of Hsp70s indicate their involvement in adaptive immune response [42e45]. However, in catfish genome, the hsp70 genes were found on the same chromosome of the MHC gene but they were not found

within the MHC complex loci. From our synntenic analysis of HSPA genes (Fig. 2), we found two MHC complex loci, MHC complex I and MHC complex II, located on the same chromosome of zebrafish hsp70.2, hsp70.3 and hspa1-like on chromosome 3 and 8 respectively. In catfish, we found one member of MHC complex, mhc1, on

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Table 3 Comparison of copy numbers of HSP70s genes among selected vertebrate genomes. The shades indicate absence of genes of that species.

Gene

Human

hspa1 hspa2 hspa6 hspa7 hspa8 hspa4 hsph1 hyou1 hspa5 hspa9 hspa12 hspa13 hspa14 Total

3 1 1 1 1 2 1 1 1 1 2 1 1 17

Bird

Amphibian

Zebrafish

Catfish

Medaka

Tilapia

Fugu

4 1

6

3

2

2

2

1

1 2 1 1 1 1 2 1 1 12

2 1 2 1 2 2 1 1 2 19

2 3 1 1 1 1 3 1 1 20

3 3

3 3

2 1

2 2

1 1 1 2 1 1 16

1 1 1 2 1 1 15

1 2 1 2 1 1 13

1 1 1 2 1 1 14

Table 4 Fold change of catfish Hsp70/110s expression in gill after F. columnare challenge. The significant genes (p value < 0.05, reads numberS 5, fold change S 1.5) and their fold changes are in bold. Gene\Time

4h

24h

48h

hsp70.2 hsp70.3 hspa8a.1 hspa8a.2 hspa8b hsc70 hspa4a hspa4b hspa4L hyou1 hspa5 hspa9 hspa12a hspa12b hspa13 hspa14

1.12 1.67 1.06 1.06 1.32 1.15 2.00 1.74 2.09 1.68 1.87 1.74 1.05 9.43 1.35 1.49

1.37 1.93 2.77 1.36 1.41 ¡2.09 2.96 2.65 2.10 4.00 6.17 2.68 1.05 6.29 1.20 3.44

1.11 1.42 2.46 1.09 1.50 ¡1.58 2.09 1.01 2.61 1.67 1.94 1.47 2.09 9.39 1.48 1.58

Table 5 Fold change of catfish Hsp70/110s expression in intestine after E. ictaluri challenge. The significant genes (p value < 0.05, reads numberS 5, fold changeS 1.5) and their fold changes are in bold. Gene\Time

3h

24h

3d

hsp70.2 hsp70.3 hspa8a.1 hspa8a.2 hspa8b hsc70 hspa4a hspa4b hspa4L hyou1 hspa5 hspa9 hspa12a hspa12b hspa13 hspa14

1.24 1.51 1.14 1.13 1.01 1.00 1.37 1.17 1.60 1.18 1.26 1.02 1.00 1.21 1.18 1.09

1.25 1.35 1.02 1.10 1.14 2.13 1.14 ¡2.23 1.52 1.63 1.28 1.14 1.97 1.47 1.18 1.59

¡1.56 1.84 1.40 1.41 1.32 2.16 1.33 1.77 2.00 1.56 1.39 1.08 1.00 1.05 1.20 1.52

the same chromosomes with the catfish orthologs, hsp70.2 and hsp70.3 on chromosome 3. And a large MHC complex loci was

found on the catfish chromosome 26. The differences in the locations of MHC complex and Hsp70 on genome may lead to the different function of Hsp70 in the immunity among organisms. We determined the expression patterns of Hsp70/110 genes after F. columnare and E. ictaluri infections, for the first time in the channel catfish. Columnaris disease, is featured by erosion or necrosis of external tissues, with the gills often being the major site of damage, and adhesion to gill is considered as the first critical step for F. columnare infection [46]. The intestinal immunology of cellular actors in teleosts was well known for the E. ictaluri infection, and the molecular processes underlying bacterial invasion and passage in the intestinal tissues has been studied, which showed that the E. ictaluri can rapidly through the intestinal barrier [38]. In this regard, we focused on the mucosal immune responses of Hsp70/110 in the early timepoints following challenge to further characterize their roles in host immune defense. 10 out of 16 Hsp70/110 genes were significantly up- or downregulated after bacterial infection, suggesting their involvement in disease responses. However, different patterns were observed in the two bacterial infections. Eight genes were found significantly up-regulated and one gene was down-regulated in gill after F. columnare infection. While after E. ictaluri infection, only two genes were significantly down-regulated (Tables 4 and 5). This suggests either pathogenesis specific pattern or tissue-specific pattern exist in channel catfish after these two bacterial infections. First, the different pathogenesis of the two bacteria may be one of the reasons that explain the different Hsp70/110 expression responses after two bacterial infections. E. ictaluri, a Gram-negative pathogen with flagella [47], is believed to gain entry through the gut. E. ictaluri can invade channel catfish with 15 min by crossing through the intestinal mucosal barrier without any damage. However, the pathogenesis of F. columnare is different, even though it is a Gram-negative bacterial too. The most critical step of F. columnare infection is adhesion on the gill of fish. The mucus from the skin and gills of channel catfish is a chemoattractant to F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills [48,49]. And the major virulence factors of F. columnare are tissue degradation enzyme and chondroitin AC lysate [2,50,51]. However, the expression level of catfish hsp70.2 and hsp70.3 were not expressed as high as expected after the E. ictaluri infection. The Hspa1, which is the homolog of them, is the most inducible gene response to disease infection and believed to be involved in antigen

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Fig. 4. Hsp70/110s expression in gill before and after F. columnare challenge. Vertical axis shows the value of RPKM.

Fig. 5. Hsp70/110s expression in intestine after E. ictaluri challenge. Vertical axis shows the value of RPKM.

presenting process by interacting with MHC complex. From the syntenic analysis, hspa1 orthologues of human and zebrafish were found located within the MHC loci. However, situation is different in catfish as only one MHC gene (mhc 1) was found on the same chromosome of catfish hspa1 orthologues (hsp70.2, hsp70.3) and in the distance of them as well. Therefore the antigen presenting process in catfish may be different with other organisms. Only two genes were significantly down-regulated at 1.5 and 2.0 fold change level in intestine after infection while 9 Hsp70/110 genes were significantly regulated in gill after infection. To compare the expression of Hsp70/110 genes in those two tissues, we took a further look at their RPKM values. The RPKM values were obtained at the same time with the meta-analysis by CLC genomic bench work software. Different catfish Hsp70/110s expression tissue-patterns have been found. As shown in Fig. 4 and Fig. 5, the RPKMs of Hsp70/110s in intestine samples after challenged with E. ictaluri are much higher than that in gill samples after challenged with F. columnare, though the fold change of Hsp70/110s in intestine samples after challenged with E. ictaluri are much less significant than that in gill samples after challenged with F. columnare (Tables 4 and 5). In gill samples, the RPKMs of Hsp70/110s are in the range of 0.1e348, while in intestine samples, the RPKMs of Hsp70/ 110s are in the range of 0e1390. Besides, hspa8a.2 has the highest expression value (RPKM) in both intestine tissue and gill tissue. Compare to hspa8a.1, hspa8a.2 act more as a house keeping gene that is constitutively expressed in channel catfish, especially in intestine sample. This indicates that the insignificant fold change in intestine after E. ictaluri challenge is not due to the low expression

or no expression of Hsp70/110s in intestine, because the RPKMs of Hsp70/110 in intestine is quite high in the samples of all the time points including the control samples. This indicates the catfish Hsp70/110s are more inducible in gill after F. columnare infection than in intestine after E. ictaluri infection. Although high correlations of our RNAseqs and their qPCR were given [38,39], our expression results are still an indication, due to a lack of biological replicates. While pooling samples obviously could have masked individual variation, our goal in the present study was to provide early insights of catfish Hsp70/110 genes response to infection. Follow-up studies can use our results here as a foundation for further biological study to prove the role of each catfish Hsp70/110s in host defense.

5. Conclusion A full set of 16 Hsp70/110s have been characterized from the genome of channel catfish. The vast majority (10 out of 16) of Hsp70/110 genes were either up- or down-regulated after bacterial infection. In detail, nine genes were found significantly expressed in gill after F. columnare infection. Two genes were found significantly expressed in intestine after E. ictaluri infection. Generally, these genes showed different patterns in different bacterial infections and tissues following infection. The RPKMs of catfish Hsp70/110s before and after two bacterial infections indicate the catfish Hsp70/110s are more inducible in gill after F. columnare infection than in intestine after E. ictaluri infection. Although further studies are warranted to explore the mechanisms of

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regulation and to understand the roles of the Hsp70/110s in host defenses against infectious diseases, the results provided here can provide the early insight of the immune functions of Hsp70/110s in catfish.

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Appendix A. Supplementary data [27]

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.12.009. [28]

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