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Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge
Fish & Shellfish Immunology 47 (2015) 751e757
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Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge Chao Li a, *, 1, Lin Song a, 1, Fenghua Tan b, Baofeng Su d, Dongdong Zhang c, Honggang Zhao c, Eric Peatman c a
Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, China School of International Education and Exchange, Qingdao Agricultural University, Qingdao 266109, China School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA d Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 August 2015 Received in revised form 16 October 2015 Accepted 17 October 2015 Available online 20 October 2015
The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gramnegative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cathepsin Catfish Infection Mucosal immunity
1. Introduction The immune system protects host against pathogen infection, but it is the mucosal barriers serve as the first line of host defense [1]. Especially for aquatic species, which are constantly colonized by a wide range of commensals and opportunistic and primary pathogens along their exposed gill and skin surfaces as well as through the intestine [2]. The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion while simultaneously mediating a variety of other critical physiological processes, including nutrient and oxygen absorption,
* Corresponding author. E-mail address: [email protected] (C. Li). 1 Both these authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.fsi.2015.10.028 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
osmoregulation, waste excretion, as well as sensing, sampling, and screening a diverse aquatic microbiota [3]. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited [4]. Moreover, while most of the studies were focused on characterizing the primary actors and mechanisms in classical immune organs [5,6], our understanding of the cellular actors and pathways governing mucosal immune responses is still lacking in a handful of vertebrates. Cysteine cathepsins, are a group of papain-like cysteine proteases that were initially considered as intracellular enzymes, responsible for intracellular and extracellular proteins [7]. As the knowledge of cysteine cathepsins are growing rapidly, they are found to be acting as important regulators and signaling molecules in different levels of biological processes, such as protein degradation and turn over, antigen processing, hormone activation and
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inflammatory responses [8,9]. In human, there are 11 groups of cysteine cathepsins including cathepsins B, C, H, F, K, L, O, S, V, W and X [10]. Among them, cathepsin B (ctsb) is considered as a unique member of the cysteine cathepsins family, because it is acting as a peptidyl-dipeptidase while the others are endopeptidases [11]. Since the first ctsb was identified from rat, it has been well studied in more and more species [12]. In fish species, ctsb has been identified in zebrafish (Danio rerio) [13], turbot (Scophthalmus maximus) [14], atlantic cod (Gadus morhua) [15], rock bream (Oplegnathus fasciatus) [16], flounder (Paralichthys olivaceus) [17], croaker (Miichthys miiuy) [18] and grouper (Epinephelus coioides) [19]. As a lysosomal cysteine protease, ctsb is involved in multiple levels of physiological and biological processes. Recently, in vertebrates, a growing body of evidence has suggested the crucial roles of ctsb for host immune defense against pathogen infection. For instance, in orange-spotted grouper, after challenged with singapore grouper iridovirus (SGIV) stimulation, ctsb was upregulated rapidly in spleen [19]. In flounder, ctsb was significantly induced following virus and LPS challenge, not only in spleen and kidney, but also in gill and intestine [20]. It is suggested that ctsb may play key roles in fish mucosal immune responses against bacterial infection, but little studies have investigated its expression patterns in mucosal tissues following challenge. Channel catfish (Ictalurus punctatus), the dominant economically aquaculture species in the America's aquaculture industry, suffers serious economic losses due to a number of bacterial pathogens. Especially, the causative agent of enteric septicemia of catfish (ESC), a Gram-negative bacteria Edwardsiella ictaluri, along with Flavobacterium columnare, which is the causative agent of columnaris disease, are the most important bacterial diseases to channel catfish industry in US [21,22]. Up to date, much efforts have been devoted to characterize cathepsins in channel catfish. Briefly, cathepsin D, H, L and S have been identified in catfish [23e25], and cathepsin D was reported to have antimicrobial activities in skin mucosa of catfish [26]. With the recognized roles of cathepsins for catfish mucosal immunity, no studies have characterized cathepsin B (ctsb) in catfish. With the availability of trancriptomic databases from our recently studies, therefore, here we identified the ctsba of channel catfish and investigated its expression patterns in mucosal tissues following E. ictaluri and F. columnare challenge. The present study is the first examination of ctsba structure and expression in catfish. Our results can provide insight for further functional studies of ctsba in catfish as well as other fish species. 2. Materials and methods 2.1. Sequence identification and analysis In order to identify the cathepsin B (ctsb) genes of channel catfish, channel catfish transcriptome databases generated by our previous studies [27e29] and the whole genome database of catfish (unpublished data) were searched using ctsb sequences from other species as queries with a cutoff E-value of 1e-10. The retrieved sequences were translated using ORF Finder (http://www.ncbi.nlm. nih.gov/gorf/gorf.html). The predicted amino acid sequences from ORF predication were further verified by BLASTP (http://blast.ncbi. nlm.nih.gov/Blast.cgi) against NCBI non-redundant protein sequence database. The simple modular architecture research tool (SMART; http://smart.embl-heidelberg.de/) was used to identify the conserved domains and signal peptides. ExPASy server was used to analyze the N-glycosylation sites [30]. 2.2. Phylogenetic analysis The amino acids coding for the conserved domains of ctsba from
channel catfish and the other organisms were selected to construct the phylogenetic tree, including human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), frog (Xenopus laevis), turtle (Chrysemys picta), medaka (Oryzias latipes), tilapia (Oreochromis niloticus), fugu (Takifugu rubripes), zebrafish and stickleback (Gasterosteus aculeatus). Multiple protein sequence alignment was conducted using the ClustalW2 program [31]. Phylogenetic and molecular evolutionary analyses were performed using the neighbor-joining method within the Molecular Evolutionary Genetics Analysis (MEGA 6) package [32]. Data was analyzed using poisson correction, and gaps were removed by complete deletion. Bootstrapping with 10,000 replications was conducted to evaluate the topological stability of the phylogenetic tree. 2.3. Bacteria challenge and sample collection In order to characterize the immune roles of ctsba genes in the host defense against bacterial infection, E. ictaluri and F. columnare challenges were conducted following the established protocols [33]. Prior to experimental challenge, channel catfish fingerlings (average body weight: 10.2 g and average body length ¼ 9.5 cm) were obtained from the Auburn University Fish Genetics Research Unit, and acclimated in the laboratory in a flow-through system for at least two weeks at a temperature of 28 C. After a pre-challenge, the bacteria was re-isolated from a symptomatic fish and biochemically confirmed before cultured. During challenge, symptomatic fish were confirmed to be infected with E. ictaluri and F. columnare, respectively. During the experiments, water circulation was off for 2 h and then was on until the end of each experiment. For sample collection, the fish were euthanized with tricaine methanesulfonate (MS-222) at 200 mg/L (buffered with sodium bicarbonate). Briefly, a virulent strain, MS-S97-773 of E. ictaluri was inoculated in a brain heart infusion (BHI) medium in a shaker incubator at 28 C overnight. A total of 250 fish were equally divided into 8 aquariums, four treated groups and their corresponding control group. The four treated groups were then assigned for four sampling time-points, namely, 4 h treatment, 24 h treatment, 3 d treatment and 7 d treatment according to the duration of treatment. For the challenge, the fish were immersed for 2 h at a final concentration of 4 108 CFU/mL, while control fish were immersed in sterilized media alone. At 4 h, 24 h, 3 d and 7 d after challenge, tissues (skin and gill) of 30 treated fish from each aquaria were dissected as well as the control group. At each time point, tissues from ten individual fish per condition were pooled as 3 replicates. The bacteria F. columnare (BGFS-27; genomovar II) was inoculated in a modified Shieh broth for 24 h in a shaker incubator (100 rpm) at 28 C. Fish (N ¼ 200, siblings from the same catfish family used for E. ictaluri experiment) were randomly divided into 6 rectangular 30-L aquaria of 3 control groups and the other 3 were designated as challenge groups for sampling at three time-points (4 h, 24 h and 60 h). For the challenge, the fish were immersed for 2 h at a final concentration of 3 106 CFU/mL. Control fish were exposed only to the sterile modified Shieh broth. Eighteen fish from each aquarium were sacrificed at 4 h, 24 h and 60 h, and samples of gill and skin were dissected and pooled (6 fish per replication) in both challenge and control groups, respectively. All samples from both experiments were flash-frozen in liquid nitrogen and then stored in a 80 C ultra-low freezer until preparation of RNA. 2.4. Total RNA extraction and cDNA synthesis Prior to RNA extraction, samples were homogenized under liquid nitrogen using mortar and pestle under liquid nitrogen. Total RNA was extracted using Trizol® Reagent (Invitrogen, USA)
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according to the supplied protocol. The quality and quantity of RNA of each sample was measured on a nanodrop 2000 (Thermo Electron North America LLC, FL). All extracted samples had an A260/280 ratio greater than 1.8, and were diluted to 500 ng/ml. First strand cDNA was synthesized by iScript™ cDNA Synthesis Kit (Bio-Rad, USA) following manufacturer's protocol (500 ng RNA per 10 ml reaction).
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Different numbers of N-glycosylation sites have been identified in ctsb from other species. For instance, only one putative N-glycosylation site was found in ctsb of yellow croaker (P. olivaceus) [20,36]. In cathepsins, the N-glycosylation site was reported to be essential for lysosomes transporting, and play key roles for immune response against pathogen infection [37]. The higher number of Nglycosylation sites may suggest ctsba play more crucial roles for catfish host immune response.
2.5. Real-time PCR analysis 3.2. Phylogenetic analyses Gene specific primers were designed using Primer3 software based on the channel catfish ctsba sequence: UpperGGTGAAGCAGTAGGAGGTCAT; Lower-CCAGCCACAATCTCAGACTCT (50 to 30 ). And channel catfish 18S rRNA gene (UpperGAGAAACGGCTACCACATCC; Lower-GATACGCTCATTCCGATTACAG; 50 to 30 ) was used as a reference gene for normalization of the levels of gene expression in the same samples. The PCR product was sequenced to confirm the specificity of these primers. Quantitative real-time PCR (qPCR) was performed on a C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) by using the SsoFast EvaGreen supermix kit (Bio-Rad) following the manufacturer's instructions. The thermal cycling profile was as follows: denaturation, 95 C/30 s, 40 cycles of 95 C/5 s, 58 C/5 s followed by dissociation curve analysis, 5 s at 65 C, then up to 95 C at a rate of 0.1 C/s increment, to verify the specificity of the amplicons. Triple RNA samples from healthy and infected tissue at each time-point were analyzed for gene expression. Crossing-point (Ct) values were exported into a Microsoft Excel sheet from Bio-Rad CRX Manager (Version 1.6.541.1028, 2008) for analysis. Results were analyzed using Relative Expression Software Tool (REST) [34] on the assumption that PCR had 100% efficiency and randomization was performed 2000 times to capture significance at the level of P < 0.05. In order to determine the gene expression patterns in catfish healthy tissues, Ct values from muscle were used as control. The mRNA expression levels of all samples were normalized to the levels of 18S ribosomal RNA gene in the same samples. A no-template control was run on all plates. 3. Results and discussion 3.1. Identification of channel catfish ctsb genes After searching the channel catfish transcriptomic and genomic databases using available ctsb sequences from other fish species as queries, a cDNA and putative amino acid sequences (ctsba) was identified. The channel catfish ctsba is 1580 bp long with a 990 bp ORF encoding 330 amino acids. After prediction, the deduced amino acid contains a 18 amino acid signal peptide, a 40 amino acid propeptide and a 250 amino acid Pept_C1 domain. In mammals, the propeptide is required for cathepsins to play roles in protein folding and degradation, structure stabilizing to external pH changes, as well as microsomal and lysosomal targeting [35]. Comparing to other species, catfish ctsba is highly homologous to other species, shared the highest identity 84% to zebrafish, and 83% to carp, as well as 72% to human. Moreover, multiple sequence alignment showed that catfish ctsba had the conserved structural motifs such as glutarnine acid (Q), cystine acid (C), histidine acid (H) and asparagine acid (N) (Fig. 1). Alignments of the genomic sequences with the cDNA sequences revealed the presence of 10 exons and 9 introns in the channel catfish and zebrafish ctsba gene (Fig. S1). The number and the size of the exons are completely conserved between channel catfish and model organism, zebrafish. However, zebrafish ctsba gene shows a little shorter gene size of 7380 bp than that of catfish, which is 7868 bp. After scanning, there were two Nglycosylation sites and nine N-myristoylation sites in catfish ctsba.
In order to determine the phylogenetic relationship of catfish ctsba with other species, a neighbor-joining phylogenetic tree was constructed using MEGA6 (Fig. 2). The phylogenetic analysis revealed that the catfish ctsba gene was first clustered with zebrafish ctsba, and then fell in the clade of the other vertebrates. The other clade was consisted by human and mouse, and then clustered with chicken, frog and turtle. With the strongest relationship to zebrafish, the catfish ctsba phylogenetic tree was consistent with their phylogenetic relationships, and all branching nodes were supported by high bootstrap values. Collectively, the phylogenetic analysis confirmed the identification of catfish ctsba gene and showed the strong orthology to their counterparts identified in other species. 3.3. Basal tissue expression of catfish cathepsin B gene The tissue distribution analysis of channel catfish ctsba gene was conducted in eight healthy channel catfish tissues, including muscle, liver, spleen, gill, skin, intestine, head kidney and trunk kidney utilizing realtime PCR. Catfish ctsba gene was expressed in all examined tissues, consistent with the studies from the other species (Fig. 3) [38]. Because the lowest expression level of ctsba was detected in muscle, the relative fold changes between different tissues were calculated by using muscle as control tissue. In detail, the highest expression level of ctsba was found in catfish trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin (Fig. 3). Similar to human, the ctsb had higher expression level in kidney [39], and was also detected very abundant in intestine [40]. Among vertebrates, the expression patterns of ctsb is quite different. Briefly, in contrast to our result, ctsb showed the highest expression level in muscle in turbot, and the lowest expression level in kidney [14]. In olive flounder, the highest expression level of ctsb was detected in gill and intestine, and was lowly expressed in liver and muscle [17]. In yellow croaker, the highest expression level of ctsb was found in spleen, while the lowest expression level was found in kidney [36]. In addition, in Chinese mitten crab, the highest expression level was also observed in muscle [41]. The different expression patterns across the different species suggested ctsb might play different roles in different species. Here, the detected high expression levels of ctsba in classical immune organ kidney indicating its important immune roles, and the high expression levels in mucosal tissues gill and intestine may indicate its roles in catfish mucosal immunity. Indeed, the further studies are needed to examine the different functions of Cathepsin B in different tissues and species. 3.4. Expression profiles of ctsba gene after bacterial challenge To elucidate whether catfish ctsba gene plays key roles in mucosal immune responses against bacterial infection, its expression pattern was examined in catfish mucosal surfaces (skin, gill and intestine), which are the critical tissues for pathogen attachment and entry, following challenge by two Gram-negative bacteria. In general, the ctsba showed a trend of up-regulated in mucosal
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Fig. 1. Alignments of the deduced amino acid sequences of channel catfish cathepsin B (ctsba) gene with other fish species. Dashes represent amino acid deletions. The predicted signal peptides were underlined. The catalytic residues were shaded. Asterisks indicate identical amino acids; colons indicate similar amino acids and empty spaces represent absence or low level of similarities.
Fig. 2. Phylogenetic tree for the cathepsin B (ctsba) gene of channel catfish. The phylogenetic tree was constructed based on the amino acid sequence of Cathepsin B from other selected species of fish and mammals, using the neighbor-joining method in MEGA 6. Gaps were removed by complete deletion and the phylogenetic tree was evaluated with 1000 bootstrap replications, and the bootstrapping values are indicated by numbers at the nodes. Dark solid circles indicated the newly characterized catfish ctsba genes.
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Fig. 3. Gene expression analysis of the ctsba gene in different healthy channel catfish tissues. Expression levels were calibrated against that in the muscle, and 18S rRNA was used as reference gene. The abbreviations are as the following: HK for head kidney and TK for trunk kidney.
Fig. 4. Real time qRT-PCR analysis for cathepsin B (ctsba) expression following Edwardsiella ictaluri infection. The ctsba expression was measured in the mucosal tissues including (A) Skin, (B) Gill, and (C) Intestine at the timepoints of 4 h, 24 h, 3 d, and 7 d post-infection. Fold change was calculated by the change in expression at a given timepoint relative to the untreated control and normalized by changes in the 18S housekeeping gene. The results are presented as mean ± SE of fold changes and * indicates statistical significance at P < 0.05.
tissues following challenge (Fig. 4A). In detail, following E. ictaluri infection, the ctsba was rapidly induced as early as 4 h in skin with the largest fold change (4 fold; Fig. 4A), and although the expression level was decreased, still significantly up-regulated at 7 d. Different from the acute response in skin, the ctsba was only induced at 7 d in gill (12 fold; Fig. 4B). Similar in intestine, the strongest up-regulation was also observed at 7 d (7 fold; Fig. 4C), while the other up-regulation was detected as early as 4 h. The same pattern was also found post F. columnare infection, the ctsba was dramatically induced in both skin and gill at 60 h. In addition, ctsba was only up-regulated at 60 h (10 fold) in skin (Fig. 5A), while it was earlier induced at 24 h (6 fold) in gill (Fig. 5B). For successful infection of host, pathogens need to penetrate the mucosal surfaces, and establish efficient colonization and replication. At the same time, the mucosal immune system needs to recognize the pathogen, prevent the pathogen invading and clean up the attached pathogen. In addition to serve as the physical barriers, the mucosal surfaces are also considered as a reservoir for many immune factors with antimicrobial functions. According to early studies, the innate immune factors including immunoglobulins, lysozymes, proteases, lectins, complement factors and antimicrobial peptides which involved in inhibition of pathogen trapping and sloughing have been widely identified in fish mucosal tissues [42e44]. Therefore, understanding of the immune activities of immune actors in fish mucosal tissues following challenge can expand our knowledge of teleost mucosal immunity and improve the development of effective strategies for disease control. In catfish, F. columnare is believed to gain entry through skin and gill [45], while the intestine was considered as the primary route for E. ictaluri infection, but its ability of entry through gill and skin was also observed [22,46,47]. Whereas several innate immune actors have been characterized in catfish, nothing is known about the breadth and function of ctsb in catfish [48e50]. Therefore, here we characterized the ctsb of channel catfish and determined its expression patterns following E. ictaluri and F. columnare infection. Following challenge with two different Gram-negative pathogens, the substantial similarity of the ctsba expression profiles was detected. ctsba was significantly up-regulated in both challenges, but showed a more acute response post E. ictaluri infection. Additionally, ctsba was more dramatically induced (more than 10 fold) in F. columnare challenge. This distinct difference consistent with the difference of our experimental challenge, while the mortality of F. columnare occurred as early as 48 h, the mortality of E. ictaluri has not been observed in the first week of challenge. Previously, cathepsins were revealed to regulate the inflammatory responses
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.10.028. References
Fig. 5. Real time qRT-PCR analysis for cathepsin B (ctsba) expression following Flavobacterium columnare infection. The ctsba expression was measured in the mucosal tissues including (A) Skin and (B) Gill, at the timepoints of 4 h, 24 h and 60 h postinfection. Fold change was calculated by the change in expression at a given timepoint relative to the untreated control and normalized by changes in the 18S housekeeping gene. The results are presented as mean ± SE of fold changes and the * indicates statistical significance at P < 0.05.
following infection, and can regulate the production of inflammatory actors in the sites of infection [51,52]. In mice, the increased activity of Ctsb along with the increased inflammation responses was observed following challenge [53]. On the other hand, inhibition of Ctsb significantly decreased the expression of inflammatory cytokines together with the inflammasome components [53]. Here, the increased expression of ctsba in mucosal surfaces should be resulted from the inflammatory response against invading pathogen. Similar in flounder, ctsb showed the strongest up-regulation in gill following viral challenge, along with the dramatic increased expression in intestine [20]. Not only in mucosal tissues, ctsb was also induced in spleen post challenge in grouper [19]. Collectively, the wide distribution and regulation across catfish tissues suggested catfish ctsba is not only involved in the acute inflammatory responses, but also played key roles in the pathogen triggered immune response after pathogen entry and recognition. In present study, the ctsba gene was first identified in catfish as well as its expression pattern in mucosal tissues following different bacteria challenge. Our results indicate the protective function of ctsba against infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may also play additional physiological and biological roles in catfish mucosal tissues.
Acknowledgments This study is supported by Advanced Talents Foundation of QAU, Grant Number 1114337.
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Short communication
Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge Chao Li a, *, 1, Lin Song a, 1, Fenghua Tan b, Baofeng Su d, Dongdong Zhang c, Honggang Zhao c, Eric Peatman c a
Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, China School of International Education and Exchange, Qingdao Agricultural University, Qingdao 266109, China School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA d Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 August 2015 Received in revised form 16 October 2015 Accepted 17 October 2015 Available online 20 October 2015
The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gramnegative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cathepsin Catfish Infection Mucosal immunity
1. Introduction The immune system protects host against pathogen infection, but it is the mucosal barriers serve as the first line of host defense [1]. Especially for aquatic species, which are constantly colonized by a wide range of commensals and opportunistic and primary pathogens along their exposed gill and skin surfaces as well as through the intestine [2]. The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion while simultaneously mediating a variety of other critical physiological processes, including nutrient and oxygen absorption,
* Corresponding author. E-mail address: [email protected] (C. Li). 1 Both these authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.fsi.2015.10.028 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
osmoregulation, waste excretion, as well as sensing, sampling, and screening a diverse aquatic microbiota [3]. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited [4]. Moreover, while most of the studies were focused on characterizing the primary actors and mechanisms in classical immune organs [5,6], our understanding of the cellular actors and pathways governing mucosal immune responses is still lacking in a handful of vertebrates. Cysteine cathepsins, are a group of papain-like cysteine proteases that were initially considered as intracellular enzymes, responsible for intracellular and extracellular proteins [7]. As the knowledge of cysteine cathepsins are growing rapidly, they are found to be acting as important regulators and signaling molecules in different levels of biological processes, such as protein degradation and turn over, antigen processing, hormone activation and
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inflammatory responses [8,9]. In human, there are 11 groups of cysteine cathepsins including cathepsins B, C, H, F, K, L, O, S, V, W and X [10]. Among them, cathepsin B (ctsb) is considered as a unique member of the cysteine cathepsins family, because it is acting as a peptidyl-dipeptidase while the others are endopeptidases [11]. Since the first ctsb was identified from rat, it has been well studied in more and more species [12]. In fish species, ctsb has been identified in zebrafish (Danio rerio) [13], turbot (Scophthalmus maximus) [14], atlantic cod (Gadus morhua) [15], rock bream (Oplegnathus fasciatus) [16], flounder (Paralichthys olivaceus) [17], croaker (Miichthys miiuy) [18] and grouper (Epinephelus coioides) [19]. As a lysosomal cysteine protease, ctsb is involved in multiple levels of physiological and biological processes. Recently, in vertebrates, a growing body of evidence has suggested the crucial roles of ctsb for host immune defense against pathogen infection. For instance, in orange-spotted grouper, after challenged with singapore grouper iridovirus (SGIV) stimulation, ctsb was upregulated rapidly in spleen [19]. In flounder, ctsb was significantly induced following virus and LPS challenge, not only in spleen and kidney, but also in gill and intestine [20]. It is suggested that ctsb may play key roles in fish mucosal immune responses against bacterial infection, but little studies have investigated its expression patterns in mucosal tissues following challenge. Channel catfish (Ictalurus punctatus), the dominant economically aquaculture species in the America's aquaculture industry, suffers serious economic losses due to a number of bacterial pathogens. Especially, the causative agent of enteric septicemia of catfish (ESC), a Gram-negative bacteria Edwardsiella ictaluri, along with Flavobacterium columnare, which is the causative agent of columnaris disease, are the most important bacterial diseases to channel catfish industry in US [21,22]. Up to date, much efforts have been devoted to characterize cathepsins in channel catfish. Briefly, cathepsin D, H, L and S have been identified in catfish [23e25], and cathepsin D was reported to have antimicrobial activities in skin mucosa of catfish [26]. With the recognized roles of cathepsins for catfish mucosal immunity, no studies have characterized cathepsin B (ctsb) in catfish. With the availability of trancriptomic databases from our recently studies, therefore, here we identified the ctsba of channel catfish and investigated its expression patterns in mucosal tissues following E. ictaluri and F. columnare challenge. The present study is the first examination of ctsba structure and expression in catfish. Our results can provide insight for further functional studies of ctsba in catfish as well as other fish species. 2. Materials and methods 2.1. Sequence identification and analysis In order to identify the cathepsin B (ctsb) genes of channel catfish, channel catfish transcriptome databases generated by our previous studies [27e29] and the whole genome database of catfish (unpublished data) were searched using ctsb sequences from other species as queries with a cutoff E-value of 1e-10. The retrieved sequences were translated using ORF Finder (http://www.ncbi.nlm. nih.gov/gorf/gorf.html). The predicted amino acid sequences from ORF predication were further verified by BLASTP (http://blast.ncbi. nlm.nih.gov/Blast.cgi) against NCBI non-redundant protein sequence database. The simple modular architecture research tool (SMART; http://smart.embl-heidelberg.de/) was used to identify the conserved domains and signal peptides. ExPASy server was used to analyze the N-glycosylation sites [30]. 2.2. Phylogenetic analysis The amino acids coding for the conserved domains of ctsba from
channel catfish and the other organisms were selected to construct the phylogenetic tree, including human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), frog (Xenopus laevis), turtle (Chrysemys picta), medaka (Oryzias latipes), tilapia (Oreochromis niloticus), fugu (Takifugu rubripes), zebrafish and stickleback (Gasterosteus aculeatus). Multiple protein sequence alignment was conducted using the ClustalW2 program [31]. Phylogenetic and molecular evolutionary analyses were performed using the neighbor-joining method within the Molecular Evolutionary Genetics Analysis (MEGA 6) package [32]. Data was analyzed using poisson correction, and gaps were removed by complete deletion. Bootstrapping with 10,000 replications was conducted to evaluate the topological stability of the phylogenetic tree. 2.3. Bacteria challenge and sample collection In order to characterize the immune roles of ctsba genes in the host defense against bacterial infection, E. ictaluri and F. columnare challenges were conducted following the established protocols [33]. Prior to experimental challenge, channel catfish fingerlings (average body weight: 10.2 g and average body length ¼ 9.5 cm) were obtained from the Auburn University Fish Genetics Research Unit, and acclimated in the laboratory in a flow-through system for at least two weeks at a temperature of 28 C. After a pre-challenge, the bacteria was re-isolated from a symptomatic fish and biochemically confirmed before cultured. During challenge, symptomatic fish were confirmed to be infected with E. ictaluri and F. columnare, respectively. During the experiments, water circulation was off for 2 h and then was on until the end of each experiment. For sample collection, the fish were euthanized with tricaine methanesulfonate (MS-222) at 200 mg/L (buffered with sodium bicarbonate). Briefly, a virulent strain, MS-S97-773 of E. ictaluri was inoculated in a brain heart infusion (BHI) medium in a shaker incubator at 28 C overnight. A total of 250 fish were equally divided into 8 aquariums, four treated groups and their corresponding control group. The four treated groups were then assigned for four sampling time-points, namely, 4 h treatment, 24 h treatment, 3 d treatment and 7 d treatment according to the duration of treatment. For the challenge, the fish were immersed for 2 h at a final concentration of 4 108 CFU/mL, while control fish were immersed in sterilized media alone. At 4 h, 24 h, 3 d and 7 d after challenge, tissues (skin and gill) of 30 treated fish from each aquaria were dissected as well as the control group. At each time point, tissues from ten individual fish per condition were pooled as 3 replicates. The bacteria F. columnare (BGFS-27; genomovar II) was inoculated in a modified Shieh broth for 24 h in a shaker incubator (100 rpm) at 28 C. Fish (N ¼ 200, siblings from the same catfish family used for E. ictaluri experiment) were randomly divided into 6 rectangular 30-L aquaria of 3 control groups and the other 3 were designated as challenge groups for sampling at three time-points (4 h, 24 h and 60 h). For the challenge, the fish were immersed for 2 h at a final concentration of 3 106 CFU/mL. Control fish were exposed only to the sterile modified Shieh broth. Eighteen fish from each aquarium were sacrificed at 4 h, 24 h and 60 h, and samples of gill and skin were dissected and pooled (6 fish per replication) in both challenge and control groups, respectively. All samples from both experiments were flash-frozen in liquid nitrogen and then stored in a 80 C ultra-low freezer until preparation of RNA. 2.4. Total RNA extraction and cDNA synthesis Prior to RNA extraction, samples were homogenized under liquid nitrogen using mortar and pestle under liquid nitrogen. Total RNA was extracted using Trizol® Reagent (Invitrogen, USA)
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according to the supplied protocol. The quality and quantity of RNA of each sample was measured on a nanodrop 2000 (Thermo Electron North America LLC, FL). All extracted samples had an A260/280 ratio greater than 1.8, and were diluted to 500 ng/ml. First strand cDNA was synthesized by iScript™ cDNA Synthesis Kit (Bio-Rad, USA) following manufacturer's protocol (500 ng RNA per 10 ml reaction).
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Different numbers of N-glycosylation sites have been identified in ctsb from other species. For instance, only one putative N-glycosylation site was found in ctsb of yellow croaker (P. olivaceus) [20,36]. In cathepsins, the N-glycosylation site was reported to be essential for lysosomes transporting, and play key roles for immune response against pathogen infection [37]. The higher number of Nglycosylation sites may suggest ctsba play more crucial roles for catfish host immune response.
2.5. Real-time PCR analysis 3.2. Phylogenetic analyses Gene specific primers were designed using Primer3 software based on the channel catfish ctsba sequence: UpperGGTGAAGCAGTAGGAGGTCAT; Lower-CCAGCCACAATCTCAGACTCT (50 to 30 ). And channel catfish 18S rRNA gene (UpperGAGAAACGGCTACCACATCC; Lower-GATACGCTCATTCCGATTACAG; 50 to 30 ) was used as a reference gene for normalization of the levels of gene expression in the same samples. The PCR product was sequenced to confirm the specificity of these primers. Quantitative real-time PCR (qPCR) was performed on a C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) by using the SsoFast EvaGreen supermix kit (Bio-Rad) following the manufacturer's instructions. The thermal cycling profile was as follows: denaturation, 95 C/30 s, 40 cycles of 95 C/5 s, 58 C/5 s followed by dissociation curve analysis, 5 s at 65 C, then up to 95 C at a rate of 0.1 C/s increment, to verify the specificity of the amplicons. Triple RNA samples from healthy and infected tissue at each time-point were analyzed for gene expression. Crossing-point (Ct) values were exported into a Microsoft Excel sheet from Bio-Rad CRX Manager (Version 1.6.541.1028, 2008) for analysis. Results were analyzed using Relative Expression Software Tool (REST) [34] on the assumption that PCR had 100% efficiency and randomization was performed 2000 times to capture significance at the level of P < 0.05. In order to determine the gene expression patterns in catfish healthy tissues, Ct values from muscle were used as control. The mRNA expression levels of all samples were normalized to the levels of 18S ribosomal RNA gene in the same samples. A no-template control was run on all plates. 3. Results and discussion 3.1. Identification of channel catfish ctsb genes After searching the channel catfish transcriptomic and genomic databases using available ctsb sequences from other fish species as queries, a cDNA and putative amino acid sequences (ctsba) was identified. The channel catfish ctsba is 1580 bp long with a 990 bp ORF encoding 330 amino acids. After prediction, the deduced amino acid contains a 18 amino acid signal peptide, a 40 amino acid propeptide and a 250 amino acid Pept_C1 domain. In mammals, the propeptide is required for cathepsins to play roles in protein folding and degradation, structure stabilizing to external pH changes, as well as microsomal and lysosomal targeting [35]. Comparing to other species, catfish ctsba is highly homologous to other species, shared the highest identity 84% to zebrafish, and 83% to carp, as well as 72% to human. Moreover, multiple sequence alignment showed that catfish ctsba had the conserved structural motifs such as glutarnine acid (Q), cystine acid (C), histidine acid (H) and asparagine acid (N) (Fig. 1). Alignments of the genomic sequences with the cDNA sequences revealed the presence of 10 exons and 9 introns in the channel catfish and zebrafish ctsba gene (Fig. S1). The number and the size of the exons are completely conserved between channel catfish and model organism, zebrafish. However, zebrafish ctsba gene shows a little shorter gene size of 7380 bp than that of catfish, which is 7868 bp. After scanning, there were two Nglycosylation sites and nine N-myristoylation sites in catfish ctsba.
In order to determine the phylogenetic relationship of catfish ctsba with other species, a neighbor-joining phylogenetic tree was constructed using MEGA6 (Fig. 2). The phylogenetic analysis revealed that the catfish ctsba gene was first clustered with zebrafish ctsba, and then fell in the clade of the other vertebrates. The other clade was consisted by human and mouse, and then clustered with chicken, frog and turtle. With the strongest relationship to zebrafish, the catfish ctsba phylogenetic tree was consistent with their phylogenetic relationships, and all branching nodes were supported by high bootstrap values. Collectively, the phylogenetic analysis confirmed the identification of catfish ctsba gene and showed the strong orthology to their counterparts identified in other species. 3.3. Basal tissue expression of catfish cathepsin B gene The tissue distribution analysis of channel catfish ctsba gene was conducted in eight healthy channel catfish tissues, including muscle, liver, spleen, gill, skin, intestine, head kidney and trunk kidney utilizing realtime PCR. Catfish ctsba gene was expressed in all examined tissues, consistent with the studies from the other species (Fig. 3) [38]. Because the lowest expression level of ctsba was detected in muscle, the relative fold changes between different tissues were calculated by using muscle as control tissue. In detail, the highest expression level of ctsba was found in catfish trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin (Fig. 3). Similar to human, the ctsb had higher expression level in kidney [39], and was also detected very abundant in intestine [40]. Among vertebrates, the expression patterns of ctsb is quite different. Briefly, in contrast to our result, ctsb showed the highest expression level in muscle in turbot, and the lowest expression level in kidney [14]. In olive flounder, the highest expression level of ctsb was detected in gill and intestine, and was lowly expressed in liver and muscle [17]. In yellow croaker, the highest expression level of ctsb was found in spleen, while the lowest expression level was found in kidney [36]. In addition, in Chinese mitten crab, the highest expression level was also observed in muscle [41]. The different expression patterns across the different species suggested ctsb might play different roles in different species. Here, the detected high expression levels of ctsba in classical immune organ kidney indicating its important immune roles, and the high expression levels in mucosal tissues gill and intestine may indicate its roles in catfish mucosal immunity. Indeed, the further studies are needed to examine the different functions of Cathepsin B in different tissues and species. 3.4. Expression profiles of ctsba gene after bacterial challenge To elucidate whether catfish ctsba gene plays key roles in mucosal immune responses against bacterial infection, its expression pattern was examined in catfish mucosal surfaces (skin, gill and intestine), which are the critical tissues for pathogen attachment and entry, following challenge by two Gram-negative bacteria. In general, the ctsba showed a trend of up-regulated in mucosal
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Fig. 1. Alignments of the deduced amino acid sequences of channel catfish cathepsin B (ctsba) gene with other fish species. Dashes represent amino acid deletions. The predicted signal peptides were underlined. The catalytic residues were shaded. Asterisks indicate identical amino acids; colons indicate similar amino acids and empty spaces represent absence or low level of similarities.
Fig. 2. Phylogenetic tree for the cathepsin B (ctsba) gene of channel catfish. The phylogenetic tree was constructed based on the amino acid sequence of Cathepsin B from other selected species of fish and mammals, using the neighbor-joining method in MEGA 6. Gaps were removed by complete deletion and the phylogenetic tree was evaluated with 1000 bootstrap replications, and the bootstrapping values are indicated by numbers at the nodes. Dark solid circles indicated the newly characterized catfish ctsba genes.
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Fig. 3. Gene expression analysis of the ctsba gene in different healthy channel catfish tissues. Expression levels were calibrated against that in the muscle, and 18S rRNA was used as reference gene. The abbreviations are as the following: HK for head kidney and TK for trunk kidney.
Fig. 4. Real time qRT-PCR analysis for cathepsin B (ctsba) expression following Edwardsiella ictaluri infection. The ctsba expression was measured in the mucosal tissues including (A) Skin, (B) Gill, and (C) Intestine at the timepoints of 4 h, 24 h, 3 d, and 7 d post-infection. Fold change was calculated by the change in expression at a given timepoint relative to the untreated control and normalized by changes in the 18S housekeeping gene. The results are presented as mean ± SE of fold changes and * indicates statistical significance at P < 0.05.
tissues following challenge (Fig. 4A). In detail, following E. ictaluri infection, the ctsba was rapidly induced as early as 4 h in skin with the largest fold change (4 fold; Fig. 4A), and although the expression level was decreased, still significantly up-regulated at 7 d. Different from the acute response in skin, the ctsba was only induced at 7 d in gill (12 fold; Fig. 4B). Similar in intestine, the strongest up-regulation was also observed at 7 d (7 fold; Fig. 4C), while the other up-regulation was detected as early as 4 h. The same pattern was also found post F. columnare infection, the ctsba was dramatically induced in both skin and gill at 60 h. In addition, ctsba was only up-regulated at 60 h (10 fold) in skin (Fig. 5A), while it was earlier induced at 24 h (6 fold) in gill (Fig. 5B). For successful infection of host, pathogens need to penetrate the mucosal surfaces, and establish efficient colonization and replication. At the same time, the mucosal immune system needs to recognize the pathogen, prevent the pathogen invading and clean up the attached pathogen. In addition to serve as the physical barriers, the mucosal surfaces are also considered as a reservoir for many immune factors with antimicrobial functions. According to early studies, the innate immune factors including immunoglobulins, lysozymes, proteases, lectins, complement factors and antimicrobial peptides which involved in inhibition of pathogen trapping and sloughing have been widely identified in fish mucosal tissues [42e44]. Therefore, understanding of the immune activities of immune actors in fish mucosal tissues following challenge can expand our knowledge of teleost mucosal immunity and improve the development of effective strategies for disease control. In catfish, F. columnare is believed to gain entry through skin and gill [45], while the intestine was considered as the primary route for E. ictaluri infection, but its ability of entry through gill and skin was also observed [22,46,47]. Whereas several innate immune actors have been characterized in catfish, nothing is known about the breadth and function of ctsb in catfish [48e50]. Therefore, here we characterized the ctsb of channel catfish and determined its expression patterns following E. ictaluri and F. columnare infection. Following challenge with two different Gram-negative pathogens, the substantial similarity of the ctsba expression profiles was detected. ctsba was significantly up-regulated in both challenges, but showed a more acute response post E. ictaluri infection. Additionally, ctsba was more dramatically induced (more than 10 fold) in F. columnare challenge. This distinct difference consistent with the difference of our experimental challenge, while the mortality of F. columnare occurred as early as 48 h, the mortality of E. ictaluri has not been observed in the first week of challenge. Previously, cathepsins were revealed to regulate the inflammatory responses
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.10.028. References
Fig. 5. Real time qRT-PCR analysis for cathepsin B (ctsba) expression following Flavobacterium columnare infection. The ctsba expression was measured in the mucosal tissues including (A) Skin and (B) Gill, at the timepoints of 4 h, 24 h and 60 h postinfection. Fold change was calculated by the change in expression at a given timepoint relative to the untreated control and normalized by changes in the 18S housekeeping gene. The results are presented as mean ± SE of fold changes and the * indicates statistical significance at P < 0.05.
following infection, and can regulate the production of inflammatory actors in the sites of infection [51,52]. In mice, the increased activity of Ctsb along with the increased inflammation responses was observed following challenge [53]. On the other hand, inhibition of Ctsb significantly decreased the expression of inflammatory cytokines together with the inflammasome components [53]. Here, the increased expression of ctsba in mucosal surfaces should be resulted from the inflammatory response against invading pathogen. Similar in flounder, ctsb showed the strongest up-regulation in gill following viral challenge, along with the dramatic increased expression in intestine [20]. Not only in mucosal tissues, ctsb was also induced in spleen post challenge in grouper [19]. Collectively, the wide distribution and regulation across catfish tissues suggested catfish ctsba is not only involved in the acute inflammatory responses, but also played key roles in the pathogen triggered immune response after pathogen entry and recognition. In present study, the ctsba gene was first identified in catfish as well as its expression pattern in mucosal tissues following different bacteria challenge. Our results indicate the protective function of ctsba against infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may also play additional physiological and biological roles in catfish mucosal tissues.
Acknowledgments This study is supported by Advanced Talents Foundation of QAU, Grant Number 1114337.
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