Molecular Insights into the Immunological Responses in Teleost under Pathological Conditions via Characterization of Four Complement Genes from Rock Bream (Oplegnathus fasciatus)

Abstract

The complement system is a group of plasma proteins that reacts with one another to opsonize pathogens via antibodies and induce a series of inflammatory responses as part of the innate immune system. Among them number of complement proteins are proteases that are activated by proteolytic cleavage by themselves. Such enzymes are called zymogens and are stored inside the cells and secreted as inactive precursor enzymes. In the complement system, the precursor zymogens are widely distributed throughout the body fluids and tissues to response for the pathogenic infection. However, they are activated locally and trigger series of potent inflammatory events at the sites of infection. The complement system activates through a triggered-enzyme cascade. In such a cascade, an active complement enzyme is generated by cleavage of its zymogen precursor then cleaved its substrate, another complement zymogen, to its active enzymatic form. The activation of an initiator complement proteins at the start of the pathway is hugely amplified by each successive enzymatic reaction, resulting in the rapid generation of a disproportionately large complement response. As might be expected, there are many regulatory mechanisms to prevent uncontrolled complement activation via the function of complement anaphylatoxin inactivators and complement inhibitors. There are three distinct pathways through which complement can be activated on pathogen surfaces. These pathways depend on different molecules for their initiation, and there are three ways in which the complement system protects against infection. First, it generates large numbers of activated complement proteins that bind covalently to pathogens, opsonizing them for engulfment by phagocytes bearing receptors for complement. Second, the small fragments of some complement proteins act as chemoattractants to recruit more phagocytes to the site of complement activation, and also to activate these phagocytes. Third, the terminal complement components damage certain bacteria by creating pores in the bacterial membrane. In mammalians, complement system is well studied in relation to the activations, direct functional characteristics, activation of other signaling pathways, drawback of the needless activations etc. However, complement components have been poorly studied in teleost fish or identified arbitrarily in several fish species. Therefore, immunological study of complement components from one of the economically important teleost fish species rock bream, is a valuable investigation for molecular immunologists. Rock bream (Oplegnathus fasciatus), is demanding aquacrop in South Korea which is prone to infectious diseases. The studies on immunological perspectives of fish and genetic background are important to gaining a detailed understanding of fish immune system and may immensely help in development of the effective disease control strategies. Understanding of molecular insights into mRNA expression of vital complement components of the complement cascade system is enormously relief, to evaluate the first line of defense mechanism. Thus, scientific studies will be able to obtained clear illustration of innate immune function of complement components and will be able to develop novel strategies to prevent diseases of rock bream fish farming in South Korea. In this study, four vital complement genes including the complement component 1r (C1r), complement component 1s (C1s), complement factor D (CfD) and carboxypeptidase N1 (CPN1) have been identified and characterized at molecular, genomic and transcriptional level as an initiator, convertor and terminator complement components, respectively. This report is divided into three main chapters based on its involvement in complement system, such as initiative components of the classical pathway (C1r and C1s), convertor molecule of the alternative pathway (CfD) and anaphylatoxin inactivator of the complement cascade system (CPN1). In chapter I, rock bream C1r (OfC1r) and C1s (OfC1s) from classical pathway were characterized at molecular and genomic level while analyzing their transcriptional modulation under pathological conditions. The complete cDNA sequences of OfC1r and OfC1s were consisted of 2533 bp and 2724 bp, respectively. Their putative polypeptide sequences were 704 aa (78 kDa) and 691 aa (76 kDa), respectively. Subsequently, the identified sequences were characterized using various bioinformatics tools, while comparing the sequences with other known similitudes. Both OfC1r and OfC1s shared similar domain architecture including a signal peptide, two CUB domains, a calcium binding EGF domain, two complement control protein (CCP) domains and serine protease trypsin domain (SPr). OfC1r and OfC1s shared highest identity with Pundamilia nyererei C1r (73.4 %) and Oryzias latipes C1s (58.2 %), respectively. Phylogenetic analysis revealed that both OfC1r and OfC1s were tightly clustered with the fish clade while they were grouped into two different main clusters. According to the genomic organization, the OfC1r coding region (CDS) was distributed across thirteen exons interrupted by twelve introns whereas; OfC1s CDS was distributed within eleven exons interrupted by ten introns. The mRNA of OfC1r was significantly expressed in blood and second most in liver. Though, the mRNA of OfC1s was highly expressed in the liver and spleen, as indicated by quantitative real-time PCR (qPCR) tissue profiling. In addition, both OfC1r and OfC1s were transcriptionally responded to immune challenges, with liver expression being significantly up-regulated in the early phase of infection with live bacteria (Edwardsiella tarda and Streptococcus iniae). These data collectively suggest that the OfC1r and OfC1s may play first line defense roles as an innate immune response to protect the rock bream from bacterial infections. Chapter II enlightened the molecular characteristics and transcriptional properties of OfCfD from alternative pathway. The CfD is one of the key molecules in alternative pathway which makes C3 convertase (C3bBb). The complete cDNA sequence of OfCfD consisted of 1100 bp with 720 bp of CDS, encoding polypeptide of 239 aa (26 kDa). Characteristic serine protease trypsin superfamily domain, three active sites (31H, 78D, 183S) and three substrate binding sites (177D, 198S, 201G) were identified in mature protein of OfCfD. CfD orthologue of Oreochromis niloticus exhibited the maximum identity (73.8 %) and similarity (78.9 %) with OfCfD. According to the multiple sequence alignment, CfD homologues showed the conservation in the trypsin superfamily domain. Phylogenetic analysis of OfCfD evidenced to its higher evolutionary proximity with orthologue from olive flounder, among the other fish similitudes. The modeled tertiary structure of OfCfD resembled the characteristic features of the folding of human CfD X-ray crystal structure. The complete genomic DNA of OfCfD was 2451 bp, consists of five exons interrupted by four introns. In spatial mRNA expression profile, OfCfD transcripts were constitutively expressed in all the tissues while highest was observed in spleen and second most in liver. In challenge experiments; OfCfD transcripts were significantly up-regulated in spleen tissue post challenge with S. iniae, lipopolysaccharide (LPS), rock bream iridovirus (RBIV) and Poly: IC. Collective findings of this chapter may suggest that the OfCfD crucial in first line defense mechanism involved in alternative pathway of the rock bream upon pathogenic infections. Third chapter describes the molecular features, genomic arrangements and immune responsive transcriptional regulation of OfCPN1 from the complement cascade as an anaphylatoxin inactivator. The full-length cDNA sequence was 1591 bp which possessed the 1347 bp CDS. The predicted molecular mass of putative polypeptide of OfCPN1 (488 aa) was 51 kDa. The domain architecture evidenced a signal peptide, Zn-binding region 1 signature, Zn-binding region 2 signature, peptidase-M14 Zn carboxypeptidase site, Zn-binding site 1, Zn- binding site 2 and N-glycosylation site. According to the phylogenetic and pairwise homology sequence comparison, OfCPN1 showed highest identity to Dicentrarchus labrax. Multiple sequence analysis revealed the highly conserved Zn binding sites at the Zn binding regions among the CPN1 homologues. Genomic arrangement discovered that OfCPN1 consisted of nine exons interrupted by eight introns. The qPCR analysis confirmed that OfCPN1 gene was constitutively expressed in all examined tissues, isolated from healthy rock bream, and the highest expression was observed in liver and second most in kidney. In temporal mRNA expression of OfCPN1 from immune challenged rock bream, significant up-regulations were detected in liver tissues upon E. tarda, S. iniae, LPS, RBIV and Poly: IC challenges. Taken together, the molecular characteristics and transcriptional responses to immune stimulants suggest that OfCPN1 is involved in immune responses upon the complement system activation in of rock bream. Collectively, this study perceives molecular perspectives of four complement genes of rock bream fish Oplegnathus fasciatus using as a model organism. Moreover, this furnished a substantial contribution to the extension of existing knowledge in innate immune mechanism and it will be important to understand their role in relation to survival of fish from challenging environment with abundant pathogenic threats. Further, these findings make significant understanding into the immunological perspectives of the teleost complement system.