Molecular dissection of Cystatin B and C from big-belly seahorse and Cyclooxygenase 2 from red-spotted grouper reveals their implications in host immune defense mechanisms

Abstract

Aquaculture has emerged as a vital fish production practice, overtaking the captive fisheries due to insufficient wild capture to meet the rising demand for fish over the past few decades. It contributes to a substantial impact on global food security as the cheapest source of human nutrition and providing livelihood. Nevertheless, aquaculture has been combatted due to frequent outbreaks of pathogen infections. Therefore, it is important to have a deep insight into fish immune defense mechanisms, and the overall objective of the current study is to perceive the immune defense mechanisms of teleosts and thereby foster sustainable aquaculture through disease prevention. In the present study, I have identified and characterized two Cystatin homologs from big-belly seahorse and Cyclooxygenase 2 from red-spotted grouper and elucidated their roles in the innate immune system of teleost. Big-belly seahorse (Hippocampus abdominalis) is the largest seahorse species, naturally inhabited in shallow coastal areas, such as seagrass beds and natural estuaries. They have good commercial value for the aquarium trade and Chinese traditional medicine, albeit they are highly vulnerable 0to their natural habitat loss and overexploitation due to many human interventions. Therefore, seahorse aquaculture has emerged as a good solution to overcome consumer demand while conserving the wild population. However, there are some shortcomings in seahorse aquaculture such as their high sensitivity towards the varying culture conditions and the frequent outbreak of pathogen infections. Red spotted grouper (Epinephelus akaara) is an ideal research model and a perfect candidate for extensive aquaculture owing to its high nutritional value, rapid growth, and high market demand. However, the frequent outbreak of viral diseases, especially, Nervous Necrosis Virus, are the major drawbacks in grouper aquaculture that has been creating a huge economic loss annually. Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin B (CSTB) is a member of the type 1 cystatin family and found to be involved in numerous pathophysiological conditions. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. In this study, the 297-bp cystatin B (HaCSTB) and 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse were cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTB is a homolog of the teleostean type 1 cystatin family. HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTB and HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in blood and ovaries respectively. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in both HaCSTB and HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 10.80-kDa of recombinant HaCSTB protein (rHaCSTB) and 14.29-kDa of recombinant HaCSTC protein (rHaCSTC) were expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by both rHaCSTB and rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, both HaCSTB and HaCSTC overexpression significantly (p < 0.05) decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, both HaCSTB and HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Cyclooxygenases (COXs) are major biosynthetic enzymes of prostaglandins, which play a key role in the generation of inflammatory response by acting on specific cellular receptors. There are two types of COX isomers, COX-1, and COX-2. COX-1 is constitutively expressed as house-keeping gene whereas COX-2 is highly inducible in response to various external stimuli. COX-2 is the most comprehensively studied mammalian dioxygenase and commonly used in anti-inflammatory drug therapies. The cDNA sequence of COX-2 with 1827 bp length, encoding 608 amino acid sequence, was cloned from Epinephelus akaara and designated as EaCOX-2. According to in silico analysis EaCOX-2 is a teleostean homolog of myeloperoxidase superfamily with prostaglandin endoperoxide synthase domain and a membrane-binding domain. The 3D structure of EaCOX-2 was a homodimer with heme binding sites at the center of the catalytic domains. The results of pairwise alignment disclosed that EaCOX-2 possessed 99% of the highest identity and 99% of similarity with Epinephelus lanceolatus. EaCOX-2 transcripts were ubiquitously expressed in all tested, red-spotted grouper tissues, with significantly (p<0.05) the highest expression in gills. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, and nervous necrosis virus caused significant (p<0.05) upregulation in EaCOX-2 transcript levels at 12 h and 24 h post-infection in gills. The overexpression of EaCOX-2 exhibited pro-inflammatory effect through reducing FHM cell viability, activation of classical M1 type macrophage polarization and increased nitric oxide (NO) production in murine macrophage cells (RAW264.7). Collectively, our findings imply the profound roles of Cystatins and Cyclooxygenase 2 against pathogen infections in teleosts by modulating fish immune responses.