Cellular and transcriptome signatures unveiled by single-cell RNA-Seq following ex vivo infection of murine splenocytes with Borrelia burgdorferi.

Introduction: Lyme disease, the most common tick-borne infectious disease in the US, is caused by a spirochetal pathogen Borrelia burgdorferi (Bb). Distinct host responses are observed in susceptible and resistant strains of inbred of mice following infection with Bb reflecting a subset of inflammatory responses observed in human Lyme disease. The advent of post-genomic methodologies and genomic data sets enables dissecting the host responses to advance therapeutic options for limiting the pathogen transmission and/or treatment of Lyme disease. Methods: In this study, we used single-cell RNA-Seq analysis in conjunction with mouse genomics exploiting GFP-expressing Bb to sort GFP+ splenocytes and GFP- bystander cells to uncover novel molecular and cellular signatures that contribute to early stages of immune responses against Bb. Results: These data decoded the heterogeneity of splenic neutrophils, macrophages, NK cells, B cells, and T cells in C3H/HeN mice in response to Bb infection. Increased mRNA abundance of apoptosis-related genes was observed in neutrophils and macrophages clustered from GFP+ splenocytes. Moreover, complement-mediated phagocytosis-related genes such as C1q and Ficolin were elevated in an inflammatory macrophage subset, suggesting upregulation of these genes during the interaction of macrophages with Bb-infected neutrophils. In addition, the role of DUSP1 in regulating the expression of Casp3 and pro-inflammatory cytokines Cxcl1, Cxcl2, Il1b, and Ccl5 in Bb-infected neutrophils were identified. Discussion: These findings serve as a growing catalog of cell phenotypes/biomarkers among murine splenocytes that can be exploited for limiting spirochetal burden to limit the transmission of the agent of Lyme disease to humans via reservoir hosts.

Coxiella burnetii Virulent Phase I and Avirulent Phase II Variants Differentially Manipulate Autophagy Pathway in Neutrophils.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever in humans. The virulent C. burnetii Nine Mile phase I (NMI) strain causes disease in animal models, while the avirulent NM phase II (NMII) strain does not. In this study, we found that NMI infection induces severe splenomegaly and bacterial burden in the spleen in BALB/c mice, while NMII infection does not. A significantly higher number of CD11b+ Ly6G+ neutrophils accumulated in the liver, lung, and spleen of NMI-infected mice than in NMII-infected mice. Thus, neutrophil accumulation correlates with NMI and NMII infection-induced inflammatory responses. In vitro studies also demonstrated that although NMII exhibited a higher infection rate than NMI in mouse bone marrow neutrophils (BMNs), NMI-infected BMNs survived longer than NMII-infected BMNs. These results suggest that the differential interactions of NMI and NMII with neutrophils may be related to their ability to cause disease in animals. To understand the molecular mechanism underlying the differential interactions of NMI and NMII with neutrophils, global transcriptomic gene expressions were compared between NMI- and NMII-infected BMNs by RNA sequencing (RNA-seq) analysis. Interestingly, several genes involved in autophagy-related pathways, particularly membrane trafficking and lipid metabolism, are upregulated in NMII-infected BMNs but downregulated in NMI-infected BMNs. Immunofluorescence and immunoblot analyses indicate that compared to NMI-infected BMNs, vacuoles in NMII-infected-BMNs exhibit increased autophagic flux along with phosphatidylserine translocation in the cell membrane. Similar to neutrophils, NMII activated LC3-mediated autophagy in human macrophages. These findings suggest that the differential manipulation of autophagy of NMI and NMII may relate to their pathogenesis.

The Feasibility of Using Coxiella burnetii Avirulent Nine Mile Phase II Viable Bacteria as a Live Attenuated Vaccine Against Q fever.

This study aimed to explore if viable C. burnetii avirulent Nine Mile phase II (NMII) can elicit protective immunity against virulent NM phase I (NMI) infection. Interestingly, mice immunized with viable NMII elicited significant protection against NMI infection at different time points post-immunization. Viable NMII induced a dose-dependent NMI-specific IgG response in mice, but all doses of NMII-immunized mice conferred a similar level of protection. Comparing different routes of immunization indicated that intranasally immunized mice showed significantly higher levels of protection than other immunization routes. The observation that viable NMII induced a similar level of long-term protection against NMI challenge as the formalin-inactivated NMI vaccine (PIV) suggests that viable NMII bacteria can induce a similar level of long-term protection against virulent NMI challenge as the PIV. Viable NMII also induced significant protection against challenge with virulent Priscilla and Scurry strains, suggesting that viable NMII can elicit broad protection. Immune sera and splenocytes from viable NMII-immunized mice are protective against NMI infection, but immune serum-receiving mice did not control NMI replication. Additionally, viable NMII conferred a comparable level of protection in wild-type, CD4+ T cell-deficient, and CD8+ T cell-deficient mice, and partial protection in B cell-deficient mice. However, NMII-immunized T cell-deficient mice were unable to prevent C. burnetii replication. Thus, both B cells and T cells are required for viable NMII-induced protective immunity but T cells may play a critical role. Collectively, this study demonstrates the feasibility of using avirulent NMII as a live attenuated vaccine against human Q fever.

Resolving the pathogenicity factors of a novel opportunistic fungus Schizophyllum commune at molecular level.

Schizophyllum commune is a well-known mushroom forming fungi which is an edible one due to its nutritive value. It exhibits a special wood degrading mechanism to grow in decay matters by releasing a series of enzymes. These enzymes might make them an opportunistic pathogen which has been reported to infect various animals and human beings too. Although these fungi were identified as human and animal pathogens, their mechanisms of pathogenesis and the key virulence factors involved in disease establishment are not known. In this study, we reported this fungal infection in freshwater fish for the first time and its morphological features. Further, we employed RNA-seq technique to identify the major virulence factors involved in the pathogenesis in fish and the network of interaction between the identified virulence factors were analysed. Also, we confirmed the virulence roles of this fungus during infection by qRT-PCR analysis. This study emphasizes the virulence nature of the common mushroom forming food fungus and the involvement of enzymes such as phosphoinositide phospholipase C, hexosaminidase and few toxins such as pesticidal and insecticidal crystal proteins which opened a new avenue in the virulence nature of edible mushrooms.

Design and characterization of a novel Arthrospira platensis glutathione oxido-reductase-derived antioxidant peptide GM15 and its potent anti-cancer activity via caspase-9 mediated apoptosis in oral cancer cells.

Glutathione oxido-reductase (GR) is a primary antioxidant enzyme of most living forms which protects the cells from oxidative metabolism by reducing glutathione (GSH) from its oxidized form (GSSG). Although the antioxidant role of the enzyme is well characterized, the specific role of conserved N' peptide sequence in antioxidant mechanism remains unclear. In this study, we have identified an RNA sequence encoding GR enzyme from spirulina, Arthrospira platensis (Ap) and the changes in its gene expression profile was analysed during H2O2 stress. Results showed that H2O2 (10 mM) stimulated the expression of ApGR throughout the timeline of study (0, 5, 10, 15 and 20 days) with highest expression at 5th day post-exposure which confirmed the antioxidant role of ApGR in spirulina during H2O2 induced oxidative stress. A dithiol containing short antioxidant peptide, 39GGTCVIRGCVPKKLM53 (GM15) from ApGR was predicted and its radicals (superoxide and hydroxyl radical) scavenging potential was confirmed by in vitro cell-free assays. GM15 (12.5 µM) reduced the intracellular generalized oxidative stress level, as measured using DCFDA assay in H2O2 exposed leucocytes without affecting any of the cellular population. Further, the biomedical application of the radical scavenging property of GM15 was validated in oral carcinoma (KB) cells where GM15 exhibited significant cytotoxicity. Also, GM15 exhibited heterogenous effects on intracellular oxidative stress level in KB cells: at lower concentration (6.25 µM), the peptide reduced oxidative stress whereas, at higher concentration (25 µM) it increased the intensity of oxidative stress. GM15 (25 µM) induced caspase-9 mediated apoptosis in KB cells along with membrane disruption and DNA degradation which are confirmed by propidium iodide (PI) internalization and comet assays, respectively. Overall, the study shows that GM15 peptide i) scavenges superoxide, hydroxyl radicals, and influences intracellular oxidative stress, and ii) has anti-cancer effect in oral cancer cells.

Gene profiling of antimicrobial peptides, complement factors and MHC molecules from the skin transcriptome of Channa striatus and its expression pattern during Aeromonas hydrophila infection.

Channa striatus is one of the economically important freshwater fish with high demand in Southeast Asia for its nutritional and medicinal values. The unique composition of skin mucus of murrel provides immunity against pathogens; however, they are susceptible to few bacterial pathogens especially Aeromonas hydrophila. Although few immune molecules such as antimicrobial peptides have already been identified from the murrel mucus, there is no report on the complete gene profile of the skin and mucosal immunity. Therefore, in this study we applied transcriptome approach to identify the mRNA sequences of various immune molecules such as antimicrobial peptides, complement factors and adaptive immune molecules from the skin tissue. Transcriptome wide search revealed unique mRNA sequences of 13 antimicrobial peptides, 11 complement components, 2 major histocompatibility complex proteins and its receptor, 6 butyrophilins, 2 leptins and its receptor. Brief bioinformatics analysis of the identified mRNA sequences and their respective putative protein sequences were performed to understand molecular information of those immune components. Further, we analysed the differential expression pattern of selected 13 mRNA sequences representing each immune group using qRT-PCR technique which highlighted the role of those genes during A. hydrophila challenge. Overall, this study revealed the complex immune response of murrel skin and the involvement of various innate and adaptive immune molecules against A. hydrophila infection.

A comparative transcriptome approach for identification of molecular changes in Aphanomyces invadans infected Channa striatus.

Snakehead murrel, Channa striatus is an economically important aquatic species in Asia and are widely cultured and captured because of its nutritious and medicinal values. Their growth is predominantly affected by epizootic ulcerative syndrome (EUS) which is primarily caused by an oomycete fungus, Aphanomyces invadans. However, the molecular mechanism of immune response in murrel against this infection is still not clear. In this study, transcriptome technique was used to understand the molecular changes involved in C. striatus during A. invadans infection. RNA from the control (CF) and infected fish (IF) groups were sequenced using Illumina Hi-seq sequencing technology. For control group, 28,952,608 clean reads were generated and de novo assembly was performed to produce 60,753 contigs. For fungus infected group, 25,470,920 clean reads were obtained and assembled to produce 58,654 contigs. Differential gene expression analysis revealed that a total of 146 genes were up-regulated and 486 genes were down regulated. Most of the differentially expressed genes were involved in innate immune mechanism such as pathogen recognition, signalling and antimicrobial mechanisms. Interestingly, few adaptive immune genes, especially immunoglobulins were also significantly up regulated during fungal infection. Also, the results were validated by qRT-PCR analysis. These results indicated the involvement of various immune genes involved in both innate and adaptive immune mechanism during fungal infection in C. striatus which provide new insights into murrel immune mechanisms against A. invadans.

Molecular insight into the metabolic activities of a protein-rich micro alga, Arthrospira platensis by de novo transcriptome analysis.

To gain genetic insights into the protein-rich microalga, the transcriptome of Arthrospira platensis was sequenced using Illumina technology and de novo assembly was carried out. A total of 6023 transcripts were present in the transcriptome among which 4616 transcripts were annotated with specific functions. Gene ontology analysis revealed that the genes are mainly involved in three major functions such as biological (16.19%), cellular (41.47%) and molecular (42.34%) processes. Pathway analysis indicated that majority of genes are involved in amino acid biosynthesis and metabolism which is depicting the protein-rich nature of spirulina. Other major pathways involved are carbohydrate metabolism, lipid metabolism, metabolism of co-factors and vitamins, antioxidant mechanism and metabolism of terpenoids and polyketides. qRT-PCR analysis was performed to confirm the potential antioxidant role of five candidate genes of spirulina in protecting the cells from oxidative stress induced by hydrogen peroxide. Moreover, these results indicated that spirulina is rich in biological resources which could be efficiently used for multiple applications such as carbon dioxide utilization, nitrogen fixation and biofuel production.

Bactericidal and fungistatic activity of peptide derived from GH18 domain of prawn chitinase 3 and its immunological functions during biological stress.

Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.

A mini review on immune role of chemokines and its receptors in snakehead murrel Channa striatus.

Chemokines are ubiquitous cytokine molecules involved in migration of cells during inflammation and normal physiological processes. Though the study on chemokines in mammalian species like humans have been extensively studied, characterization of chemokines in teleost fishes is still in the early stage. The present review provides an overview of chemokines and its receptors in a teleost fish, Channa striatus. C. striatus is an air breathing freshwater carnivore, which has enormous economic importance. This species is affected by an oomycete fungus, Aphanomyces invadans and a Gram negative bacteria Aeromonas hydrophila is known to cause secondary infection. These pathogens impose immune changes in the host organism, which in turn mounts several immune responses. Of these, the role of cytokines in the immune response is immense, due to their involvement in several activities of inflammation such as cell trafficking to the site of inflammation and antigen presentation. Given that importance, chemokines in fishes do have significant role in the immunological and other physiological functions of the organism, hence there is a need to understand the characteristics, activities and performace of these small molecules in details.

Gene expression and in silico analysis of snakehead murrel interleukin 8 and antimicrobial activity of C-terminal derived peptide WS12.

Chemokines have been known for their wide range of functions including chemoattractant property in humans and other vertebrate organisms. They act as a bridge between innate and adaptive immune system. In the present study, we have identified a CXC chemokine from the cDNA library of C. striatus; on the basis of orthology study, it was found highly identical to interleukin 8 (IL8). The bioinformatics analysis of the chemokine revealed the presence of a typical γ-core domain and a CXC motif at the N-terminal region of the molecule. Based on the amphipathic nature at the C terminal helical region of CstIL8 and their antimicrobial propensity observed during bioinformatics analysis, a short peptide namely WS12 comprising 12 amino acid residues was predicted and synthesized to determine its antimicrobial activity. The peptide WS12 was active against Bacillus cereus, a Gram positive bacterium. Scanning electron microscopy (SEM) results showed bleb-like formation on the surface of the bacteria after the treatment of WS12. Additionally, WS12 did not exhibit any cytotoxic activity against the fish leukocytes. Further, the gene expression studies also revealed that CstIL8 was expressed significantly in liver of Channa striatus (Cst) at basal level. The immune challenge studies with pathogens and immune-stimulants revealed an increase in the mRNA levels at different time points post-challenge. Hence, it is possible to conclude that WS12 was a potent antimicrobial agent and it was significantly expressed during the pathogen stress.

Application and development of a TaqMan real-time PCR for detecting infectious spleen and kidney necrosis virus in Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) is one of the major epidemiological agents that had caused great economic loss in Chinese perch (Siniperca chuatsi). In this study, a specific TaqMan real-time PCR was developed using a pair of primers and a TaqMan probe specific to the ORF007 gene of ISKNV to rapidly detect ISKNV copies in Chinese perch samples. This assay was optimized to produce linearity from 8.75 × 108 to 8.75 × 101 copies in standard curve with an efficiency of 98% and a R2 value of 0.9999. Moreover, the minimum detection limit of this assay was 10,000 times more sensitive than that of conventional PCR method. The coefficients of variation of intra- and inter-assay repeatability were less than 2.4% and 3.3%, respectively. The viral distribution in different tissues of diseased Chinese perch was evaluated by TaqMan real-time PCR method and the highest level of viral copies was detected in spleen. Among the 76 diseased Chinese perch clinical samples, 35 and 29 were positive samples based on the TaqMan real-time PCR and conventional PCR methods, respectively, indicating that the TaqMan real-time PCR was more sensitive than conventional PCR. Therefore, the TaqMan real-time PCR should be a useful tool for the early surveillance and quantitation of ISKNV.

Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan.

Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.

Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin.

Antimicrobial peptides (AMPs) are innate molecules that are found in a wide variety of species ranging from bacteria to humans. In recent years, excessive usage of antibiotics resulted in development of multi-drug resistant pathogens which made researchers to focus on AMPs as potential substitute for antibiotics. Lily type mannose-binding lectin is an extended super-family of structurally and evolutionarily related sugar binding proteins. These lectins are well-known AMPs which play important roles in fish defense mechanism. Here, we report a full-length lily type lectin-2 (LTL-2) identified from the cDNA library of striped murrel, Channa striatus (Cs). CsLTL-2 protein contained B-lectin domain along with three carbohydrate binding sites which is a prominent characteristic functional feature of LTL. The mRNA transcripts of CsLTL-2 were predominantly expressed in gills and considerably up-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila). To evaluate the antimicrobial activity of the carbohydrate binding region of CsLTL-2, the region was synthesized (QP13) and its bactericidal activity was analyzed. In addition, QP13 was labeled with fluorescein isothiocyanate (FITC) and its binding affinity with the bacterial cell membranes was analyzed. Minimum inhibitory concentration assay revealed that QP13 inhibited the growth of Escherichia coli at a concentration of 80 µM/ml. Confocal microscopic observation showed that FITC tagged QP13 specifically bound to the bacterial membrane. Fluorescence assisted cell sorter (FACS) assay showed that QP13 reduced the bacterial cell count drastically. Therefore, the mechanism of action of QP13 on E. coli cells was determined by propidium iodide internalization assay which confirmed that QP13 induced bacterial membrane disruption. Moreover, the peptide did not show any cytotoxicity towards fish peripheral blood leucocytes. Taken together, these results support the potentiality of QP13 that can be used as an antimicrobial agent against the tested pathogens.

Pellino-1 derived cationic antimicrobial prawn peptide: Bactericidal activity, toxicity and mode of action.

The antimicrobial peptides (AMPs) are multifunctional molecules which represent significant roles in the innate immune system. These molecules have been well known for decades because of their role as natural antibiotics in both invertebrates and vertebrates. The development of multiple drug resistance against conventional antibiotics brought a greater focus on AMPs in recent years. The cationic peptides, in particular, proven as host defense peptides and are considered as effectors of innate immunity. Among the various innate immune molecules, functions of pellino-1 (Peli-1) have been recently studied for its remarkable role in specific immune functions. In our study, we have identified Peli-1 from the cDNA library of freshwater prawn Macrobrachium rosenbergii (Mr) and analyzed its features using various in-silico methods. Real time PCR analysis showed an induced expression of MrPeli-1 during white spot syndrome virus (WSSV), bacteria (Vibrio harveyi) and lipopolysaccharide (LPS) from Escherichia coli challenge. Also, a cationic AMP named MrDN was derived from MrPeli-1 protein sequence and its activity was confirmed against various pathogenic bacteria. The mode of action of MrDN was determined to be its membrane permeabilization ability against Bacillus cereus ATCC 2106 as well as its DNA binding ability. Further, scanning electron microscopic (SEM) images showed the membrane disruption and leakage of cellular components of B. cereus cells induced by MrDN. The toxicity of MrDN against normal cells (HEK293 cells) was demonstrated by MTT and hemolysis assays. Overall, the results demonstrated the innate immune function of MrPeli-1 with a potential cationic AMP in prawn.

Coagulation profile, gene expression and bioinformatics characterization of coagulation factor X of striped murrel Channa striatus.

A transcriptome wide analysis of the constructed cDNA library of snakehead murrel Channa striatus revealed a full length cDNA sequence of coagulation factor X. Sequence analysis of C. striatus coagulation factor X (CsFX) showed that the cDNA contained 1232 base pairs (bp) comprising 1209 bp open reading frame (ORF). The ORF region encodes 424 amino acids with a molecular mass of 59 kDa. The polypeptide contains γ-carboxyglutamic acid (GLA) rich domain and two epidermal growth factor (EGF) like domains including EGF-CA domain and serine proteases trypsin signature profile. CsFX exhibited the maximum similarity with fish species such as Stegastes partitus (78%), Poecilia formosa (76%) and Cynoglossus semilaevis (74%). Phylogenetically, CsFX is clustered together with the fish group belonging to Actinopterygii. Secondary structure of factor X includes alpha helix 28.54%, extended strand 20.75%, beta turn 7.78% and random coil 42.92%. A predicted 3D model of CsFX revealed a short α-helix and a Ca(2+) (Gla domain) binding site in the coil. Four disulfide bridges were found in serine protease trypsin profile. Obviously, the highest gene expression (P < 0.05) was noticed in blood. Further, the changes in expression of CsFX was observed after inducing with bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) infections and other synthetic immune stimulants. Variation in blood clotting time (CT), prothrombin time (PT) and activated prothromboplastin time (APTT) was analyzed and compared between healthy and bacterial infected fishes. During infection, PT and APTT showed a declined clotting time due to the raised level of thrombocytes.

Multifunctional murrel caspase 1, 2, 3, 8 and 9: Conservation, uniqueness and their pathogen-induced expression pattern.

Caspases are evolutionarily conserved proteases which play fundamental role in apoptosis. Invasion of pathogen triggers the activation of caspases-mediated pro-inflammatory and pro-apoptotic pathways, where multifunctional caspases are involved. In striped murrel Channa striatus, epizootic ulcerative syndrome (EUS) causes endemics resulting in huge economic loss. Aphanomyces invadans, an oomycete is the primary causative agent of EUS which further induces secondary bacterial infections especially Aeromonas hydrophila. In order to get insights into the caspase gene family in C. striatus during EUS infection, we performed various physicochemical and structural analyses on the cDNA and protein sequences of five different murrel caspases namely CsCasp 1, 2, 3, 8 and 9. Sequence analysis of murrel caspase proteins showed that in spite of the conserved CASC domain, each caspase embraces some unique features which made them functionally different. Tissue distribution analysis showed that all the murrel caspases are highly expressed in one of the immune organs such as liver, kidney, spleen and blood cells. Further, to understand the role of caspase during EUS infection, modulation in expression of each caspase gene was analysed after inducing fungal and bacterial infection in C. striatus. Pathogen-induced gene expression pattern revealed an interesting fact that the expression of all the caspase genes reached a maximum level at 24 h post-infection (p.i) in case of bacteria, whereas it was 48 h in fungus. However, the initiation of elevated expression differed between each caspase based on their role such as pro-inflammatory, initiator and executioner caspase. Overall, the results suggested that the caspases in murrel are diverse in their structure and function. Here, we discuss the similarities and differences of five different murrel caspases.

A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

Fish chemokines 14, 20 and 25: A comparative statement on computational analysis and mRNA regulation upon pathogenic infection.

In this study, we reported a molecular characterization of three CC chemokines namely, CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 which are were identified from the established cDNA library of striped murrel Channa striatus. Multiple sequence alignment of all the three chemokines revealed the presence of gene specific domains and motifs including small cytokine domain, IL8 like domain, receptor binding site and glycosaminoglycan (GAG) binding sites. Three dimensional structures of the chemokines under study showed an important facet on their anti-microbial property. Tissue specific mRNA expression showed that the CsCC-Chem14 is highly expressed in spleen, CsCC-Chem20 in liver and CsCC-Chem25 in trunk kidney. On challenge C. striatus with oomycete fungus Aphanomyces invadans, both CsCC-Chem20 and CsCC-Chem25 showed significant (P < 0.05) up-regulation compared to CsCC-Chem14. The increase in the expression levels of CsCC-Chem20 and CsCC-Chem25 due to infection showed that they are antimicrobial proteins. But considering the CsCC-Chem14 expression, it is found to be a constitutive chemokine and is involved in homeostatic function in spleen of C. striatus. C. striatus challenged with bacteria Aeromonas hydrophila also exhibited different up-regulation pattern in all the three chemokines at various time points. However, extensive studies are required to determine the functional activities of CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 in vitro and in vivo to gain more knowledge at the molecular and proteomic levels.