Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella, Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species.
The innate immune system provides the primary vertebrate defence system against pathogen invasion, but it is energetically costly and can have immune pathological effects. A previous study in sticklebacks found that intermediate major histocompatibility complex (MHC) diversity correlated with a lower leukocyte coping capacity (LCC), compared to individuals with fewer, or many, MHC alleles. The organization of the MHC genes in mammals, however, differs to the highly duplicated MHC genes in sticklebacks by having far fewer loci. Using European badgers (Meles meles), we therefore investigated whether innate immune activity, estimated functionally as the ability of an individual's leukocytes to produce a respiratory burst, was influenced by MHC diversity. We also investigated whether LCC was influenced by factors such as age-class, sex, body condition, season, year, neutrophil and lymphocyte counts, and intensity of infection with five different pathogens. We found that LCC was not associated with specific MHC haplotypes, MHC alleles, or MHC diversity, indicating that the innate immune system did not compensate for the adaptive immune system even when there were susceptible MHC alleles/haplotypes, or when the MHC diversity was low. We also identified a seasonal and annual variation of LCC. This temporal variation of innate immunity was potentially due to physiological trade-offs or temporal variation in pathogen infections. The innate immunity, estimated as LCC, does not compensate for MHC diversity suggests that the immune system may function differently between vertebrates with different MHC organizations, with implications for the evolution of immune systems in different taxa.
Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.
Despite the importance of innate immunity in invertebrates, the diversity and function of innate immune cells in invertebrates are largely unknown. Using single-cell RNA-seq, we identified prohemocytes, monocytic hemocytes, and granulocytes as the three major cell-types in the white shrimp hemolymph. Our results identified a novel macrophage-like subset called monocytic hemocytes 2 (MH2) defined by the expression of certain marker genes, including Nlrp3 and Casp1. This subtype of shrimp hemocytes is phagocytic and expresses markers that indicate some conservation with mammalian macrophages. Combined, our work resolves the heterogenicity of hemocytes in a very economically important aquatic species and identifies a novel innate immune cell subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.
A total of 78 alleles and 29 loci were detected from nine microsatellite and three minisatellite markers, respectively across 26 blast and ufra disease resistant genotypes. For blast resistant genotypes, the Polymorphic Information Content (PIC) values ranged from 0.280 to 0.726 and RM21 was considered as the best marker. PIC values ranged from 0.5953 to 0.8296 for ufra resistant genotypes and RM23 was the best marker for characterization of ufra resistant genotypes. The genetic similarity analysis using UPGMA clustering generated nine clusters with coefficient of 0.66 for blast resistant genotypes while five genetic clusters with similarity coefficient of 0.42 for ufra resistant genotypes. In order to develop resistant varieties of two major diseases of rice, hybridisation should be made using the parents, BR29 and NJ70507, BR36 and NJ70507 for blast, while BR11 and Aokazi, BR3 and Aokazi, Rayda and BR3 and Rayda and BR11 for ufra.
The occurrences of multiple drug-resistant strains have been relentlessly increasing in recent years. The aquaculture industry has encountered major disease outbreaks and crucially affected by this situation. The usage of non-specific chemicals and antibiotics expedites the stimulation of resistant strains. Triggering the natural defense mechanism would provide an effective and safest way of protecting the host system. Hence, we have investigated the innate immune function of serine/threonine-protein kinase (STPK) in Macrobrachium rosenbergii (Mr). The in-silico protein analysis resulted in the identification of cationic antimicrobial peptide, MrSL-19, with interesting properties from STPK of M. rosenbergii. Antimicrobial assay, FACS and SEM analysis demonstrated that the peptide potentially inhibits Staphylococcus aureus by interacting with its membrane. The toxic study on MrSL-19 demonstrated that the peptide is not toxic against HEK293 cells as well as human erythrocytes. This investigation showed the significant innate immune property of an efficient cationic antimicrobial peptide, MrSL-19 of STPK from M. rosenbergii.
Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.
Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.
Dengue virus (DENV) is the etiological agent of dengue fever. Severe dengue could be fatal and there is currently no effective antiviral agent or vaccine. The only licensed vaccine, Dengvaxia, has low efficacy against serotypes 1 and 2. Cellular miRNAs are post-transcriptional regulators that could play a role in direct regulation of viral genes. Host miRNA expressions could either promote or repress viral replications. Induction of some cellular miRNAs could help the virus to evade the host immune response by suppressing the IFN-α/β signaling pathway while others could upregulate IFN-α/β production and inhibit the viral infection. Understanding miRNA expressions and functions during dengue infections would provide insights into the development of miRNA-based therapeutics which could be strategized to act either as miRNA antagonists or miRNA mimics. The known mechanisms of how miRNAs impact DENV replication are diverse. They could suppress DENV multiplication by directly binding to the viral genome, resulting in translational repression. Other miRNA actions include modulation of host factors. In addition, miRNAs that could modulate immunopathogenesis are discussed. Major hurdles lie in the development of chemical modifications and delivery systems for in vivo delivery. Nevertheless, advancement in miRNA formulations and delivery systems hold great promise for the therapeutic potential of miRNA-based therapy, as supported by Miravirsen for treatment of Hepatitis C infection which has successfully completed phase II clinical trial.
The prophenoloxidase activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase, is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases. In this study, a cDNA encoding a prophenoloxidase activating enzyme-III from the giant freshwater prawn Macrobrachium rosenbergii, designated as MrProAE-III, was identified and characterized. The full-length cDNA contains an open reading frame of 1110 base pair (bp) encoding a predicted protein of 370 amino acids including an 22 amino acid signal peptide. The MrProAE-III protein exhibits a characteristic sequence structure of a long serine proteases-trypsin domain and an N- and C-terminal serine proteases-trypsin family histidine active sites, respectively, which together are the characteristics of the clip-serin proteases. Sequence analysis showed that MrProAE-III exhibited the highest amino acid sequence similarity (63%) to a ProAE-III from Atlantic blue crab, Callinectes sapidus. MrProAE-III mRNA and enzyme activity of MrProAE-III were detectable in all examined tissues, including hepatopancreas, hemocytes, pleopods, walking legs, eye stalk, gill, stomach, intestine, brain and muscle with the highest level of both in hepatopancreas. This is regulated after systemic infectious hypodermal and hematopoietic necrosis virus infection supporting that it is an immune-responsive gene. These results indicate that MrProAE-III functions in the proPO system and is an important component in the prawn immune system.
Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
One of the major steps in the innate immune response of shrimp includes the activation of serine proteinases of the pro-phenoloxidase pathway by the prophenoloxidase activation enzyme (PPAF). In this study, the cDNA encoding a serine proteinase homologue (SPH) with prophenoloxidase activating activity of Penaeus monodon (PmPPAF) was cloned and characterized. PmPPAF cDNA consists of 1444 nucleotides encoding a protein with 394 amino acid residues. The estimated molecular weight of PmPPAF is 43.5 kDa with an isoelectric point of 5.19. PmPPAF consists of a signal peptide, a CLIP domain and a carboxyl-terminal trypsin-like serine protease domain. It is highly similar to the masquerade-like protein 2A (61% similarity) of the crayfish Pacifastacus leniusculus, other serine proteases (42.9-67% identity) of P. monodon, and the PPAF of the crab (61% similarity). Unlike other SPH of P. monodon, which express mainly in the hemocytes, PmPPAF transcripts were detected in the hemocytes, eyestalk, hypodermis, gill, swimming leg and brain. Similar to the crab PPAF, PmPPAF transcript level is high in shrimp at the premolt stages and PmPPAF expression is up-regulated in shrimp infected with white spot syndrome virus (WSSV). Gene silencing of PmPPAF decreased expression of a prophenoloxidase-like gene and injection of Anti-PmPPAF antibody causes a decrease in PO activity. Taken together, these results provided evidence that PmPPAF is a serine proteinase homologue, and is involved in the pro-PO activation pathway of the shrimp innate immune system.
At present, very little is known about how Burkholderia pseudomallei (B. pseudomallei) interacts with its host to elicit melioidosis symptoms. We established a murine acute-phase melioidosis model and used DNA microarray technology to investigate the global host/pathogen interaction. We compared the transcriptome of infected liver and spleen with uninfected tissues over an infection period of 42 hr to identify genes whose expression is altered in response to an acute infection.