C-type Lectin Gene Research Articles

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Comparative transcriptome analysis reveals the different responding mechanisms of ovary and hepatopancreas following polyI:C challenge in Macrobrachium nipponense

The ovary in mammals has developed specialized mechanisms for protection against pathogen infections; however, the understanding of the innate immune system in the ovary of crustaceans is still limited. To elucidate the ovary's defense mechanisms in response to viral challenges, we subjected oriental river prawns (Macrobrachium nipponense) to poly I:C, a double-stranded RNA analog that emulates viral dsRNA, and analyzed the ovary's transcriptome profiles. Concurrently, RNA-seq analysis was performed on the hepatopancreas, a well-recognized immune-related tissue, following poly I:C challenge to investigate the distinct response mechanisms of the ovary and hepatopancreas and to gain a comprehensive understanding of the immune responses in both tissues. The results indicate that 1368 genes are differentially expressed in the ovary, with 903 genes upregulated and 465 genes downregulated. Subsequent analysis reveals that these differentially expressed genes (DEGs) include numerous genes associated with innate immunity, such as members of the C-type lectin, fibrinogen-related protein (Frep), Toll-like receptor, and NOD-like receptor (NLR) gene families, as well as acid phosphatase, scavenger receptor, crustin, Down syndrome cell adhesion molecule (Dscam), hemocyanin, and lipopolysaccharide and beta-1,3-glucan binding protein (LGBP). Furthermore, the DEGs include several genes related to ovary development, such as sox8, vitellogenin, progranulin, cyclin-dependent kinase, ecdysone receptor, frizzled, and members of the Fox gene family. In the hepatopancreas, a total of 729 DEGs were identified. Comparison of the DEGs in both tissues indicates that only 91 genes are common to both groups, highlighting significant tissue-specific responses to poly I:C stimulation. This study aims to enhance our understanding of the immune protective mechanisms employed by the ovary in response to pathogen exposure and establishes a foundation for investigating ovarian reproductive immunity in crustaceans.

A novel C-type lectin protein (BjCTL5) interacts with apoptosis stimulating proteins of p53 (ASPP) to activate NF-κB signaling pathway in primitive chordate

C-type lectin proteins (CTLs), a group of pattern recognition receptors (PRRs), play pivotal roles in immune responses. However, the signal transduction and regulation of CTLs in cephalochordates have yet to be explored. In this study, we examined the composition of CTLs in Branchiostoma japonicum, identifying a total of 272 CTLs. These CTLs underwent further analysis concerning domain arrangement, tandem and segmental duplication events. A multidomain C-type lectin gene, designated as BjCTL5, encompassing CLECT, KR, CUB, MAM, and SR domains, was the focal point of our investigation. BjCTL5 exhibits ubiquitous expression across all detected tissues and is responsive to stimulation by LPS, mannose, and poly (I:C). The recombinant protein of BjCTL5 can bind to Escherichia coli and Staphylococcus aureus, inducing their agglutination and inhibiting the proliferation of S. aureus. Yeast two-hybrid, CoIP, and confocal immunofluorescence experiments revealed the interaction between BjCTL5 and apoptosis-stimulating proteins of p53, BjASPP. Intriguingly, BjCTL5 was observed to induce the luciferase activity of the NF-κB promoter in HEK293T cells. These results suggested a potential interaction between BjCTL5 and BjASPP, implicating that they involve in the activation of the NF-κB signaling pathway, which provides an evolutionary viewpoint on NF-κB signaling pathway in primitive chordate.

A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.

A CTL − Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

BackgroundGut bacteria are beneficial to the host, many of which must be passed on to host offspring. During metamorphosis, the midgut of holometabolous insects undergoes histolysis and remodeling, and thus risks losing gut bacteria. Strategies employed by holometabolous insects to minimize this risk are obscure. How gut bacteria affect host insects after entering the hemocoel and causing opportunistic infections remains largely elusive.ResultsWe used holometabolous Helicoverpa armigera as a model and found low Lactobacillus load, high level of a C-type lectin (CTL) gene CD209 antigen-like protein 2 (CD209) and its downstream lysozyme 1 (Lys1) in the midgut of the wandering stage. CD209 or Lys1 depletion increased the load of midgut Lactobacillus, which further translocate to the hemocoel. In particular, CD209 or Lys1 depletion, injection of Lactobacillus plantarum, or translocation of midgut L. plantarum into the hemocoel suppressed 20-hydroxyecdysone (20E) signaling and delayed pupariation. Injection of L. plantarum decreased triacylglycerol and cholesterol storage, which may result in insufficient energy and 20E available for pupariation. Further, Lysine-type peptidoglycan, the major component of gram-positive bacterial cell wall, contributed to delayed pupariation and decreased levels of triacylglycerols, cholesterols, and 20E, in both H. armigera and Drosophila melanogaster.ConclusionsA mechanism by which (Lactobacillus-induced) opportunistic infections delay insect metamorphosis was found, namely by disturbing the homeostasis of lipid metabolism and reducing 20E production. Moreover, the immune function of CTL − Lys was characterized for insect metamorphosis by maintaining gut homeostasis and limiting the opportunistic infections.

Differing Expression and Potential Immunological Role of C-Type Lectin Receptors of Two Different Chicken Breeds against Low Pathogenic H9N2 Avian Influenza Virus.

Diverse immune responses in different chicken lines can result in varying clinical consequences following avian influenza virus (AIV) infection. We compared two widely used layer breeds, Lohmann Brown (LB) and Lohmann White (LW), to examine virus replication and immune responses against H9N2 AIV infection. The transcription profile in the spleen of H9N2-infected chickens was compared using a microarray. Confirmatory real-time RT-PCR was used to measure the expression of C-type lectin, OASL, and MX1 genes. Additionally, to investigate the role of chicken lectin receptors in vitro, two C-type lectin receptors (CLRs) were expressed in DF-1 cells, and the early growth of the H9N2 virus was evaluated. The LB chickens shed a lower amount of virus from the cloaca compared with the LW chickens. Different expression levels of C-type lectin-like genes were observed in the transcription profile, with no significant differences in OASL or MX gene expression. Real-time RT-PCR indicated a sharp decrease in C-type lectin levels in the spleen of H9N2-infected LW chickens. In vitro studies demonstrated that cells overexpressing CLR exhibited lower virus replication, while silencing of homeostatic CLR had no effect on AIV replication. This study demonstrated distinct immune responses to H9N2 avian influenza in LB and LW chickens, particularly with differences in C-type lectin expression, potentially leading to lower virus shedding in LB chickens.

Efficacy of White Spot Syndrome Virus Protein VP28-Expressing Chlorella vulgaris as an Oral Vaccine for Shrimp.

The white spot syndrome virus (WSSV) is the causative agent of white spot disease, which kills shrimp within a few days of infection. Although WSSV has a mortality rate of almost 100% and poses a serious threat to the shrimp farming industry, strategies for its prevention and treatment are extremely limited. In this study, we examined the efficacy of VP28, a recombinant WSSV protein expressed in Chlorella vulgaris (C. vulgaris), as an oral shrimp vaccine. When compared with the control group, in which WSSV had a cumulative mortality of 100%, shrimp treated with 5% VP28-expressing C. vulgaris in their feed only had a 20% cumulative mortality rate 12 days after the WSSV challenge. When compared with the nonvaccinated group, the transcription of anti-lipopolysaccharide factor, C-type lectin, and prophenoloxidase genes, which are involved in shrimp defense against WSSV infection, was upregulated 29.6 fold, 15.4 fold, and 11.5 fold, respectively. These findings highlight C. vulgaris as a potential host for industrial shrimp vaccine production.

A Genomic and Transcriptomic Analysis of the C-Type Lectin Gene Family Reveals Highly Expanded and Diversified Repertoires in Bivalves

C-type lectins belong to a widely conserved family of lectins characterized in Metazoa. They show important functional diversity and immune implications, mainly as pathogen recognition receptors. In this work, C-type lectin-like proteins (CTLs) of a set of metazoan species were analyzed, revealing an important expansion in bivalve mollusks, which contrasted with the reduced repertoires of other mollusks, such as cephalopods. Orthology relationships demonstrated that these expanded repertoires consisted of CTL subfamilies conserved within Mollusca or Bivalvia and of lineage-specific subfamilies with orthology only between closely related species. Transcriptomic analyses revealed the importance of the bivalve subfamilies in mucosal immunity, as they were mainly expressed in the digestive gland and gills and modulated with specific stimuli. CTL domain-containing proteins that had additional domains (CTLDcps) were also studied, revealing interesting gene families with different conservation degrees of the CTL domain across orthologs from different taxa. Unique bivalve CTLDcps with specific domain architectures were revealed, corresponding to uncharacterized bivalve proteins with putative immune function according to their transcriptomic modulation, which could constitute interesting targets for functional characterization.

Molecular characterization and functional analysis of interferon regulatory factor-4 in the Red Claw Crayfish (Cherax quadricarinatus)

Vibrio parahaemolyticus (V. parahaemolyticus) is a primary pathogen in crustaceans and causes acute hepatopancreatic necrosis disease. Interferon regulatory factor-4 (IRF4) belongs to the regulatory factor family and plays a key role in the innate immunity of crustaceans. To better understand the IRF4 regulatory mechanism and its immunomodulatory role in response to challenge with Vibrio in crustaceans, the cDNA of Cherax quadricarinatus (C. quadricarinatus) IRF4 (CqIRF4) was isolated and the immune effect of this protein in the C. quadricarinatus response to V. parahaemolyticus was studied. The length of CqIRF4 was 2354 bp, with a 1251 bp open reading frame encoding 417 amino acids. After infection with V. parahaemolyticus, the expression levels of CqIRF4 in the hepatopancreas and hemolymph of C. quadricarinatus were significantly upregulated (P < 0.05), while no significant fluctuations were detected in muscle or gill tissues. Additionally, histopathological damage was detected in the hepatopancreas, such as loss of the stellate structure, significant vacuolization of cells, intercellular hemorrhaging, hypertrophy, and necrosis in some tissues. After injecting recombinant CqIRF4 protein in vivo, colonization of the hepatopancreas by V. parahaemolyticus decreased dramatically. The transcription levels of the interleukin-like genes IL-4, -6, -23, -12, and -15, as well as the lysozyme, AlP, ALF, SOD1, laccase, and C-type lectin genes, were significantly upregulated (P < 0.05). This study shows that the CqIRF4 as an immunomodulatory factor, was highly activated by a Vibrio challenge, and inhibited V. parahaemolyticus colonization in the hepatopancreas of C. quadricarinatus by regulating the corresponding immune genes.

In the battle of the disease: a transcriptomic analysis of European foulbrood-diseased larvae of the Western honey bee (Apis mellifera)

BackgroundEuropean foulbrood is a significant bacterial brood disease of Apis sp. and can cause severe and devastating damages in beekeeping operations. Nevertheless, the epidemiology of its causative agent Melissococcus plutonius has been begun to uncover but the underlying mechanisms of infection and cause of disease still is not well understood. Here, we sought to provide insight into the infection mechanism of EFB employing RNAseq in in vitro reared Apis mellifera larvae of two developmental stages to trace transcriptional changes in the course of the disease, including Paenibacillus alvei secondary infected individuals.ResultsIn consideration of the progressing development of the larva, we show that infected individuals incur a shift in metabolic and structural protein-encoding genes, which are involved in metabolism of crucial compounds including all branches of macronutrient metabolism, transport protein genes and most strikingly chitin and cuticle associated genes. These changes underpin the frequently observed developmental retardation in EFB disease. Further, sets of expressed genes markedly differ in different stages of infection with almost no overlap. In an earlier stage of infection, a group of regulators of the melanization response cascade and complement component-like genes, predominantly C-type lectin genes, are up-regulated while a differential expression of immune effector genes is completely missing. In contrast, late-stage infected larvae up-regulated the expression of antimicrobial peptides, lysozymes and prominent bacteria-binding haemocyte receptor genes compared to controls. While we clearly show a significant effect of infection on expressed genes, these changes may partly result from a shift in expression timing due to developmental alterations of infection. A secondary infection with P. alvei elicits a specific response with most of the M. plutonius associated differential immune effector gene expression missing and several immune pathway genes even down-regulated.ConclusionWe conclude that with progressing infection diseased individuals undergo a systemic response with a change of metabolism and their activated immune defence repertoire. Moreover, larvae are capable of adjusting their response to a secondary invasion in late stage infections.

Bombyx mori C-Type Lectin (BmIML-2) Inhibits the Proliferation of B. mori Nucleopolyhedrovirus (BmNPV) through Involvement in Apoptosis.

C-type lectins (CTLs) are widely distributed in mammals, insects, and plants, which act as pattern recognition receptors (PRRs) to recognize pathogens and initiate immune responses. In this study, we identified a C-type lectin gene called BmIML-2 from the silkworm Bombyx mori. Its open reading frame (ORF) encodes 314 amino acids, which contain dual tandem C-type lectin-like domain (CTLD). BmIML-2 is highly expressed in the fat body and is significantly induced at 24 h after BmNPV infection. Moreover, overexpression of BmIML-2 dramatically inhibited the proliferation of BmNPV, and knockdown assay via siRNA further validated the inhibition of BmIML-2 on viral proliferation. In addition, transcript level detection of apoptosis-related genes and observation of apoptosis bodies implied that overexpression of BmIML-2 promoted BmNPV-induced apoptosis. Immunofluorescence analysis indicated that BmIML-2 distributed throughout the cytoplasm and was slightly concentrated in the cell membrane. Taken together, our results suggest that BmIML-2 could inhibit in the proliferation of BmNPV by facilitating cell apoptosis.

The Dual Functions of a Bracovirus C-Type Lectin in Caterpillar Immune Response Manipulation.

Parasitoids are widespread in natural ecosystems and normally equipped with diverse viral factors to defeat host immune responses. On the other hand, parasitoids can enhance the antibacterial abilities and improve the hypoimmunity traits of parasitized hosts that may encounter pathogenic infections. These adaptive strategies guarantee the survival of parasitoid offspring, yet their underlying mechanisms are poorly understood. Here, we focused on Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella, and found that C. vestalis parasitization decreases the number of host hemocytes, leading to disruption of the encapsulation reaction. We further found that one bracovirus C-type lectin gene, CvBV_28-1, is highly expressed in the hemocytes of parasitized hosts and participates in suppressing the proliferation rate of host hemocytes, which in turn reduces their population and represses the process of encapsulation. Moreover, CvBV_28-1 presents a classical bacterial clearance ability via the agglutination response in a Ca2+-dependent manner in response to gram-positive bacteria. Our study provides insights into the innovative strategy of a parasitoid-derived viral gene that has dual functions to manipulate host immunity for a successful parasitism.

Parallel Evolution of C-Type Lectin Domain Gene Family Sizes in Insect-Vectored Nematodes.

The dispersal stage of pathogens is crucial for the successful spread and infection of their hosts. Some plant-parasitic nematodes (PPNs) have evolved specialized dispersal stages to reach healthy hosts by being carried out by insect vectors. Because gene gain and loss is a major factor contributing to the evolution of novel characteristics, it is essential to clarify the gene family characteristics among nematodes with different dispersal modes to disentangle the evolution of insect-mediated dispersal. Here, the size of the C-type lectin (CTL) family genes of insect-vectored nematodes was found to be drastically reduced compared with those of self-dispersing nematodes, whereas the diversity of their functional domains was significantly higher. The gene family sizes of vector-dispersed nematodes were only a twentieth of the size of that of a self-dispersing (i.e., without a biotic vector) nematode model Caenorhabditis elegans, and these genes were inactive during the dispersal stage. Phylogenetic analysis showed that some CTL genes of vector-borne PPNs shared higher homology to the animal parasitic nematodes compared with other PPNs. Moreover, homology modeling predicted that the CTLs of insect-vectored nematodes bear remarkable structural similarity to the lectin genes of their vector's immune system. Because CTL genes are important sugar-binding proteins for the innate immune response of C. elegans, the loss of some CTL genes of vector-transmitted PPNs might be responsible for their parallel adaptations to a mutualistic relationship with their vector. These results expand our understanding of the evolutionary benefits of vector-mediated transmission for the nematode and vector-nematode co-evolution.

Immunotoxicity pathway and mechanism of benzo[a]pyrene on hemocytes of Chlamys farreri in vitro

Benzo[a]pyrene (B[a]P), a typical PAHs widely existing in the marine environment, has been extensively studied for its immunotoxicity due to its persistence and high toxicity. Nevertheless, the immunotoxicity mechanism remain incompletely understood. In this study, isolated hemocytes of Chlamys farreri were exposed at three concentrations of B[a]P (5, 10 and 15 μg/mL), and the effects of B[a]P on detoxification metabolism, signal transduction, humoral immune factors, exocytosis and phagocytosis relevant proteins and immune function at 0, 6, 12, 24 h were studied. Results illustrated the AhR, ARNT and CYP1A1 were significantly induced by B[a]P at 12 h. Additionally, the content of B[a]P metabolite BPDE increased in a dose-dependent manner with pollutants. Under B[a]P stimulation, the expressions of PTK (Src, Fyn) and PLC-Ca2+-PKC pathway gene increased significantly, while the transcription level of AC-cAMP-PKA pathway gene decreased remarkably. Additionally, the expressions of nuclear transcription factors (CREB, NF-κB), complement system genes and C-type lectin genes up-regulated obviously. The gene expressions of phagocytosis and exocytosis related proteins were also notably affected. 5 μg/mL B[a]P could promote phagocytosis in a transitory time, but with the increase of exposure time and concentration of B[a]P, the phagocytosis, antibacterial and bacteriolytic activities gradually decreased. These results indicated that similar to vertebrates, BPDE, the metabolite of B[a]P, mediated downstream signal transduction via PTK in bivalves. The declined of the immune defense ability of hemocytes might be closely related to the inhibition of AC-cAMP-PKA pathway and the imbalance of intracellular Ca2+ pathway. In addition, the results manifested that complement and lectin systems play a significant role in regulating immune response. In this study, the direct relationship between detoxification metabolism and immune signal transduction in bivalves under B[a]P stress was demonstrated for the first time, which provided important information for the potential molecular mechanism of B[a]P-induced immune system disorder in bivalves.

Genome-Wide Analysis of Gene Families of Pattern Recognition Receptors in Fig Wasps (Hymenoptera, Chalcidoidea).

Pattern recognition receptors (PRRs) play important roles in detecting pathogens and initiating the innate immune response. Different evolutionary histories of pollinators and non-pollinators may result in different immune recognition systems. A previous study had reported that there were significant differences in peptidoglycan recognition proteins (PGRPs) between pollinators and non-pollinators in gene number and lineage of specific genes. In this study, based on the genomic data of 12 fig wasp species, with seven pollinators and five non-pollinators, we investigated the evolution patterns of PRRs, such as Gram-negative bacteria-binding proteins (GNBPs), C-type lectins (CTLs), scavenger receptors class B (SCRBs), fibrinogen-related proteins (FREPs), galectins, and thioester-containing proteins (TEPs). Our results showed that pollinators had no GNBP, but non-pollinators all had two gene members, which were clustered into two different clades in the phylogenetic tree, with each clade having specific domain and motif characteristics. The analysis of CTL and SCRB gene families also showed that there were lineage-specific genes and specific expansion in non-pollinators. Our results showed that there were significant differences in immune recognition between pollinators and non-pollinators, and we concluded that they had undergone flexible adaptive evolution in different environments. Our study can provide more molecular evidence for future functional studies on the immune system of fig wasps.

Molecular characterization and expression analysis of a novel C-type lectin (CTL) gene in yellow catfish Pelteobagrus fulvidraco

C-type lectin (CTL) proteins contain special carbohydrate-recognition domains (CRDs) and play vital roles in activating the innate immune system. In this study, a CTL gene from freshwater yellow catfish (Pelteobagrus fulvidraco) was identified and named as PfCTL. Analysis revealed that the complete PfCTL cDNA was 718 bp in size, including a 42-bp 5′-untranslated region (UTR), 61-bp 3′-UTR, and 615-bp open reading frame (ORF) encoding a 204 amino acid polypeptide, which contained a signal peptide and CRD. The PfCTL protein contained two casein kinase II phosphorylation sites, four protein kinase C phosphorylation sites, and two N-myristoylation sites. The deduced PfCTL protein sequence has 37–83 % in comparison to CTL proteins from other fish. Further phylogenetic analysis revealed that PfCTL was closely related to the CTL of Ictalurus punctatus. Based on quantitative real-time polymerase chain reaction, the PfCTL gene was expressed in all tested tissues at different levels, with highest distribution in the spleen, followed by the head kidney, and lowest distribution in blood. After challenge with lipopolysaccharide and polyriboinosinic polyribocytidylic acid, the expression of PfCTL was up-regulated in the liver, spleen, head kidney, and blood at different time points. These results imply that this CTL gene may be an important pathogen pattern recognition molecule in the innate immune system of yellow catfish under pathogen stimulation.

A Pattern Recognition Receptor C-type Lectin-S6 (CTL-S6) is Involved in the Immune Response in the Silkworm (Lepidoptera: Bombycidae).

Insect innate immunity is initiated by the special recognition and binding of the foreign pathogens, which is accomplished by the pattern recognition receptors (PRRs). As an important type of PRRs, C-type lectins (CTLs) play various roles in insect innate immunity, including pathogen recognition, stimulation of prophenoloxidase, regulation of cellular immunity and so on. In this study, we have cloned the full-length cDNA of a CTL gene named CTL-S6 from the silkworm, Bombyx mori. The open reading frame (ORF) of B. mori CTL-S6 encodes 378 amino acids, which contain a secretion signal peptide. The mRNA of CTL-S6 exhibited the highest transcriptional level in the midgut. Its transcriptional level increased dramatically in fat body and hemocytes upon Escherichia coli or Micrococcus luteus challenge. Purified recombinant CTL-S6 could bind to bacterial cell wall components, including peptidoglycan (PGN, from Bacillus subtilis) and lipopolysaccharide (LPS, from E. coli 0111:B4), and recombinant CTL-S6 was involved in the encapsulation and melanization of hemocytes. Furthermore, the addition of recombinant CTL-S6 to the hemolymph of silkworm resulted in a significant increase in phenoloxidase activity. Overall, our results indicated that B. mori CTL-S6 may serve as a PRR for the recognition of foreign pathogens, prophenoloxidase pathway stimulation and involvement in the innate immunity.

Immune activation by a multigene family of lectins with variable tandem repeats in oriental river prawn (Macrobrachium nipponense).

Genomic regions with repeated sequences are unstable and prone to rapid DNA diversification. However, the role of tandem repeats within the coding region is not fully characterized. Here, we have identified a new hypervariable C-type lectin gene family with different numbers of tandem repeats (Rlecs; R means repeat) in oriental river prawn (Macrobrachium nipponense). Two types of repeat units (33 or 30 bp) are identified in the second exon, and the number of repeat units vary from 1 to 9. Rlecs can be classified into 15 types through phylogenetic analysis. The amino acid sequences in the same type of Rlec are highly conservative outside the repeat regions. The main differences among the Rlec types are evident in exon 5. A variable number of tandem repeats in Rlecs may be produced by slip mispairing during gene replication. Alternative splicing contributes to the multiplicity of forms in this lectin gene family, and different types of Rlecs vary in terms of tissue distribution, expression quantity and response to bacterial challenge. These variations suggest that Rlecs have functional diversity. The results of experiments on sugar binding, microbial inhibition and clearance, regulation of antimicrobial peptide gene expression and prophenoloxidase activation indicate that the function of Rlecs with the motif of YRSKDD in innate immunity is enhanced when the number of tandem repeats increases. Our results suggest that Rlecs undergo gene expansion through gene duplication and alternative splicing, which ultimately leads to functional diversity.

Caenorhabditis elegans mounts a p38 MAPK pathway-mediated defence to Cutibacterium acnes infection.

Cutibacterium acnes is capable of inducing inflammation in acne and can lead to a chronic prostatic infection. The diverse pathogenicity among different strains of C. acnes has been presented, but simple appropriate animal models for the evaluation of this bacterium are lacking. In this study, the nematode Caenorhabditis elegans was used as an invertebrate infection model. We revealed that C. acnes type strain ATCC 6919 caused lethal infections to C. elegans in solid and liquid culture media (p < .0001). Compared with the strain ATCC 6919, the antibiotic-resistant strain HM-513 was more virulent, resulting in reduced survival (p < .0001). Four different C. acnes strains killed worms with a p value of less than .0001 when provided to C. elegans at 4.8 × 108 CFU/ml. The infection model was also employed to explore host defence responses. An increase in numerous immune effectors in response to C. acnes was detected. We focused on nine C-type lectins, including: clec-13, clec-17, clec-47, clec-52, clec-60, clec-61, clec-70, clec-71 and clec-227. The induced expression of these C-type lectin genes was down-regulated in mutant worms deficient in the p38 mitogen-activated protein kinase (MAPK) pathway. Meanwhile, PMK-1 (MAPK) was phosphorylated and activated at the onset of C. acnes infection. By monitoring the survival of mutant worms, we found that PMK-1, SEK-1 (MAPKK) and TIR-1 (MAPKKK) were critical in responding to C. acnes infection. C. elegans pmk-1 and tir-1 mutants exhibited higher mortality to C. acnes infection (p < .0001). In conclusion, C. elegans serves as a simple and valuable model to study C. acnes virulence and facilitates improvements in understanding of host innate immune responses.

The first identification of a C-type lectin gene (CqCTL) in Cherax quadricarinatus: sequence features and expression profiles

As pattern recognition receptors (PRRs), C-type lectins (CTLs) have important roles in the recognition and clearance of pathogens by the innate immune system. In the present study, the first Cherax quadricarinatus CTL gene (designated CqCTL) was cloned and characterized. The complete cDNA sequence of CqCTL contained an open reading frame (ORF) of 543 bp, which encoded a protein of 180 amino acids. A carbohydrate recognition domain (CRD) containing four conserved cysteines (Cys48, Cys59, Cys76, Cys177) and the EPD (Glu80-Pro81-Asn82) and QPD (Gln146-Pro147-Asn148) motifs were identified in the deduced amino acid sequence of CqCTL. The deduced tertiary structure of CqCTL revealed two α helices，five β sheets and two disulfide bonds. CqCTL exhibited high similarity with previously identified CTLs from other species. The mRNA transcripts of CqCTL were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. These results provide useful information on the potential role of CqCTL in the innate immune system of C. quadricarinatus, and lay the foundation for further studies on the CTLs of crustacean.