Pattern Recognition Proteins Research Articles

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Effects of Taiwanese indigenous cinnamon (Cinnamomum osmophloeum) leaf hot-water extract on nonspecific immune responses, resistance against Vibrio parahaemolyticus, nonviable cells, and haemocyte subpopulations in white shrimp (Penaeusvannamei)

This study investigated the effects of Cinnamomum osmophloeum leaf hot-water extract (CLWE) on nonspecific immune responses and resistance to Vibrio parahaemolyticus in white shrimp (Penaeus vannamei). Firstly, a cell viability assay demonstrated that the CLWE is safe to white shrimp heamocytes in the concentration of 0–500 mg L−1. Haemocytes incubated in vitro with 10 and 50 mg L−1 of CLWE showed significantly higher response in superoxide anion production, PO activity, and phagocytic activity.In the in vivo trials, white shrimp were fed with 0, 0.5, 1, 5, and 10 g kg−1 CLWE supplemented feeds (designated as CLWE 0, CLWE 0.5, CLWE 1, CLWE 5, and CLWE 10, respectively) over a period of 28 days. In vivo experiments demonstrated that CLWE 0.5 feeding group resulted in the highest total haemocyte count, superoxide anion production, phenoloxidase activity, and phagocytic activity. Moreover, CLWE 0.5 supplemented feed significantly upregulated the clotting system, antimicrobial peptides, pattern recognition receptors, pattern recognition proteins, and antioxidant defences in white shrimp. Furthermore, the shrimp were infected with V. parahaemolyticus injections after 14 days of feeding as challenge test. Based on the challenge test result, both CLWE 0.5 and CLWE 5 demonstrated a strong resistance to V. parahaemolyticus. These two dosages effectively reduced the number of nonviable cells and activated different haemocyte subpopulations. These findings indicated that treatment with CLWE 0.5 could promote nonspecific immune responses, immune-related gene expression, and resistance to V. parahaemolyticus in white shrimp.

Gut bacterium Burkholderia cepacia (BsNLG8) and immune gene Defensin A contribute to the resistance against Nicotine-induced stress in Nilaparvata lugens (Stål)

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Immunological lectins in shrimp Penaeus vannamei challenged with infectious myonecrosis virus (IMNV) under low-salinity conditions

Lectins are proteins capable of recognizing and binding to glycan in a specific way. In invertebrates, lectins are a crucial group of Pattern Recognition Proteins (PRRs), activating cellular and humoral responses in the innate immune system. The shrimp Penaeus vannamei is the main crustacean cultivated worldwide, however, the productivity of cultures is strongly affected by diseases, mainly viral ones, such as Infectious Myonecrosis (IMN). Thus, we investigated the participation of five lectins (LvAV, LvCTL4, LvCTL5, LvCTLU, and LvLdlrCTL) in IMNV-challenged shrimp. We verified upregulation gene profiles of lectins after IMNV-challenge, especially in hepatopancreas and gills, in addition to an increase in total hemocytes count (THC) after to 12 h post-infection (hpi). The bioinformatics characterization also revealed several sites of post-translational modification (PTM), such as phosphorylation and glycosylation, which possibly influence the action and stabilization of these lectins. We conclude that LvLdlrCTL and LvCTL5 are the lectins with greater participation in the activation of the immune system against IMNV, showing the greatest potential for PTM, higher upregulation levels, and overlapping with the THC and IMNV viral load.

Molecular characterization and functional analysis of a beta-1,3-glucan recognition protein from oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

The beta-1,3-glucan recognition protein (BGRP) is an important pattern recognition protein (PRP), which plays an important role in immune recognition and signaling pathway of insect innate immunity. Herein, a BGRP gene was obtained from the transcriptome of Grapholita molesta and its expression was verified by PCR. The full cDNA of the GmBGRP gene was 1691 bp encoding 486 amino acid residues. The calculated molecular mass of the mature protein was 54.83 kDa with an estimated pI of 6.14. The amino acid sequence of GmBGRP was highly homologous to BGRPs of other lepidopterans including Leguminivora glycinivorella BGRP-3. Expression profile of GmBGRP at different developmental stages and different tissues of 5th instar larvae showed that the expression level of this gene tends to slightly increase and then decrease at the adult stage, with the highest at the pupa stage; and mainly expressed in the epidermis, fat body and hemocytes compared with other tissues. In addition, we investigated the transcription levels of other immune-related genes, such as Serine-1, Serine-2, Serine-3, Serpin, SRCB (scavenger receptor gene), Toll, PPO (prophenoloxidase) upon GmBGRP gene silencing, indicating that GmBGRP expression is associated with immune responses of G. molesta. This was further supported by the upregulation of the mRNA level of GmBGRP following fungal infection. Taken together, these results provide experimental evidence for the role of GmBGRP gene in immune defense in G. molesta larvae.

Mechanisms and roles of the first stage of nodule formation in lepidopteran insects.

Nodule formation is a process of cellular immunity in insects and other arthropods with open circulatory systems. Based on histological observations, nodule formation occurs in 2 stages. The first stage occurs immediately after microbial inoculation and includes aggregate formation by granulocytes. The second stage occurs approximately 2-6 h later and involves the attachment of plasmatocytes to melanized aggregates produced during the first stage. The first stage response is thought to play a major role in the rapid capture of invading microorganisms. However, little is known regarding how granulocytes in the hemolymph form aggregates, or how the first stage of the immunological response protects against invading microorganisms. Since the late 1990s, our understanding of the molecules and immune pathways that contribute to nodule formation has improved. The first stage of nodule formation involves a hemocyte-induced response that is triggered by pathogen-associated molecular pattern (PAMP) recognition proteins in the hemolymph regulated by a serine proteinase cascade and cytokine (Spätzle) and Toll signaling pathways. Hemocyte agglutination proceeds through stepwise release of biogenic amine, 5-HT, and eicosanoids that act downstream of the Toll pathway. The first stage of nodule formation is closely linked to melanization and antimicrobial peptide (AMP) production, which is critical for insect humoral immunity. Nodule formation in response to artificial inoculation with millions of microorganisms has long been studied. It has recently been suggested that this system is the original natural immune system, and enables insects to respond to a single invading microorganism in the hemocoel.

Phylogenetic analyses of the proteins involved in encapsulation signaling pathways in ants

"One of the major evolutionary transitions is the shift from solitary to social lifestyle, which involved a plethora of behavioral and physiological changes in social entities. Group living has several advantages as the evolution of collective defense mechanisms. It may also affect the individual immune system either due to the efficiency of social immune defenses or because of the high transmission frequency of pathogens. Individual defense consists of the innate and acquired immune components. In insects, there are two signaling pathways (Toll and Jak/Stat) that result in the expression of specific immune genes, which, in their turn, encode peptides, proteins and activate innate immune responses like encapsulation. The main aim of our study was to verify whether transition to eusocial lifestyle is reflected in proteins involved in immune responses. We carried out phylogenetic analyses of 15 proteins involved in encapsulation signaling pathways in ants. We also included four other social insect groups, bees, sweat bees, social wasps, and termites, and three solitary insect groups, as fruit flies, braconid wasps, and megachilid bees. Ants grouped separately from other insect groups in most cases, however, there were some notable exceptions mostly in the case of pattern recognition proteins, probably correlating with differences in potential pathogens. No major differences were revealed though between solitary and social insects with respect to proteins involved in encapsulation. Keywords: eusociality, evolution, immune response, innate immunity, insects, social immunity"

Involvement of an Enhanced Immunity Mechanism in the Resistance to Bacillus thuringiensis in Lepidopteran Pests.

Bacillus thuringiensis (Bt) is the safest, economically successful entomopathogen to date. It is extensively produced in transgenic crops or used in spray formulations to control Lepidopteran pests. The most serious threat to the sustainable usage of Bt is insect resistance. The resistance mechanisms to Bt toxins depend not only on alterations in insect receptors, but also on the enhancement of insect immune responses. In this work, we review the current knowledge of the immune response and resistance of insects to Bt formulations and Bt proteins, mainly in Lepidopteran pests. We discuss the pattern recognition proteins for recognizing Bt, antimicrobial peptides (AMPs) and their synthetic signaling pathways, the prophenoloxidase system, reactive oxygen species (ROS) generation, nodulation, encapsulation, phagocytosis, and cell-free aggregates, which are involved in immune response reactions or resistance to Bt. This review also analyzes immune priming, which contributes to the evolution of insect resistance to Bt, and puts forward strategies to improve the insecticidal activity of Bt formulations and manage insect resistance, targeting the insect immune responses and resistance.

Investigating host-gut microbial relationship in Penaeus monodon upon exposure to Vibrio harveyi

To understand the host-gut microbial relationship, we used multidisciplinary platforms, metagenome, transcriptome, and metabolome analyses, to determine shrimp and intestinal microbial interactions upon 0, 6, 12, 24, and 48 h exposure to 107 CFU/mL of Vibrio harveyi, a shrimp pathogen. The bacterial communities in the intestine of shrimp were disrupted during exposure to V. harveyi. The abundance of Vibrio ASVs in the Harveyi and Vulnificus clades was significantly increased after exposure to the pathogen (6, 12, and 24 h) and decreased later after 48 h. Conversely, Pseudoalteromonas was found in lower abundance (6, 12, and 24 h) but significantly increased after 48 h of the bacterial challenge. Gene expression analysis revealed that genes belonging to several immune-related pathways, including the Toll pathway, the immune deficiency (IMD) pathway, and pattern recognition proteins (PRPs), were significantly upregulated. Early responses in the first 6 h after exposure were the genes involved in phagocytosis, pattern recognition proteins (PRPs), and signal transduction (spätzle and ankyrin). Late responses (12–48 h) were genes related to proteinases and proteinase inhibitors (PPIs), antimicrobial peptides (AMPs), and oxidative stress. Genes related to lipid metabolisms such as fatty acid metabolism and choline metabolism were also upregulated. Metabolomics analysis also showed an increase in phospholipids, including phosphocholine groups, in the intestine of shrimp exposed to the pathogen. Taken together, the gene expression and metabolomics analyses suggest the importance of lipid metabolism in the defense mechanism against bacterial invasion. Moreover, our metabolomic analysis showed a decrease in tryptophan and indole-3-acrylic acid, metabolites related to intestinal immune homeostasis, after bacterial infection. Our observations suggest that pathogenic Vibrio disrupted biological processes in the shrimp intestine, resulting in a decrease in indole-3-acrylic acid and its derivatives which in turn compromised intestinal immunity. In addition, shrimp responded to the bacterial invasion by activating metabolites related to eicosanoid and phospholipid biosynthesis. Our findings on the interactions between shrimp and intestinal microbiota will be an indispensable gateway to systems biology to better understand the function of shrimp gut microbiota and its influence on shrimp innate immunity.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 μg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 μg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 μg/body weight) was administered than the group fed with a lower concentration of LPS (4 μg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

OfGNBP silencing enhances the toxicity of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki)

The immunity of insects plays a vital role in their survival. Our experiments found that lipopolysaccharide (LPS) and glucono-δ-lactone (GDL) could influence the virulence of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki) by affecting the immunity. Gram-negative binding proteins (GNBPs) are an important pattern recognition proteins that play a crucial role in the innate immune system. Therefore, two OfGNBPs were cloned in O. formosanus. The expression of OfGNBPs was significantly changed by LPS,SM1 and GDL, not prick. In addition, the immune-related gene expression, the phenoloxidase activity and antibacterial activity of donor termites and recipient termites were significantly induced by SM1. Furthermore, the knockdown of OfGNBP by RNA interference reduced not only individual immunity but also social immunity in O. formosanus, which increased the virulence of SM1 to O. formosanus. Importantly, dsOfGNBP alone also had good control effect on O. formosanus. In summary, we concluded that dsOfGNBPs are important termite immunosuppressants.

A new SVWC protein functions as a pattern recognition protein in antibacterial responses in Chinese mitten crab (Eriocheirsinensis)

Because invertebrates lack acquired immunity, they rely primarily on the innate immune system to defend themselves against viral and bacterial infections. SVWC, also called Vago, is a class of small-molecule proteins characterized by a single von Willebrand factor C-domain and appears to be restricted to arthropods. It has been reported that SVWC is involved in antiviral immunity in invertebrates, but whether it is involved in antimicrobial immunity and the mechanism of its involvement in antimicrobial immunity remains unclear. In this study, we identified a novel SVWC gene in Eriocheir sinensis and named it EsSVWC. EsSVWC was found to respond positively to bacterial stimulation and to regulate the expression of related antimicrobial peptides (AMPs). The EsSVWC protein recognized and bound to a variety of pathogen-associated molecular patterns (PAMPs) but did not exhibit direct bactericidal effects. Thus, the EsSVWC protein in crabs helps resist bacterial infection and improve survival rates. In summary, EsSVWC may regulate the innate immune system of crabs in response to microbial invasion in an indirect manner.

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

CLL-162 PTX3 is Constitutively Active in CLL Cells

The pentraxin-related PTX3, commonly produced by myeloid and endothelial cells, is a humoral pattern-recognition protein of the innate immune system. Because CLL patients' PTX3 plasma levels are high and most circulating cells in patients with chronic lymphocytic leukemia (CLL) are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from 7 out of 7 CLL patients produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes co-expressing CD19 and CD5, and confocal microscopy demonstrated that PTX3 co-localized with STAT3. Because signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL cells and we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 antibodies precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an electromobility shift assay that we established and a dual-reporter luciferase assay, we confirmed that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 induced apoptotic disassembly of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

Calcium-dependent reversible coaggregation activity of C-reactive protein and M-ficolin

C-reactive protein (CRP) and M-ficolin are the pattern recognition proteins of the innate immune system. In this report, a mixture of CRP and M-ficolin reversibly co-aggregated in a calcium-dependent manner. This coaggregation was enhanced at low pH (6.5) or low salt (35 mM NaCl) concentrations. The co-aggregate was dissolved by adding EDTA and reformed by adding calcium. The M-ficolin fibrinogen-like domain (FD1), the ligand-binding domain of M-ficolin, also showed calcium-dependent coaggregation with CRP, indicating that reversible coaggregation is caused by CRP interacting with FD1. Interestingly, adding phosphocholine (PC), the ligand of CRP, to a CRP-FD1 mixture abolished the reversible coaggregation activity. PC also inhibited the interaction between CRP and FD1. These results indicate that CRP retains PC-binding activity in the coaggregation state and that FD1 binds specifically to the PC-binding site on CRP but does not fully occupy the five PC-binding sites on a CRP pentamer as judged by SDS-PAGE analysis of precipitates. Coaggregation analysis using FD1 mutants showed that FD1 also retains ligand-binding activity in the coaggregation state and that coaggregation requires the trimeric form of FD1. It was also found that modifications to the ligand-binding site of FD1 affect coaggregation efficiency. Although the biological functions of the coaggregation activity of CRP and M-ficolin remain unresolved, the co-aggregates may function as bacteria-trapping particles with affinities for ligands of CRP and M-ficolin. In addition, coaggregation may be involved in CRP deposition in the lesions of several arterial diseases, such as atherosclerosis.

Gene polymorphism of mannose-binding lectin-associated serine protease (MASP2) in indigenous populations of the Russian Arctic Territories

Mannose-binding lectin-associated serine proteases (MASP) are among of the key components in the lectin pathway (LP) of the complement activation. MASP-2 is the most studied agent among specific enzymes activating both mannose-binding lectin (MBL) and ficolins, pattern-recognition proteins involved in the elimination of pathogenic microorganisms through LP complement activation. There are some mutations in MASP2, with the most significant identified as rs72550870 (p.D120G). The homozygous GG rs72550870 is associated with congenital MASP-2 deficiency and characterized by a total lack of serum protease activity, which leads to impaired binding to lectins. This, in turn, results in severe course of infectious diseases with a high risk of adverse outcome. There seem to be some marked populational differences in the genotype and haplotype prevalence in MASP2 gene polymorphisms. To date, no data are available on the genotype distribution for the MASP2 gene in the indigenous populations of the Russian Arctic regions. The aim of the work was to study the prevalence and ethnic specificity in the distribution of allelic variants of MASP2 rs72550870 in the populations of the Taymyr Dolgan-Nenets District of the Krasnoyarsk Territory (Nenets, Dolgans, Nganasans) as well as the city of Krasnoyarsk (Russians). MASP2 genotyping was performed by using real-time PCR. The frequencies of the AG genotype associated with low MASP-2 level was 6.6% for ethnic Russian newborns in the Eastern Siberia. The prevalence of the AG genotype was significantly lower in newborns of the Arctic populations than in the Russians, being 0.3% and 0.9% for the Nenets and the Dolgan-Nganasans, respectively, which is close to the prevalence values identified for Asian and African populations (0%). No homozygous GG rs72550870 associated with congenital MASP-2 deficiency in newborns of the indigenous populations of the Taymyr Dolgan-Nenets region of Krasnoyarsk Territory (Nenets and Dolgan-Nganasans) and ethnic Caucasian subjects of the Krasnoyarsk city was detected. The frequency of the rare allelic variant G rs72550870 in ethnic Russian subjects was 3.3%, being close to the frequencies in the European populations of the world (4.0%), whereas it was 0.5% in the indigenous inhabitants of the Arctic Region. We have suggested that isolated Arctic populations encounter some intracellular infections historically later and, as contrasted with Caucasoid populations, retained a high activity in the lectin pathway of the complement activation established at the early stage of human evolution.

STAT3 Activates the Pentraxin 3 Gene in Chronic Lymphocytic Leukemia Cells.

Pentraxin-related protein 3 (PTX3), commonly produced by myeloid and endothelial cells, is a humoral pattern recognition protein of the innate immune system. Because PTX3 plasma levels of patients with chronic lymphocytic leukemia (CLL) are high and most circulating cells in patients with CLL are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from seven of seven patients with CLL produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes coexpressing CD19 and CD5, and confocal microscopy showed that PTX3 is present in the cytoplasm of CLL cells. Because STAT3 is constitutively activated in CLL cells, and because we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 Abs precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an EMSA, we established and used a dual-reporter luciferase assay to confirm that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 enhanced apoptosis of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae.

The emergence of Vibrio diseases, including acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp., had resulted in heavy losses in global shrimp production. Biofloc technology is a closed aquaculture system developed as one of the sustainable solutions to increase system resilience in the shrimp industry. In this study, biofloc was formed externally (ex situ biofloc) with probiotics Bacillus sp. strain BME and Bacillus sp. strain BCE, diatom microalgae Chaetoceros calcitrans, and a consortium of nitrifying bacteria, in the ratio of 1:1:6:6 as a starter. The study showed that the ex situ biofloc supplementation in Pacific whiteleg shrimp (L. vannamei) postlarvae culture can increase the shrimp culture performance (shrimp survival and growth), reduce Vibrio counts in the water and shrimp body, and provide stimulation of the shrimp immune response through humoral immune responses, such as pattern recognition protein (C-type lectin) and melanization process (proPO). Overall, the results indicate that the supplementation of ex situ biofloc provided protection to shrimp under Vibrio infection, regardless of the timing of addition (before, simultaneously, or after addition of Vibrio sp. strain VPA). This suggests that the ex situ biofloc can be effective as a preventive and a supportive treatment against potential AHPND infection in L. vannamei postlarvae culture. Taken together, the ability of the ex situ biofloc to modulate immune-related gene expression and resistance of L. vannamei against potentially AHPND-causing Vibrio sp. strain makes it an effective aquaculture technology for infectious disease control in shrimp production with high-density and minimal water exchange culture. KEY POINTS: • Supplementation of ex situ produced biofloc in shrimp postlarvae culture. • Ex situ biofloc reduces Vibrio counts in the water and shrimp body. • Ex situ biofloc stimulates shrimp humoral immune responses and survival.

Response: Commentary: Pattern Recognition Proteins: First Line of Defense Against Coronaviruses.

Response: Commentary: Pattern Recognition Proteins: First Line of Defense Against Coronaviruses.