Bacterial Binding Research Articles

Inhibitory and bactericidal activities analysis of a type IIa crustin from the swimming crab, Portunus trituberculatus

Crustin is an important antibacterial peptide in crustaceans. In this study, a type IIa crustin was cloned from Portunus trituberculatus and named as PtCrustin5. The PtCrustin5 contained the typical domains of type II crustin such as a signal peptide, a cysteine-rich domain (CRD), a whey acidic protein (WAP) domain and a glycine rich region (GRR). The PtCrustin5 showed the highest transcription in hepatopancreas, and its expression level in hepatopancreas, gills and hemocytes increased after challenging with Vibrio alginolyticus for 3 h and 6 h. The recombinant protein of PtCrustin5 (rPtCrustin5) was obtained and the GRR of PtCrustin5 (PtCrustin5-GRR) was synthesized. The bacterial binding assay showed that PtCrustin5 preferred to bind to the three tested gram-positive bacteria including Staphylococcus aureus, Corynebacteri umglutamicum and Micrococcus lysodeikticus. Moreover, rPtCrustin5 and PtCrustin5-GRR displayed inhibitory activity against the three tested gram-positive bacteria. The inhibitory and bactericidal activities of rPtCrustin5 and PtCrustin5-GRR under different conditions were investigated. The rPtCrustin5 and PtCrustin5-GRR displayed inhibitory activity against S. aureus and C. umglutamicum at relatively low concentration under all the tested conditions, while the inhibitory activity of rPtCrustin5 and PtCrustin5-GRR against M. lysodeikticus was affected by pH and salinity. This study enriched our understanding of the immune functions of type II crustin in defending against bacteria and provided theoretical basis for the application of PtCrustin5 and PtCrustin5-GRR in preventing and treating bacteriosis.

Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 μM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

Hydrophobicity Determines the Bacterial Killing Rate of α-Helical Antimicrobial Peptides and Influences the Bacterial Resistance Development.

Rapid antimicrobial action is an important advantage of antimicrobial peptides (AMPs) over antibiotics, which is also a reason for AMPs being less likely to induce bacterial resistance. However, the structural parameters and underlying mechanisms affecting the bacterial killing rate of AMPs remain unknown. In this study, we performed a structure-activity relationship (SAR) study using As-CATH4 and 5 as templates. We revealed that hydrophobicity, rather than other characteristics, is the critical structural parameter determining the bacterial killing rate of α-helical AMPs. With the hydrophobicity increase, the action rates of AMPs including bacterial binding, lipopolysaccharides neutralization, and outer and inner membrane permeabilization increased. Additionally, the higher hydrophobic AMPs with enhanced bacterial killing rates possess better in vivo therapeutic potency and a lower propensity to induce bacterial resistance. These findings revealed the importance of the bacterial killing rate for AMPs and are of great significance to the design and optimization of AMP-related drugs.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.

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.

Fluorescence Resonance Energy Transfer Based Biosensor from Thermophilic Bacterial Periplasmic Binding Protein to Measure Branched-chain and Aromatic Amino Acids

Aim: Exploring the ligand binding capacity of leucine-isoleucine-valine binding protein from Thermotoga maritima.
 Background: The Fischer ratio, ratio of concentrations between branched-chain and aromatic amino acids, has been considered as an indicator of hepatic disease involving metabolic dysfunctions since its instigation in the 1970's. These amino acids are usually measured by high performance liquid chromatography, gas chromatography and enzymatic spectrophotometry. Considering the future potential of periplasmic binding proteins in clinical diagnosis, leucine-isoleucine-valine binding protein from a thermophilic bacterium Thermotoga maritima was purified and characterized, and evaluated its binding properties with twenty amino acids. The protein's stereo-specificity was also tested due to its importance in astrobiology research. Amino acids and carbohydrates are both known to exist in extraterrestrial environments, and stereo-chemistry is the key aspect for distinguishing between abiotic and biotic origin.
 Results: Single amino acid substitutions were generated by overlapping polymerase chain reaction mediated mutagenesis using mutagenic primers to construct two mutants of leucine-isoleucine-valine binding protein (LIVBP). The first mutant of LIVBP in which phenylalanine (F) at 118 position was replaced with cysteine (C) was termed as LIVBP-F118C and the resultant plasmid was named as pKM242. Another mutant of LIVBP in which aspartic acid (D) at 221 position was replaced with cysteine was termed as LIVBP-D221C and the resultant plasmid was named as pKM244. Since the LIVBP-F118C showed higher labeling and fluorescence resonance energy transfer efficiency compared to that of D221C, the LIVBP-F118C was used for further study.
 Conclusion: Fluorescence resonance energy transfer technology was applied to measure dissociation constant (Kd) where chimeric isoleucine-valine binding protein (LIVBP) was engineered to conjugate a donor fluorophore at amino-terminal and an acceptor fluorophore at the incorporated cysteine residue. Ligand binding studies revealed that LIVBP from Thermotoga maritima was able to bind with fourteen amino acids out of twenty including branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) with Kd values ranging from 10-6 to 10-9 M. The Kd values of BCAAs, AAAs, methionine and cysteine were observed at nM level. Highest Kd value was observed for L-leucine (37.2 nM) and L-phenylalanine (44.6 nM). These results indicated that LIVBP from Thermotoga maritima has a broader substrate specificity than previously reported for LIVBP from other organisms, which might be helpful for the development of a miniaturized and noninvasive biosensor to measure BCAAs and AAAs.

Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol.

Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP–BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16–520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30–500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.

Dipole‐Dipole and Anion‐π+ Interaction Manipulation Synergistically Enhance Intrinsic Antibacterial Activities of AIEgens

Pathogenic bacteria infections, especially multidrug resistant bacteria infections have aroused worldwide attention due to their severe threats to human beings. Thus, the development of highly effective antibacterial reagents is very important. However, the design of antimicrobials is still quite challenging for the lack of a universal design strategy. Here, a synergistic manipulation strategy of dipole-dipole and anion-π+ interaction is proposed for constructing highly efficient antimicrobials with aggregation-induced emission (AIE) feature. Firstly, four anion-π+ -type AIE luminogens were designed and synthesized. Due to the electron-donating and hydrophilic characteristic of methoxy groups, 3MOTPO containing three methoxy groups showed the largest dipole moment (5.06 Debye) and dual anion-π+ interactions in the solid state. Driven by both dipole-dipole and anion-π+ interactions, 3MOTPO showed the strongest bacterial binding ability and the best antibacterial activities (MIC90 =3.76 μM). The work offers a deep insight into the rational design of highly efficient antimicrobials for luminescence-guided antibacterial study.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate.

Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia (Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.

Immunoglobulin A antibody composition is sculpted to bind the self gut microbiome.

Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual's own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found that the binding specificity of bulk fecal and serum IgA toward resident gut bacteria resolves well at the species level and has modest strain-level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, whereas a small portion displayed cross-reactivity. Orally administered hybridoma-produced IgAs still retained bacterial antigen binding capability, implying the potential for a new class of therapeutic antibodies. Species-specific IgAs had a range of strain specificities. Given the distinctive bacterial species and strain composition found in each individual's gut, our findings suggest the IgA antibody repertoire is shaped uniquely to bind "self" gut bacteria.

Identification of heptapeptides targeting a lethal bacterial strain in septic mice through an integrative approach

Effectively killing pathogenic bacteria is key for the treatment of sepsis. Although various anti-infective drugs have been used for the treatment of sepsis, the therapeutic effect is largely limited by the lack of a specific bacterium-targeting delivery system. This study aimed to develop antibacterial peptides that specifically target pathogenic bacteria for the treatment of sepsis. The lethal bacterial strain Escherichia coli MSI001 was isolated from mice of a cecal ligation and puncture (CLP) model and was used as a target to screen bacterial binding heptapeptides through an integrative bioinformatics approach based on phage display technology and high-throughput sequencing (HTS). Heptapeptides binding to E. coli MSI001 with high affinity were acquired after normalization by the heptapeptide frequency of the library. A representative heptapeptide VTKLGSL (VTK) was selected for fusion with the antibacterial peptide LL-37 to construct the specific-targeting antibacterial peptide VTK-LL37. We found that, in comparison with LL37, VTK-LL37 showed prominent bacteriostatic activity and an inhibitive effect on biofilm formation in vitro. In vivo experiments demonstrated that VTK-LL37 significantly inhibited bacterial growth, reduced HMGB1 expression, alleviated lesions of vital organs and improved the survival of mice subjected to CLP modeling. Furthermore, membrane DEGP and DEGQ were identified as VTK-binding proteins by proteomic methods. This study provides a novel strategy for targeted pathogen killing, which is helpful for the treatment of sepsis in the era of precise medicine.

The In Vitro Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages.

ABSTRACTAchromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo. This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology.IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo. This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

Antimicrobial effects of bacterial binding to a dialkylcarbamoyl chloride-coated wound dressing: an in vitro study.

Wound dressings that inactivate or sequestrate microorganisms, such as those with a hydrophobic, bacteria-binding dialkylcarbamoyl chloride (DACC) surface, can reduce the risk of clinical infections. This 'passive' bioburden control, avoiding bacterial cell wall disruption with associated release of bacterial endotoxins aggravating inflammation, is advantageous in hard-to-heal wounds. Hence, the full scope of DACC dressings, including the potential impact of higher inoculum densities, increased protein load and different pH on antibacterial activity, needs to be evaluated. The Japanese Industrial Standard (JIS) L 1902 challenge test was used to evaluate the antimicrobial activity of the DACC-coated dressing against several World Health Organization (WHO)-prioritised wound pathogens (e.g., meticillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, microorganisms with extended-spectrum beta-lactamases and Acinetobacter baumannii), the effect of repeated bacterial challenge in an adverse wound environment, and antimicrobial performance at wound-related pH. High antibacterial activity of the DACC-coated dressing against the WHO-prioritised bacteria strains by its irreversible binding and inhibition of growth of bound bacteria was confirmed using JIS L 1902. At increased inoculation densities, compared to standard conditions, the DACC-coated dressing still achieved strong-to-significant antibacterial effects. Augmenting the media protein content also affected antibacterial performance; a 0.5-1 log reduction in antibacterial activity was observed upon addition of 10% fetal calf serum. The pH did not influence antibacterial performance. The DACC-coated dressing also sustained antibacterial activity over subsequent reinfection steps. It can be assumed that the DACC-coated dressing exerts beneficial effects in controlling the wound bioburden, reducing the overall demand placed on antibiotics, without using antimicrobial substances.

Natural and commercial antibiotic comparison with drugs modeling cell integrity cell stability of Bio-Kinetics changes under morphological topographies cells with lower toxicological characteristics for multidrug resistances problem

Antibacterial drug-resistant strains are a serious problem of bacterial treatments nowadays and have a concern. The plant exacts of Adhatoda vasica and Calotropis procera are well-known for their role as antibiotic agents. The extraction of novel antibiotic compounds was done by HPLC-DAD, their yield is quantified by numerous solvents. The complete biological activity with antioxidants, bio-kinematicof four compounds of B-Sitosteryl linoleate, Myristyl diglucoside, D-Triglucopyranoside, and S- allylcysteine acids were studied. The supercritical fluid extraction techniques were the best strategies for higher yield, accuracy clarity, and inter, intra process of all four compounds. A. vasica and C. procera samples and investigated in six different solvents. D-Triglucopyranoside (13.81 ± 0.48%), Myristyl diglucoside (11.81 ± 0.41%), B- Sitosteryl linoleate (12.81 ± 0.48%), and s-allylcysteine acids (14.81 ± 0.31%) were higher. The design and action of compounds were applied to proper compartmental pharmacokinetic modelling for in-depth design understanding. The morphology and structure of bacterial cells with the extracted compounds upheld the permeability of cell membranes, membrane integrity, and membrane potential and lower the bacterial binding capacity the infectious index was measured in transmission electron microscopy (TEM) and their alteration process. Plants have well upheld the cellular permeability The toxicity test was performed on both extracted samples with concentrations (1, 0.4, and 0.8%). The areas under plasma half-life of compounds with their solubility, abortion level were higher in four compounds showed the potential of novel antibiotics. The novel medicinal plants used as antibiotics could be the best sources of infection control as a source of future medicines with antibacterial potential solving multidrug issues of bacteria in the world.

Identification and functional analysis of Mannose receptor in Asian swamp eel (Monopterus albus) in response to bacterial infection

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely MaMR). The open reading frame of MaMR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that MaMR shared the highest similarity with that of Paralichthys olivaceus. The expression of MaMR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda, the transcript level of MaMR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of MaMR was significantly induced post stimulated with LPS and LTA. Then the MaMR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that rMaMR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that MaMR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1β and tnf-α in EPC cells. Taken together, these results reveal that MaMR plays an important role in the immune response of fish to pathogen infections.

Human Milk Oligosaccharides Impact Cellular and Inflammatory Gene Expression and Immune Response.

Human milk harbors complex carbohydrates, including human milk oligosaccharides (HMOs), the third most abundant component after lactose and lipids. HMOs have been shown to impact intestinal microbiota, modulate the intestinal immune response, and prevent pathogenic bacterial binding by serving as decoy receptors. However, the direct effect of HMOs on intestinal function and immunity remains to be elucidated. To address this knowledge gap, 21-day-old germ-free mice (C57BI/6) were orally gavaged with 15 mg/day of pooled HMOs for 7 or 14 days and euthanized at day 28 or 35. A set of mice was maintained until day 50 to determine the persistent effects of HMOs. Control groups were maintained in the isolators for 28, 35, or 50 days of age. At the respective endpoints, intestinal tissues were subjected to histomorphometric and transcriptomic analyses, while the spleen and mesenteric lymph nodes (MLNs) were subjected to flow cytometric analysis. The small intestine (SI) crypt was reduced after HMO treatment relative to control at days 28 and 35, while the SI villus height and large intestine (LI) gland depth were decreased in the HMO-treated mice relative to the control at day 35. We report significant HMO-induced and location-specific gene expression changes in host intestinal tissues. HMO treatment significantly upregulated genes involved in extracellular matrix, protein ubiquitination, nuclear transport, and mononuclear cell differentiation. CD4+ T cells were increased in both MLNs and the spleen, while CD8+ T cells were increased in the spleen at day 50 in the HMO group in comparison to controls. In MLNs, plasma cells were increased in HMO group at days 28 and 35, while in the spleen, only at day 28 relative to controls. Macrophages/monocytes and neutrophils were lower in the spleen of the HMO group at days 28, 35, and 50, while in MLNs, only neutrophils were lower at day 50 in the 14-day HMO group. In addition, diphtheria toxoid and tetanus toxoid antibody–secreting cells were higher in HMO-supplemented group compared to controls. Our data suggest that HMOs have a direct effect on gastrointestinal tract metabolism and the immune system even in the absence of host microbiota.

Antibacterial activity of an anti-lipopolysaccharide factor (MjALF-D) identified from kuruma prawn (Marsupenaeus japonicus)

Antimicrobial peptides (AMPs) play important roles in host innate immune systems. Anti-lipopolysaccharide factor (ALF), which is a primary AMP in crustaceans, is active against bacteria, fungi and some viruses. MjALF-D, an anionic peptide, is a group D ALF isolated from Marsupenaeus japonicus. In the present study, a series of experiments were performed to study its antibacterial spectrum and further explore its antibacterial and bacterial binding activities. Liquid growth inhibition data demonstrated that recombinant MjALF-D (rMjALF-D) possessed strong antibacterial activity against the gram-positive bacterium Micrococcus luteus and the gram-negative bacterium Photobacterium damselae, with a minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) lower than 1.25 μM. The kinetic analysis showed that the antibacterial activity of rMjALF-D was dose- and time-dependent. Additionally, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations the potential bactericidal process. rMjALF-D treatment resulted in a large number of unidentified filamentous structures wrapped around the bacteria, and during the incubation, the cell surface became obviously rough and disrupted. rMjALF-D showed distinct binding ability after direct incubation with M. luteus and P. damselae but no binding ability to Escherichia coli, which was weakly inhibited by rMjALF-D. These data suggest that MjALF-D displays modest antibacterial activity and may provide more insights into the function and role of ALF in shrimp immunity.

A C-type lectin containing two carbohydrate recognition domains participates in the antibacterial response by regulating the JNK pathway and promoting phagocytosis

C-type lectins (CTLs) are important immune-related molecules in crustaceans. However, the immunologic mechanism by which CTLs eliminate invading pathogens is still unclear. In this study, we studied the antimicrobial mechanism of a CTL containing two carbohydrate recognition domains (DClec). After Aeromonas hydrophila challenge, several antimicrobial peptides (ALF1, ALF4, ALF5 and lys-i2) were upregulated. The transcript levels of ALF1, ALF4 and ALF5 were downregulated after A. hydrophila challenge in groups with DClec interference or inhibition compared with the control group. Similar results were obtained after c-Jun N-terminal kinase (JNK) interference. This finding indicates that DClec might regulate the JNK signalling pathway and subsequently adjust antimicrobial peptide (AMP) expression. Additionally, we found that DClec was secreted into the hemolymph. Recombinant protein DClec (rDClec) agglutinated gram-positive or gram-negative bacteria. Both rDClec and the native DClec in hemolymph bound to different bacteria. In this process, Ca2+ promoted the rDClec bacterial binding ability. After DClec interference, the phagocytosis ability of hemocytes was lower than that of the control group. Therefore, DClec can facilitate bacterial elimination by promoting AMPs expression and hemocyte phagocytosis.

A Teleost CXCL10 Is Both an Immunoregulator and an Antimicrobial

Chemokines are a group of cytokines that play important roles in cell migration, inflammation, and immune defense. In this study, we identified a CXC chemokine, CXCL10, from Japanese flounder Paralichthys olivaceus (named PoCXCL10) and investigated its immune function. Structurally, PoCXCL10 possesses an N-terminal coil, three β-strands, and a C-terminal α-helix with cationic and amphipathic properties. PoCXCL10 expression occurred in multiple tissues and was upregulated by bacterial pathogens. Recombinant PoCXCL10 (rPoCXCL10) promoted the migration, cytokine expression, and phagocytosis of flounder peripheral blood leukocytes (PBLs). rPoCXCL10 bound to and inhibited the growth of a variety of common Gram-negative and Gram-positive fish pathogens. rPoCXCL10 killed the pathogens by causing bacterial membrane permeabilization and structure destruction. When introduced in vivo, rPoCXCL10 significantly inhibited bacterial dissemination in fish tissues. A peptide derived from the C-terminal α-helix exhibited bactericidal activity and competed with rPoCXCL10 for bacterial binding. Deletion of the α-helix affected the in vitro bactericidal activity but not the chemotaxis or in vivo antimicrobial activity of PoCXCL10. Together, these results indicate that PoCXCL10 exerts the role of both an immunoregulator and a bactericide/bacteriostatic via different structural domains. These findings provide new insights into the immune function and working mechanism of fish CXC chemokines.