Identification of a novel amphioxus leucine-rich repeat receptor involved in phagocytosis reveals a role for Slit2-N-type LRR in bacterial elimination.

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

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.

Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai).

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet-activating factor receptor.

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host-pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet-activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community-acquired pneumonia in the elderly, we explored the role of A1 in the age-driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial-host interactions.

Molecular characterization and functional study of a tandem-repeat Galectin-9 from Japanese flounder (Paralichthys olivaceus).

Galectin-9 is a ß-galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, one Galectin-9 (named PoGalectin-9) was identified from Japanese flounder Paralichthys olivaceus. PoGalectin-9 belongs to the tandem-repeat type, containing one 127-amino acids CRD domain within N terminal and one 122-amino acids CRD domain within C-terminal. The open reading frame of PoGalectin-9 cDNA was 921 bp encoding 306 amino acids. Sequence similarity comparison confirmed that PoGalectin-9 shared high homology with other Galectin-9. The tissue distribution and expression profiles after bacterial infection were also investigated. PoGalectin-9 was widely distributed in all of the examined tissues of Japanese flounder but was predominantly expressed in the spleen, kidney and intestine. After Edwardsiella tarda challenge, the expression of PoGalectin-9 was up-regulated in spleen and down regulated in kidney. ELISA experiment showed that recombinant PoGalectin-9 (rPoGalectin-9) exhibit binding capacity to lipopolysaccharide (LPS) and peptidoglycan (PGN), which is significantly correlated with the concentration of rPoGalectin-9. Meanwhile, the rPoGalectin-9 protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Bacterial binding experiments showed that rPoGalectin-9 could bind all examined bacteria. In conclusion, the present study indicate that PoGalectin-9 might play important roles during the immune responses of Japanese flounder against bacterial pathogens.

In vitro evaluation of 99m Tc-sultamicillin for infection imaging.

Early detection of the site of infection non-invasively with radiolabeled molecules is important for the success of treatment. Technetium-99m labeled antibiotics have the potential to discriminate between bacterial infection and sterile inflammation. Sultamicillin is the tosylate salt of the double ester of sulbactam plus ampicillin. In this study, sultamicillin was labeled with 99m Tc according to the stannous chloride method. Quality control studies of radiolabeled sultamicillin were performed by radiochromatographic methods. In vitro binding assays were performed in live and heat-killed gram-positive Staphylococcus aureus and gram-negative Escherichia coli strains. The radiolabeling yield of 99m Tc-sultamicillin was determined as 97.8% ± 3.1% (n = 5). The maximum bacterial uptake of 99m Tc-sultamicillin was 80.7% ± 11.00% at 4 h for living S. aureus and 93.2% ± 4.40% at 2 h for E. coli. Bacterial uptake study results show that sultamicillin has the potential to be a nuclear imaging agent, especially in infections caused by gram-negative E. coli and gram-positive S. aureus.

Antimicrobial stewardship strategies in wound care: evidence to support the use of dialkylcarbamoyl chloride (DACC)- coated wound dressings.

BACKGROUND: Traditionally, infections are treated with antimicrobials (for example, antibiotics, antiseptics, etc), but antimicrobial resistance (AMR) has become one of the most serious health threats of the 21st century (before the emergence of COVID-19). Wounds can be a source of infection by allowing unconstrained entry of microorganisms into the body, including antimicrobial-resistant bacteria. The development of new antimicrobials (particularly antibiotics) is not keeping pace with the evolution of resistant microorganisms and novel ways of addressing this problem are urgently required. One such initiative has been the development of antimicrobial stewardship (AMS) programmes, which educate healthcare workers, and control the prescribing and targeting of antimicrobials to reduce the likelihood of AMR. Of great importance has been the European Wound Management Association (EWMA) in supporting AMS by providing practical recommendations for optimising antimicrobial therapy for the treatment of wound infection. The use of wound dressings that use a physical sequestration and retention approach rather than antimicrobial agents to reduce bacterial burden offers a novel approach that supports AMS. Bacterial-binding by dressings and their physical removal, rather than active killing, minimises their damage and hence prevents the release of damaging endotoxins. AIM: Our objective is to highlight AMS for the promotion of the judicious use of antimicrobials and to investigate how dialkylcarbamoyl chloride (DACC)-coated dressings can support AMS goals. METHOD: MEDLINE, Cochrane Database of Systematic Reviews, and Google Scholar were searched to identify published articles describing data relating to AMS, and the use of a variety of wound dressings in the prevention and/or treatment of wound infections. The evidence supporting alternative wound dressings that can reduce bioburden and prevent and/or treat wound infection in a manner that does not kill or damage the microorganisms (for example, by actively binding and removing intact microorganisms from wounds) were then narratively reviewed. RESULTS: The evidence reviewed here demonstrates that using bacterial-binding wound dressings that act in a physical manner (for example, DACC-coated dressings) as an alternative approach to preventing and/or treating infection in both acute and hard-to-heal wounds does not exacerbate AMR and supports AMS. CONCLUSION: Some wound dressings work via a mechanism that promotes the binding and physical uptake, sequestration and removal of intact microorganisms from the wound bed (for example, a wound dressing that uses DACC technology to successfully prevent/reduce infection). They provide a valuable tool that aligns with the requirements of AMS (for example, reducing the use of antimicrobials in wound treatment regimens) by effectively reducing wound bioburden without inducing/selecting for resistant bacteria.

Estrategias de protección antimicrobiana en el cuidado de heridas: evidencia para el uso de apósitos recubiertos con DACC.

BACKGROUND: Antimicrobial resistance (AMR) is one of the most serious health threats globally. The development of new antimicrobials is not keeping pace with the evolution of resistant microorganisms, and novel ways of tackling this problem are required. One of such initiatives has been the development of antimicrobial stewardship programmes (AMS). The use of wound dressings that employ a physical sequestration and retention approach to reduce bacterial burden offers a novel approach to support AMS. Bacterial-binding by dressings and their physical removal can minimise their damage and prevent the release of harmful endotoxins. OBJECTIVE: To highlight AMS to promote the correct use of antimicrobials and to investigate how dialkylcarbamyl chloride (DACC)-coated dressings can support AMS. METHOD: MEDLINE, Cochrane Database of Systematic Reviews, and Google Scholar were searched to identify articles relating to AMS, and the use of wound dressings in the prevention and treatment of wound infections. The evidence supporting alternative wound dressings that can reduce bioburden and prevent wound infection in a way that does not kill or damage the microorganisms were reviewed. RESULTS: The evidence demonstrated that using bacterial-binding wound dressings that act in a physical manner (eg, DACC-coated dressings) to preventing infection in both acute and hard-to-heal wounds does not exacerbate AMR and supports AMS. CONCLUSION: Some wound dressings work via a mechanism that promotes the binding and physical sequestration and removal of intact microorganisms from the wound bed (eg, a wound dressing that uses DACC technology to prevent/reduce infection). They provide a valuable tool that aligns with the requirements of AMS by effectively reducing wound bioburden without inducing/selecting for resistant bacteria.

ANTECEDENTES: Normalmente, las infecciones son tratadas con antimicrobianos (antibióticos, antisépticos, etc.). La resistencia antimicrobiana (AMR, por sus siglas en inglés) se ha convertido en una de las amenazas del siglo XXI más graves para la salud mundial. Las heridas pueden ser una fuente de infección al permitir la entrada libre de microorganismos dentro del cuerpo, incluyendo bacterias resistentes a antimicrobianos. El desarrollo de nuevos antimicrobianos (especialmente, antibióticos) no está siguiendo el ritmo de la evolución de microorganismos resistentes y de formas novedosas de abordar este problema con la urgencia que demanda. Una de estas iniciativas ha sido el desarrollo de programas de protección antimicrobiana (AMS, por sus siglas en inglés), que brindan capacitación a los trabajadores del área de la salud y controlan la prescripción, enfocándose en los antimicrobianos para reducir la probabilidad de que se produzca AMR. El uso de apósitos para herida que utilizan el aislamiento físico y el abordaje de retención, en vez de agentes antimicrobianos, para reducir la carga bacteriana ofrecen un abordaje novedoso para apoyar a los AMS. La fijación bacteriana por los apósitos y su retiro físico, en lugar de la muerte activa, minimiza su daño y, además, previene la liberación de endotoxinas dañinas. OBJETIVO: Resaltar los AMS para la promoción del uso correcto de antimicrobianos e investigar cómo los apósitos recubiertos con cloruro de dialquilcarbamilo (DACC) pueden ayudar a cubrir las metas de los AMS. MÉTODO: Se realizaron búsquedas en las bases de datos de revisiones sistemáticas, Medline, Cochrane y Google Scholar con el fin de identificar artículos publicados que describan los datos relacionados con los AMS, y el uso de una gran variedad de apósitos para heridas para la prevención y/o tratamiento de infecciones de la herida. La evidencia que respalda a los apósitos para heridas alternativos que pueden reducir la biocarga y prevenir y/o tratar la infección de heridas de forma tal que no maten ni dañen a los microorganismos (por ejemplo, fijándose activamente y retirando intactos a los microorganismos de las heridas) fue posteriormente revisada de forma oral. CONCLUSIÓN: Algunos apósitos para heridas actúan a través de mecanismos que promueven la fijación y absorción física, aislamiento y retiro de microorganismos intactos de la base de la herida (por ejemplo, un apósito para heridas que utiliza la tecnología DACC para prevenir/reducir la infección). Esta es una herramienta valiosa que cumple con los requisitos del AMS (por ejemplo, reducción del uso de antimicrobianos en esquemas de tratamiento de heridas) al reducir la biocarga de la herida sin inducir/seleccionar bacterias resistentes.

A Fish Galectin-8 Possesses Direct Bactericidal Activity.

Galectins are a family of animal lectins with high affinity for ß-galactosides. Galectins are able to bind to bacteria, and a few mammalian galectins are known to kill the bound bacteria. In fish, no galectins with direct bactericidal effect have been reported. In the present study, we identified and characterized a tandem repeat galectin-8 from tongue sole Cynoglossus semilaevis (designated CsGal-8). CsGal-8 possesses conserved carbohydrate recognition domains (CRDs), as well as the conserved HXNPR and WGXEE motifs that are critical for carbohydrate binding. CsGal-8 was constitutively expressed in nine tissues of tongue sole and up-regulated in kidney, spleen, and blood by bacterial challenge. When expressed in HeLa cells, CsGal-8 protein was detected both in the cytoplasm and in the micro-vesicles secreted from the cells. Recombinant CsGal-8 (rCsGal-8) bound to lactose and other carbohydrates in a dose dependent manner. rCsGal-8 bound to a wide range of gram-positive and gram-negative bacteria and was co-localized with the bound bacteria in animal cells. Lactose, fructose, galactose, and trehalose effectively blocked the interactions between rCsGal-8 and different bacteria. Furthermore, rCsGal-8 exerted potent bactericidal activity against some gram-negative bacterial pathogens by directly damaging the membrane and structure of the pathogens. Taken together, these results indicate that CsGal-8 likely plays an important role in the immune defense against some bacterial pathogens by direct bacterial interaction and killing.

A C-type lectin, Nattectin-like protein (CaNTC) in Qihe crucian carp Carassius auratus: Binding ability with LPS, PGN and various bacteria, and agglutinating activity against bacteria.

C-type lectins (CTLs), as the members of pattern-recognition receptors (PRRs), play the significant roles in innate immunity through binding with pathogen-associated molecular patterns (PAMPs) on the surface of microbe. In the present study, a novel CTL, Nattectin-like protein (named as CaNTC), was investigated in Qihe crucian carp Carassius auratus. The full-length cDNA of CaNTC was composed of 776 bp, with a 152 bp 5'-untranslated region (UTR), a 492 bp ORF encoding a 163-aa protein, and a 132 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of CaNTC contained a signal peptide, a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys57-Cys150, Cys126-Cys142), and an EPN/WND motif required for carbohydrate-binding specificity. With regard to the mRNA transcript of CaNTC, it was predominately expressed in liver. The temporal expressions of CaNTC were obviously up-regulated in liver, spleen and head-kidney after challenged by Aeromonas hydrophila and poly I: C, respectively, and the change pattern was in the time-depended manner. The recombinant CaNTC (rCaNTC) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. With regard to the agglutinating activity of rCaNTC, rCaNTC was able to agglutinate rabbit erythrocytes and three kinds of bacteria (Gram-negative bacteria, Escherichia coli and A. hydrophila, and Gram-positive bacteria Staphylococcus aureus) in a Ca2+-dependent manner. These findings collectively demonstrated that CaNTC, as a PRR, could be involved in the innate immunity and play an important role in immune defense of C. auratus.

Exploitation of SPR to Investigate the Importance of Glycan Chains in the Interaction between Lactoferrin and Bacteria.

Bovine lactoferrin (LF) has been shown to prevent adhesion to and invasion of mammalian cell lines by pathogenic bacteria, with evidence for direct bacterial binding by the milk glycoprotein. However, the glycosylation pattern of LF changes over the lactation cycle. In this study, we aim to investigate the effect that this variation has on the milk glycoprotein's ability to interact with pathogens. Surface plasmon resonance technology was employed to compare the binding of LF from colostrum (early lactation) and mature milk (late lactation) to a panel of pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella typhimurium). Novel interactions with LF were identified for C. sakazakii, S. pneumoniae and P. aeruginosa with the highest binding ability observed for mature milk LF in all cases, with the exception of S. typhimurium. The difference in bacterial binding observed may be as a result of the varying glycosylation profiles. This work demonstrates the potential of LF as a functional food ingredient to prevent bacterial infection.

Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense.

Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis).

Recognizing the presence of invading pathogens by pattern recognition receptors (PRRs) is key to mounting an effective innate immune response. Mammalian CD302 is an unconventional C-type lectin like receptor (CTLR) involved in the functional regulation of immune cells. However, the role of CD302 in fish remains unclear. In this study, we characterized a novel CD302 gene from ayu (Plecoglossus altivelis), which was tentatively named PaCD302. The cDNA sequence of PaCD302 is 1893 nucleotides in length, and encodes a polypeptide of 241 amino acids with molecular weight 27.1 kDa and pI 4.69. Sequence comparison and phylogenetic tree analysis showed that PaCD302 is a type I transmembrane CTLR devoid of the known amino acid residues essential for Ca(2+)-dependent sugar binding. PaCD302 mRNA expression was detected in all tissues and cells tested, with the highest level in the liver. Following Vibrio anguillarum infection, PaCD302 mRNA expression was significantly upregulated in all tissues tested. For further functional analysis, we generated a recombinant protein for PaCD302 (rPaCD302) by prokaryotic expression and raised a specific antibody against rPaCD302. Western blot analysis revealed that the native PaCD302 is glycosylated. Refolded rPaCD302 was unable to bind to five monosaccharides (l-fucose, d-galactose, d-glucose, d-mannose and N-acetyl glucosamine) or two other polysaccharides (lipopolysaccharide and peptidoglycan). It was able to bind to three Gram-positive and seven Gram-negative bacteria, but show no bacterial agglutinating activity. PaCD302 function blocking using anti-PaCD302 IgG resulted in inhibition of phagocytosis and bactericidal activity of ayu monocytes/macrophages (MO/MΦ), suggesting that PaCD302 regulates the function of ayu MO/MΦ. In summary, our study demonstrates that PaCD302 may participate in the immune response of ayu against bacterial infection via modulation of MO/MΦ function.

Ceramic Dressings-A New Non-Pharmacological Therapeutic Option in the Management of Chronic Wounds?

A new ceramic dressing, free from active antimicrobial or pharmaceutical agents, uses physical binding mechanisms for its absorption capacities and bacterial-binding properties. The purpose of this study was to evaluate wound healing, bacterial-related retention, and diagnostic properties of ceramic dressings in patients with stagnated chronic wounds. METHODS: In this monocentric, intra-individually controlled, prospective study, patients with conservatively treated refractory chronic wounds were enrolled. One week before the start of the application with ceramic dressing, it was ensured during a screening phase that chronic wounds showed less than a 10% reduction in wound size. During the 4-week ceramic dressing treatment wound size measurements, wound scoring, measurement of wound exudate amount, wound swabs, and ceramic dressing sonication (low-intensity ultrasound) were carried out. The sonication fluid of the removed ceramic dressing was used for analysis of bacterial retention and compared to wound swabs. RESULTS: A total of 20 patients with a mean age of 64.6 years (±26.2) and 21 chronic wounds were included in this study. After a 4-week treatment, a significant reduction of median wound size from 1178 mm2 (range 104-6300) to 751.5 mm2 (range 16-4819) and better total wound scores were observed (p < 0.001). The sensitivity of bacteria detection was 90.7% in the sonication fluid from the ceramic dressings, while only 76.9% in the conventional wound swabs. CONCLUSION: The new ceramic dressing seems to have a positive impact on wound healing in chronic wounds. Bacteria-binding characteristics of the investigated ceramic dressing, in combination with its debridement, absorption, and detoxification properties, could contribute to its healing abilities. Based on those results, the investigated ceramic dressing seems to be a promising new treatment option for chronic wounds without the use of any active antimicrobial or pharmacological agents. Moreover, ceramic dressings can also be considered for microbiological diagnostic purposes.

Molecular mechanism of the NOS/NOX regulation of antibacterial activity in Eriocheir sinensis.

To elucidate the role of nitric oxide synthase (NOS), which produces the free radical nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate oxidase (NOX), which produces the superoxide anion (O2-), in the innate immunity of Eriocheir sinensis, the full lengths of the NOS and NOX genes were cloned via rapid amplification of the cDNA ends and then expressed in the prokaryotic form to obtain the recombinant proteins, NOS-HIS and NOX-HIS. Through bacterial binding and stimulation experiments, the molecular mechanisms of NOS and NOX in the innate immunity of E. sinensis were explored. Based on the results, NOS and NOX were 5900 bp and 4504 bp long, respectively, and were evolutionarily conserved. Quantitative real-time PCR revealed that NOS and NOX were expressed in all studied tissues, and both were expressed in the highest amounts in hemocytes. NOS-HIS and NOX-HIS could bind to bacteria with different binding powers; their binding ability to gram-positive bacteria was higher than that of binding to gram-negative bacteria. After stimulation with Aeromonas hydrophila, NOS expression was significantly up-regulated at 3, 6, and 48 h, and NOX expression was significantly down-regulated at 3, 12, 24, and 48 h. After bacterial stimulation, the NOS enzyme activity in the serum of E. sinensis was also significantly up-regulated at 6 and 48 h, and the NOX enzyme activity was significantly down-regulated at 12 and 48 h, aligning with the gene expression trend. Moreover, the related free radical molecules, NO, O2-, and H2O2, tended to decrease after bacterial stimulation. Overall, the gene expression and enzyme activity of NOS and NOX had been changed respectively, and the contents of a series of free radical molecules (NO, O2- and H2O2) were induced in E. sinensis after bacterial stimulation, which then exert antibacterial immunity.

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus.

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity.

Antibacterial activity of four recombinant carbohydrate recognition domain proteins identified from the kuruma shrimp Marsupenaeus japonicus.

The carbohydrate recognition domain (CRD) is the key component of C-type lectins (CTLs) with the capacity to recognize and eliminate invading pathogens. Herein, the recombinant proteins of four CRDs identified from the kuruma shrimp, Marsupenaeus japonicus, were produced and purified by an Escherichia coli expression system and affinity chromatography. Bacterial binding and antibacterial assays showed that the four CRDs displayed various bacterial binding and antibacterial activities against different bacteria. Among the four recombinant CRDs, His-CRD2-3 exhibited the broadest spectrum of bacterial binding and antibacterial activities against gram-negative bacteria (Vibrio parahaemolyticus, V. alginolyticus and V. harveyi) and gram-positive bacteria (Staphylococcus aureus and Micrococcus lysodeikticus). Moreover, the four recombinant CRDs showed different capacities to regulate the expression of several immune effector genes (MjCTL3, MjCTL4, MjCTL, Mjily and Mjsty), among which His-CRD2-3 displayed broader and stronger inductive effects on these immune effector genes. This study indicated that the four CRDs participated in immune defense by binding and killing bacteria and regulating the transcription of other immune effector genes. In addition, our results suggested that His-CRD2-3 might be a promising agent for the prevention and treatment of bacteriosis.

Molecular identification of peptidoglycan recognition protein 5 and its functional characterization in innate immunity of large yellow croaker, Larimichthys crocea.

Fish peptidoglycan recognition proteins (PGRPs) play important roles in microbial recognition, and bacterial elimination. In the present study, a short-type PGRP from large yellow croaker, LcPGRP5 was cloned and its functions were characterized. LcPGRP5 gene encodes a protein containing conserved PGRP domain, but no signal peptide. Phylogenetic analysis shows that LcPGRP5 is clustered with other short PGRPs identified in other teleosts. LcPGRP5 is constitutively expressed in all tissues examined, with the highest expression being detected in the head kidney. Recombinant LcPGRP5 protein features amidase activity and bactericidal activity. Notably, LcPGRP5 could enhance the phagocytosis of the bacteria by large yellow croaker macrophage, with higher phagocytic capacity being observed in Staphylococcus aureus compared to Escherichia coli. Moreover, overexpression of LcPGRP5 suppresses pro-inflammatory effects elicited by bacterial exposure in the macrophage cell line. Overall, the present results clearly indicate the important roles of LcPGRP5 played in the innate immune responses against bacterial infection.

A C-type lectin with dual-CRD from Tribolium castaneum is induced in response to bacterial challenge.

BACKGROUND: C-type lectins (CTLs), a group of pattern recognition receptors, are involved in regulating the immune response of insects and could be used as potential targets for pest control. However, information about roles of CTLs in the innate immunity of Tribolium castaneum, a serious, worldwide pest that damages stored grain products, is relatively scarce. RESULTS: Here, a CTL with dual carbohydrate recognition domains (CRDs) containing a highly conserved WHD (Trp53 -His54 -Asp55 ) motif was identified in T. castaneum and named as TcCTL3. Spatiotemporal analysis showed that TcCTL3 was highly expressed in all developmental stages except early eggs, and mainly distributed in central nervous system and hemolymph. The transcript levels of TcCTL3 were significantly increased after lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. Recombinant TcCTL3 was able to bind directly to LPS, PGN and all tested bacteria and induce a broad spectrum of microbial agglutination in the presence of Ca2+ . The binding was shown mainly through CRD1 domain of TcCTL3. When TcCTL3 was knocked down by RNA interference, expression of nine antimicrobial peptides (AMPs) (attacin1, attacin2, attacin3, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2 and cecropins3) and four transcription factors (TFs) (dif1, dif2, relish and jnk) were significantly decreased under LPS and PGN stimulation, leading to increased mortality of T. castaneum when infected with Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli infection. CONCLUSION: TcCTL3 could mediate the immune response in T. castaneum via the pattern recognition, agglutination and AMP expression. These findings indicate a potential mechanism of TcCTL3 in resisting bacteria and provide an alternative molecular target for pest control. © 2020 Society of Chemical Industry.

Binding of PmClipSP2 to microbial cell wall components and activation of the proPO-activating system in the black tiger shrimp Penaeus monodon.

Clip domain serine proteinases (ClipSPs) play an important role in the prophenoloxidase-activating (proPO) system. In the shrimp Penaeus monodon, the ClipSP PmClipSP2 has been previously shown to bind to microbial polysaccharides (LPS and ß-1,3-glucan) and likely activates the proPO system. To reveal the binding site of the PmClipSP2 protein, the N-terminal clip domain (Clip-PmClipSP) and C-terminal SP domain (SP-PmClipSP2) were separately cloned. The recombinant proteins were then assayed for their binding properties and involvement in proPO activation. According to the ELISA-based binding assay, rSP-PmClipSP2, but not rClip-PmClipSP, can bind immobilized LPS and ß-1,3-glucan as well as significantly activate PO activity. The binding site at the SP domain is proposed to have a pattern sequence (X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X) that is located at the C-terminal region of the SP domain of PmClipSP2. Deletion of the pattern sequence abolished binding to LPS and ß-1,3-glucan. Conversely, a recombinant protein containing the pattern sequence (rPT-PmClipSP2-TRX) had the ability to bind to cell wall components, confirming that the pattern sequence at the C-terminus of PmClipSP2 is responsible for binding to microbes, subsequently leading to activation of the proPO cascade.