Immunological functional differentiation of two transmembrane variants of Dscam in Chinese mitten crab.

Down syndrome cell adhesion molecule (Dscam), also called hypervariable Dscam (Dscam-hv), is an important player in arthropod alternative splicing that connects neurons and immune regulation, acting as a pathogen-specific recognition molecule. Dscam-hv has two forms: transmembrane (TM) Dscam (mDscam) and soluble Dscam (sDscam). Herein, we investigated two transmembrane variants of mDscam resulting from alternative splicing of the transmembrane domain, focusing on differences in their immune regulation. We characterized the Dscam[TM1] and Dscam[TM2] genes of Chinese mitten crab (Eriocheir sinensis) through bioinformatics analysis. Both genes are expressed in the gill, intestine, and other immune tissues. Following gram-positive and gram-negative bacteria stimulation, EsDscam[TM1] and EsDscam[TM2] mRNA expression levels increased significantly in hemocytes. Sequencing showed that EsDscam[TM1] was more abundant in hemocytes than EsDscam[TM2]. Additionally, the two subtypes differ in their regulation of antimicrobial peptides, the proportion of exon 33 carried, and bacterial phagocytosis.

Molecular Mechanisms of Mast Cell Activation by Cholesterol-Dependent Cytolysins.

Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity.

Ex vivo tumor necrosis factor-alpha response of blood leukocytes in Danish Holstein-Friesian cows stimulated by Gram-positive and Gram-negative bacteria isolated from mastitic milk.

A whole blood stimulation assay was used to investigate the effects of parity, number of weeks after calving and Gram-positive and Gram-negative bacteria on the ex vivo TNF-α responsiveness of Danish Holstein-Friesian cows of first to third lactation (n = 28). Blood samples were collected in weeks 2, 3, 5 and 8 after parturition and stimulated with Escherichia coli LPS (10 µg/mL), Staphylococcus aureus peptidoglycan (PGN, 10 µg/mL) and dead Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Streptococcus dysgalactiae at a concentration of 2.5 × 106/mL. The antibiotic polymyxin-B (100 µg/mL) was added to the Gram-positive bacteria to avoid the influence of environmental endotoxin by ELISA test. Overall, parity had no effect, whereas number of weeks after calving altered the TNF-α responsiveness of the majority of the stimulants. Ex vivo, Gram-positive bacteria always resulted in a higher TNF-α response than Gram-negative bacteria with large differences within the individual cows. High correlations were found within the Gram-negative stimulants panel (r = 0.83) and within the Gram-positive (r = 0.81 to 0.86) stimulants panel except PGN. The higher TNF-α responsiveness by Gram-positive bacteria is in agreement with in vitro studies in human but in contrast to the in vivo TNF-α responsiveness in bovine udder.

More than a Pore: Nonlytic Antimicrobial Functions of Complement and Bacterial Strategies for Evasion.

The complement system is an evolutionarily ancient defense mechanism against foreign substances. Consisting of three proteolytic activation pathways, complement converges on a common effector cascade terminating in the formation of a lytic pore on the target surface. The classical and lectin pathways are initiated by pattern recognition molecules binding to specific ligands, while the alternative pathway is constitutively active at low levels in circulation. Complement-mediated killing is essential for defense against many Gram-negative bacterial pathogens, and genetic deficiencies in complement can render individuals highly susceptible to infection, for example, invasive meningococcal disease. In contrast, Gram-positive bacteria are inherently resistant to the direct bactericidal activity of complement due to their thick layer of cell wall peptidoglycan. However, complement also serves diverse roles in immune defense against all bacteria by flagging them for opsonization and killing by professional phagocytes, synergizing with neutrophils, modulating inflammatory responses, regulating T cell development, and cross talk with coagulation cascades. In this review, we discuss newly appreciated roles for complement beyond direct membrane lysis, incorporate nonlytic roles of complement into immunological paradigms of host-pathogen interactions, and identify bacterial strategies for complement evasion.

A novel electrochemical biosensor based on peptidoglycan and platinum-nickel-copper nano-cube for rapid detection of Gram-positive bacteria.

A novel non-enzyme electrochemical biosensor for the rapid detection of Gram-positive bacteria has been constructed that relys on a stable and efficient combination between the peptidoglycan layer and platinum-nickel-copper nanocubes (Pt-Ni-Cu NCs). Briefly, bacteria were first captured by a specific antibody. Then, the electrochemical signal materials (Pt-Ni-Cu NCs) were bound to the bacteria peptidoglycan layer using specific structural and surface features. The rapid and sensitive bacterial detection was then achieved using intrinsic electrochemical characteristics and superoxidase-like activity of the Pt-Ni-Cu NCs. Moreover, the nature of peptidoglycan covering the whole bacteria provided the premise for signal amplification. Under optimal conditions, the electrochemical signal variation was proportional to the concentration of bacteria ranging from 1.5 × 102 to 1.5 × 108 CFU/mL with a detection limit of 42 CFU/mL using a working potential of - 0.4 V. This electrochemical biosensor has been successfully applied to detect bacteria concentrations in urine samples, and the recoveries range from 90.4 to 107%. The proposed biosensor could be applied for broad-spectrum detection of Gram-positive bacteria since most Gram-positive bacteria possess a thick peptidoglycan layer. The developed electrochemical biosensing strategy might be used as a potential tool for clinical pathogenic bacteria detection and point-of-care testing (POCT).

Beyond the CRISPR-Cas safeguard: PICI-encoded innate immune systems protect bacteria from bacteriophage predation.

Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host.

Structural and Functional Analysis of PGRP-LC Indicates Exclusive Dap-Type PGN Binding in Bumblebees.

Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Bees possess fewer PGRPs than Drosophila melanogaster and Anopheles gambiae but retain two important immune pathways, the Toll pathway and the Imd pathway, which can be triggered by the recognition of Dap-type PGN by PGRP-LCx with the assistance of PGRP-LCa in Drosophila. There are three isoforms of PGRP-LC including PGRP-LCx, PGRP-LCa and PGRP-LCy in Drosophila. Our previous study showed that a single PGRP-LC exists in bumblebees. In this present study, we prove that the bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) can respond to an infection with Gram-negative bacterium Escherichia coli through binding to the Dap-type PGNs directly, and that E. coli infection induces the quick and strong upregulation of PGRP-LC, abaecin and defensin. Moreover, the Bl-PGRP-LC exhibits a very strong affinity for the Dap-type PGN, much stronger than the affinity exhibited by the PGRP-LC from the more eusocial honeybee Apis mellifera (Am-PGRP-LC). In addition, mutagenesis experiments showed that the residue His390 is the anchor residue for the binding to the Dap-type PGN and forms a hydrogen bond with MurNAc rather than meso-Dap, which interacts with the anchor residue Arg413 of PGRP-LCx in Drosophila. Therefore, bumblebee PGRP-LC possesses exclusive characteristics for the immune response among insect PGRPs.

The Genetic Basis of Natural Variation in Drosophila melanogaster Immune Defense against Enterococcus faecalis.

Dissecting the genetic basis of natural variation in disease response in hosts provides insights into the coevolutionary dynamics of host-pathogen interactions. Here, a genome-wide association study of Drosophila melanogaster survival after infection with the Gram-positive entomopathogenic bacterium Enterococcus faecalis is reported. There was considerable variation in defense against E. faecalis infection among inbred lines of the Drosophila Genetics Reference Panel. We identified single nucleotide polymorphisms associated with six genes with a significant (p < 10-08, corresponding to a false discovery rate of 2.4%) association with survival, none of which were canonical immune genes. To validate the role of these genes in immune defense, their expression was knocked-down using RNAi and survival of infected hosts was followed, which confirmed a role for the genes krishah and S6k in immune defense. We further identified a putative role for the Bomanin gene BomBc1 (also known as IM23), in E. faecalis infection response. This study adds to the growing set of association studies for infection in Drosophila melanogaster and suggests that the genetic causes of variation in immune defense differ for different pathogens.

Titration of Igs contained in an intravenous IgM-enriched preparation against selected pathogens involved in sepsis.

The goal of our work was to titer the IgG, IgM and IgA in Pentaglobin® (a preparation enriched in IgM), targeting specific surface antigens of Gram-positive and Gram-negative bacteria as well as a C. albicans strain. Lipopolysaccharides from Gram-negative bacteria, peptidoglycan and lipoteichoic acid from the other microorganisms were extracted and used in several ELISA assays in order to determine the titration of immunoglobulins in Pentaglobin® directed towards the aforementioned surface antigens. Our results showed an overall immunoglobulin titer of at least 103 in Pentaglobin® with some exceptions for the IgA titers and for some immunoglobulin titers against E. faecalis, K. pneumoniae and P. aeruginosa. According to these results, Pentaglobin® can be considered as a potential adjuvant for antimicrobial therapy.

Effectiveness of Two Types of Photostimulable Phosphor Plate Plastic Barrier Envelopes for Prevention of Microbiological Contamination

Abstract Objective: To compare the effectiveness of two types of commercially available photostimulable phosphor plate (PSP) protective barrier envelopes to prevent microbiological contamination. Material and Methods: In this cross-sectional study, 80 barrier envelopes were tested in 40 volunteers. The PSP plates were placed individually in Asia Teb and Soredex protective barrier envelopes and were placed in the mouth for two minutes, similar to periapical films. The protective barrier envelopes were then removed under sterile conditions, and the sensors were placed on different culture media. The number of colonies on each plate was counted. Data were analyzed using SPSS via McNemar and Wilcoxon tests. Results: Bacterial growth was noted in 17.5% of PSPs with Soredex, and 32.5% of PSPs with Asia Teb barrier envelopes. Gram-positive bacilli were the most commonly isolated bacteria. The difference between the Asia Teb and Soredex barrier envelopes for the protection of microbiological contamination was not significant (p>0.05). Conclusion: The use of different types of protective barrier envelopes was not sufficient for prevention of microbiological contamination of PSP plates, and some adjunct modalities were required to decrease microbiological contamination of PSP plates.

Transgenerational Immune Priming in the Field: Maternal Environmental Experience Leads to Differential Immune Transfer to Oocytes in the Marine Annelid Hediste diversicolor.

Transgenerational immune priming (TGIP) is an intriguing form of parental care which leads to the plastic adjustment of the progeny's immunity according to parental immune experience. Such parental effect has been described in several vertebrate and invertebrate taxa. However, very few empirical studies have been conducted from the field, with natural host-parasite systems and real ecological settings, especially in invertebrates. We investigated TGIP in wild populations of the marine annelid Hediste diversicolor. Females laid eggs in a mud tube and thus shared the local microbial threats with the first developmental stages, thus meeting expectations for the evolution of TGIP. We evidenced that a maternal bacterial challenge led to the higher antibacterial defense of the produced oocytes, with higher efficiency in the case of Gram-positive bacterial challenge, pointing out a prevalent role of these bacteria in the evolutionary history of TGIP in this species. Underlying mechanisms might involve the antimicrobial peptide hedistin that was detected in the cytoplasm of oocytes and whose mRNAs were selectively stored in higher quantity in mature oocytes, after a maternal immune challenge. Finally, maternal immune transfer was significantly inhibited in females living in polluted areas, suggesting associated costs and the possible trade-off with female's protection.

IFN-mediated negative feedback supports bacteria class-specific macrophage inflammatory responses.

Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection. Stimulation beyond a TLR4 threshold and Gram-negative bacteria-induced responses were characterized by a rapid type I IFN-dependent decline in inflammatory cytokine production, independent of IL-10, whereas inflammatory responses to Gram-positive species were more sustained due to the absence of this IFN-dependent regulation. Thus, disparate triggering of a cytokine negative feedback loop promotes tuning of macrophage responses in a bacteria class-specific manner and provides context-dependent regulation of inflammation dynamics.

Pediocin-Like Antimicrobial Peptides of Bacteria.

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Vaccine Approaches To Protect against Group A Streptococcal Pharyngitis.

Streptococcal pharyngitis (or strep throat) is a common childhood disease affecting millions of children each year, but it is one of the only childhood diseases for which a vaccine does not exist. While for decades the development of a vaccine has been the center of attention in many laboratories worldwide, with some successes, no corporate development has yet to be initiated. The reason for this probably lies in our inability to conclusively identify the streptococcal molecule or molecules responsible for the heart cross-reactive antibodies observed in the serum of rheumatic fever patients. Without this specific knowledge, any streptococcal vaccine antigen is suspect and thus not the target for a billion-dollar investment, despite the fact that the exact role of cross-reactive antibodies in rheumatic fever is still questionable. This article will describe the development of several approaches to protect against Streptococcus pyogenes infections over the past several decades.

Macrobrachium rosenbergii Cu/Zn superoxide dismutase (Cu/Zn SOD) expressed in Saccharomyces cerevisiae and evaluation of the immune function to Vibrio parahaemolyticus.

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms, and copper/zinc SOD (Cu/ZnSOD) is one of the most important SODs. In the present study, Macrobrachium rosenbergii Cu/Zn-SOD was expressed in a yeast eukaryotic system. The open reading frame (ORF) of MrCu/ZnSOD was cloned into the plasmid vector pHAC181, and the recombinant plasmid was integrated into the downstream region of the GAL1 promoter in Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination. The resulting recombinant MrCu/ZnSOD consisted of a 3 × HA-tag at its C-terminal. Via western blot, the molecular weight of the recombinant MrCu/ZnSOD was estimated at about 30 kDa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this recombinant MrCu/ZnSOD ranged from 0.556 to 0.840 µM, and from 0.967 to 2.015 µM, respectively. The recombinant MrCu/ZnSOD protein was able to agglutinate four Gram-negative bacterial strains, as well as two of three Gram-positive strains (except Staphylococcus aureus). This demonstrated that the recombinant protein possessed some antimicrobial activity against certain Gram-positive and Gram-negative bacteria. M. rosenbergii were fed with the recombinant yeast strain MrCu/ZnSOD for 4 weeks and then challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. This group of prawns presented lower mortality, higher enzymatic activity, and higher expression of the mRNA of immune-related genes than that in the control groups. Taken together, these results suggest that MrCu/ZnSOD is an antioxidant enzyme and antimicrobial peptide involved in the crustacean innate immune system and offers protection to the host against pathogenic bacteria.

Commensal and Pathogenic Bacteria Indirectly Induce IL-22 but Not IFNγ Production From Human Colonic ILC3s via Multiple Mechanisms.

Innate lymphoid cells (ILCs) are a diverse family of cells that play critical roles in mucosal immunity. One subset of the ILC family, Group 3 ILCs (ILC3s), has been shown to aid in gut homeostasis through the production of IL-22. IL-22 promotes gut homeostasis through its functional effect on the epithelial barrier. When gut epithelial barrier integrity is compromised, such as in Human Immunodeficiency Virus (HIV) infection and inflammatory bowel disease (IBD), microbes from the gut lumen translocate into the lamina propria, inducing a multitude of potentially pathogenic immune responses. In murine models of bacterial infection, there is evidence that bacteria can induce pro-inflammatory IFNγ production in ILC3s. However, the impact of diverse translocating bacteria, particularly commensal bacteria, in dictating IFNγ versus IL-22 production by human gut ILC3s remains unclear. Here, we utilized an in vitro human lamina propria mononuclear cell (LPMC) model to evaluate ILC3 cytokine production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria and determined potential mechanisms by which these cytokine responses were induced. The percentages of IL-22-producing ILC3s, but not IFNγ-producing ILC3s, were significantly increased after LPMC exposure to both Gram-positive and Gram-negative commensal or pathogenic bacterial stimuli. Stimulation of IL-22 production from ILC3s was not through direct recognition of bacterial antigen by ILC3s, but rather required the help of accessory cells within the LPMC population. CD11c+ myeloid dendritic cells generated IL-23 and IL-1ß in response to enteric bacteria and contributed to ILC3 production of IL-22. Furthermore, ligation of the natural cytotoxicity receptor NKp44 on ILC3s in response to bacteria stimulation also significantly increased the percentage of IL-22-producing ILC3s. Overall, these data demonstrate that human gut microbiota, including commensal bacteria, indirectly modulate colonic ILC3 function to induce IL-22, but additional signals are likely required to induce IFNγ production by colonic ILC3s in the setting of inflammation and microbial translocation.

A C-type lectin with a single carbohydrate-recognition domain involved in the innate immune response of Tribolium castaneum.

C-type lectins are one of the pattern-recognition proteins involved in innate immunity in invertebrates. Although there are 16 C-type lectin genes that have been identified in the genome of Tribolium castaneum, their functions and mechanisms in innate immunity remain unknown. Here, we identified one C-type lectin orthologue, TcCTL6 (TC003708), by sequencing random clones from the cDNA library of the coleopteran beetle, T. castaneum. TcCTL6 contains a 654 bp open reading frame encoding a protein of 217 amino acids that includes a single carbohydrate-recognition domain. The expression of TcCTL6 was significantly induced by Escherichia coli, Staphylococcus aureus and stimulation with carbohydrates, including lipopolysaccharide and peptidoglycan. A binding assay suggested that the recombinant TcCTL6 not only bound to lipopolysaccharide and peptidoglycan but also bound to Gram-positive (S. aureus, Bacillus subtilis and Bacillus thuringiensis) and Gram-negative bacteria (E. coli and Pseudomonas aeruginosa) in the presence of calcium ions. Furthermore, when TcCTL6 was knocked down by RNA interference, four antimicrobial peptides (attacin1, attacin2, coleoptericin1 and coleoptericin2) were significantly decreased. These results demonstrate that TcCTL6 plays a vital role in the immune response towards pathogen infection by influencing the expression of antimicrobial peptides and the agglutination of bacteria in the presence of calcium ions in T. castaneum.

CK11, a Teleost Chemokine with a Potent Antimicrobial Activity.

CK11 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to both mammalian CCL27 and CCL28 chemokines, strongly transcribed in skin and gills in homeostasis, for which an immune role had not been reported to date. In the current study, we have demonstrated that CK11 is not chemotactic for unstimulated leukocyte populations from central immune organs or mucosal tissues but instead exerts a potent antimicrobial activity against a wide range of rainbow trout pathogens. Our results show that CK11 strongly inhibits the growth of different rainbow trout Gram-positive and Gram-negative bacteria, namely Lactococcus garvieae, Aeromonas salmonicida subsp. salmonicida, and Yersinia ruckeri and a parasitic ciliate Ichthyophthirius multifiliis Similarly to mammalian chemokines and antimicrobial peptides, CK11 exerted its antimicrobial activity, rapidly inducing membrane permeability in the target pathogens. Further transcriptional studies confirmed the regulation of CK11 transcription in response to exposure to some of these pathogens in specific conditions. Altogether, our studies related to phylogenetic relations, tissue distribution, and biological activity point to CK11 as a potential common ancestor of mammalian CCL27 and CCL28. To our knowledge, this study constitutes the first report of a fish chemokine with antimicrobial activity, thus establishing a novel role for teleost chemokines in antimicrobial immunity that supports an evolutionary relationship between chemokines and antimicrobial peptides.

Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster.

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism.

Beyond the antibody: B cells as a target for bacterial infection.

It is well established that B cells play an important role during infections beyond antibody production. B cells produce cytokines and are APCs for T cells. Recently, it has become clear that several pathogenic bacterial genera, such as Salmonella, Brucella, Mycobacterium, Listeria, Francisella, Moraxella, and Helicobacter, have evolved mechanisms such as micropinocytosis induction, inflammasome down-regulation, inhibitory molecule expression, apoptosis induction, and anti-inflammatory cytokine secretion to manipulate B cell functions influencing immune responses. In this review, we summarize our current understanding of B cells as targets of bacterial infection and the mechanisms by which B cells become a niche for bacterial survival and replication away from extracellular immune responses such as complement and antibodies.