Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

Immunorthodontics: Role of HIF-1α in the Regulation of (Peptidoglycan-Induced) PD-L1 Expression in Cementoblasts under Compressive Force.

Patients with periodontitis undergoing orthodontic therapy may suffer from undesired dental root resorption. The purpose of this in vitro study was to investigate the molecular mechanisms resulting in PD-L1 expression of cementoblasts in response to infection with Porphyromonas gingivalis (P. gingivalis) peptidoglycan (PGN) and compressive force (CF), and its interaction with hypoxia-inducible factor (HIF)-1α molecule: The cementoblast (OCCM-30) cells were kinetically infected with various concentrations of P. gingivalis PGN in the presence and absence of CF. Western blotting and RT-qPCR were performed to examine the protein expression of PD-L1 and HIF-1α as well as their gene expression. Immunofluorescence was applied to visualize the localization of these proteins within cells. An HIF-1α inhibitor was added for further investigation of necroptosis by flow cytometry analysis. Releases of soluble GAS-6 were measured by ELISA. P. gingivalis PGN dose dependently stimulated PD-L1 upregulation in cementoblasts at protein and mRNA levels. CF combined with P. gingivalis PGN had synergistic effects on the induction of PD-L1. Blockade of HIF-1α inhibited the P. gingivalis PGN-inducible PD-L1 protein expression under compression, indicating an HIF-1α dependent regulation of PD-L1 induction. Concomitantly, an HIF-1α inhibitor decreased the GAS-6 release in the presence of CF and P. gingivalis PGN co-stimulation. The data suggest that PGN of P. gingivalis participates in PD-L1 up-regulation in cementoblasts. Additionally, the influence of compressive force on P. gingivalis PGN-induced PD-L1 expression occurs in HIF-1α dependently. In this regard, HIF-1α may play roles in the immune response of cementoblasts via immune-inhibitory PD-L1. Our results underline the importance of molecular mechanisms involved in bacteria-induced periodontics and root resorption.

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling.

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.

Resveratrol ameliorates lipid accumulation and inflammation in human SZ95 sebocytes via the AMPK signaling pathways in vitro.

BACKGROUND: Acne vulgaris is a prevalent skin disease lacking effective and well-tolerated treatment. An earlier study indicated that resveratrol (RVT) has therapeutic effects in acne patients through unknown mechanisms. OBJECTIVES: To evaluate the effects of RVT on linoleic acid (LA)-induced lipogenesis and peptidoglycan (PGN)-induced inflammation in cultured SZ95 sebocytes in vitro, and to investigate the underlying mechanisms. METHODS: RNA-sequencing was used to analyze the whole transcriptome. Nile red staining was used to detect intracellular neutral lipids, whereas lipidomics was used to investigate changes in the lipid profile in sebocytes. Interleukin (IL)-1ß and IL-6 mRNA and protein levels were assessed through quantitative real-time PCR and Enzyme-linked immunosorbent assay, respectively. Western blot was used to evaluate the expression of lipogenesis-related proteins, the inflammatory signaling pathway, and the AMP-activated protein kinase (AMPK) pathway. Further, specific small interfering RNA (siRNA) was used to knockdown sirtuin-1 (SIRT1) expression. RESULTS: RVT inhibited the lipogenesis-related pathway and nuclear factor-kappa B (NF-κB) signaling pathway in SZ95 sebocytes. It also downregulated LA-induced lipogenesis, the expression of lipid-related proteins, and the contents of unsaturated fatty acids. Besides, RVT promoted SIRT1 expression and deacetylation of the NF-κB p65 subunit, thereby lowering IL-1ß and IL-6 secretion under PGN induction. Furthermore, pretreatment with AMPK inhibitor Compound C abolished RVT-mediated sebosuppressive and anti-inflammation effects. Meanwhile, SIRT1 silencing abrogated the anti-inflammatory potential of RVT. CONCLUSION: In human SZ95 sebocytes, RVT exhibits sebosuppressive and anti-inflammatory effects partially through the AMPK pathway, which may justify the role of RVT treatment in acne vulgaris.

Differential binding of human and murine IgGs to catalytic and cell wall binding domains of Staphylococcus aureus peptidoglycan hydrolases.

Staphylococcus aureus is an opportunistic pathogen causing high morbidity and mortality. Since multi-drug resistant S. aureus lineages are nowadays omnipresent, alternative tools for preventive or therapeutic interventions, like immunotherapy, are urgently needed. However, there are currently no vaccines against S. aureus. Surface-exposed and secreted proteins are regarded as potential targets for immunization against S. aureus infections. Yet, many potential staphylococcal antigens of this category do not elicit protective immune responses. To obtain a better understanding of this problem, we compared the binding of serum IgGs from healthy human volunteers, highly S. aureus-colonized patients with the genetic blistering disease epidermolysis bullosa (EB), or immunized mice to the purified S. aureus peptidoglycan hydrolases Sle1, Aly and LytM and their different domains. The results show that the most abundant serum IgGs from humans and immunized mice target the cell wall-binding domain of Sle1, and the catalytic domains of Aly and LytM. Interestingly, in a murine infection model, these particular IgGs were not protective against S. aureus bacteremia. In contrast, relatively less abundant IgGs against the catalytic domain of Sle1 and the N-terminal domains of Aly and LytM were almost exclusively detected in sera from EB patients and healthy volunteers. These latter IgGs may contribute to the protection against staphylococcal infections, as previous studies suggest that serum IgGs protect EB patients against severe S. aureus infection. Together, these observations focus attention on the use of particular protein domains for vaccination to direct potentially protective immune responses towards the most promising epitopes within staphylococcal antigens.

Strategies for Using Muramyl Peptides - Modulators of Innate Immunity of Bacterial Origin - in Medicine.

The spread of infectious diseases is rampant. The emergence of new infections, the irrational use of antibiotics in medicine and their widespread use in agriculture contribute to the emergence of microorganisms that are resistant to antimicrobial drugs. By 2050, mortality from antibiotic-resistant strains of bacteria is projected to increase up to 10 million people per year, which will exceed mortality from cancer. Mutations in bacteria and viruses are occurring faster than new drugs and vaccines are being introduced to the market. In search of effective protection against infections, new strategies and approaches are being developed, one of which is the use of innate immunity activators in combination with etiotropic chemotherapy drugs. Muramyl peptides, which are part of peptidoglycan of cell walls of all known bacteria, regularly formed in the body during the breakdown of microflora and considered to be natural regulators of immunity. Their interaction with intracellular receptors launches a sequence of processes that ultimately leads to the increased expression of genes of MHC molecules, pro-inflammatory mediators, cytokines and their soluble and membrane-associated receptors. As a result, all subpopulations of immunocompetent cells are activated: macrophages and dendritic cells, neutrophils, T-, B- lymphocytes and natural killer cells for an adequate response to foreign or transformed antigens, manifested both in the regulation of the inflammatory response and in providing immunological tolerance. Muramyl peptides take part in the process of hematopoiesis, stimulating production of colony-stimulating factors, which is the basis for their use in the treatment of oncological diseases. In this review we highlight clinical trials of drugs based on muramyl peptides, as well as clinical efficacy of drugs mifamurtide, lycopid, liasten and polimuramil. Such a multifactorial effect of muramyl peptides and a well-known mechanism of activity make them promising drugs in the treatment and preventing of infectious, allergic and oncological diseases, and in the composition of vaccines.

Marginal Zone B Cells Assist With Neutrophil Accumulation to Fight Against Systemic Staphylococcus aureus Infection.

In addition to regulating immune responses by producing antibodies that confer humoral immunity, B cells can also affect these responses by producing cytokines. How B cells participate in the clearance of pathogenic infections via functions other than the production of pathogen-specific antibodies is still largely unknown. Marginal zone (MZ) B cells can quickly respond to bacterial invasion by providing the initial round of antibodies. After a bloodborne bacterial infection, neutrophils promptly migrate to the MZ. However, the mechanisms regulating neutrophil accumulation in the MZ during the initial phase of infection also remain obscure. Here, we found that MZ B cell-deficient mice are more susceptible to systemic Staphylococcus aureus (S. aureus) infection compared with wildtype mice. The expression levels of interleukin (IL)-6 and CXCL1/CXCL2 in MZ B cells increased significantly in mice at 3-4 h after infection with S. aureus, then decreased at 24 h post-infection. After systemic S. aureus infection, splenic neutrophils express increased CXCR2 levels. Our results from confocal microscopy imaging of thick-section staining demonstrate that neutrophils in wildtype mice form cell clusters and are in close contact with MZ B cells at 3 h post-infection. This neutrophil cluster formation shortly after infection was diminished in both MZ B cell-deficient mice and IL-6-deficient mice. Blocking the action of CXCL1/CXCL2 by injecting anti-CXCL1 and anti-CXCL2 antibodies 1 h before S. aureus infection significantly suppressed the recruitment of neutrophils to the MZ at 3 h post-infection. Compared with peptidoglycan stimulation alone, peptidoglycan stimulation with neutrophil co-culture further enhanced MZ B-cell activation and differentiation. Using a Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET) analysis, we observed evidence of a direct interaction between neutrophils and MZ B cells after peptidoglycan stimulation. Furthermore, neutrophil depletion in mice resulted in a reduced production of S. aureus-specific immunoglobulin (Ig)M at 24 h post-infection. Together, our results demonstrate that MZ B cells regulate the rapid neutrophil swarming into the spleen during the early phase of systemic S. aureus infection. Interaction with neutrophils assists MZ B cells with their differentiation into IgM-secreting cells and contributes to the clearance of systemic bacterial infections.

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A.

Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.IMPORTANCE The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.

Transient systemic inflammation in adult male mice results in underweight progeny.

PROBLEM: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes. METHOD OF STUDY: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality. RESULTS: Our findings demonstrated a significant induction of testicular TNF-α, IL-1ß and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier. CONCLUSION: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development.

Molecular cloning and binding analysis of polymeric immunoglobulin receptor in largemouth bass (Micropterus salmoides).

The polymeric immunoglobulin receptor (pIgR) is an important molecule in the mucosal immunity of teleosts. Previous studies have shown that pIgR can bind and transport polymeric immunoglobulins (pIgs), but few studies have focused on the binding of teleost pIgR to bacteria. In this study, we identified a gene encoding pIgR in largemouth bass (Micropterus salmoides). The pIgR gene contained two Ig-like domains (ILDs), which were homologous to ILD1 and ILD5 of mammalian pIgR. Our results showed that largemouth bass pIgR-ILD could combine with IgM. Moreover, we also found that largemouth bass pIgR-ILD could bind to Aeromonas hydrophila and Micrococcus luteus. Further analysis showed that largemouth bass pIgR-ILD could also combine with lipopolysaccharide (LPS), peptidoglycan (PGN) and various saccharides, and reduced binding to bacteria was observed with LPS and PGN treatment, indicating that largemouth bass pIgR could bind to bacteria to prevent infection and that saccharide binding is an important interaction mechanism between pIgR and bacteria. These results collectively demonstrated that largemouth bass pIgR not only combines with IgM but also binds to bacteria by various saccharides.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

Effect of D-Ala-Ended Peptidoglycan Precursors on the Immune Regulation of Lactobacillus plantarum Strains.

The resistance of Lactobacillus plantarum to vancomycin depends on its peptidoglycan composition. Vancomycin has poor binding affinity with peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) but binds strongly to peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala), resulting in resistance and sensitivity, respectively. The ligase Ddl, which generates D-Ala-D-Lac or D-Ala-D-Ala incorporated into the peptidoglycan precursor chain, is responsible for this specificity. To study the effect of peptidoglycan precursors on immunity, we constructed several strains of L. plantarum expressing the ddl gene of Lactococcus lactis to change their peptidoglycan precursors. The change in the termini of the peptidoglycan precursors was determined by the sensitivity of the strains to vancomycin. The overexpression of ddl increased the susceptibility of the strains to vancomycin. We further explored the regulation of the macrophage inflammatory response pathway by the wild-type and constructed strains, and found that these strains induced the MyD88-dependent TRAF6/MAPK pathway, and the increase in D-Ala L. plantarum peptidoglycan precursors increased the secretion of the inflammatory factors IL-6, IL-1ß and TNF-α. These results indicate that D-Ala-ended peptidoglycan precursors play a central role in the variable immunomodulatory ability of L. plantarum.

Uptake, recognition and responses to peptidoglycan in the mammalian host.

Microbiota, and the plethora of signalling molecules that they generate, are a major driving force that underlies a striking range of inter-individual physioanatomic and behavioural consequences for the host organism. Among the bacterial effectors, one finds peptidoglycan, the major constituent of the bacterial cell surface. In the steady-state, fragments of peptidoglycan are constitutively liberated from bacterial members of the gut microbiota, cross the gut epithelial barrier and enter the host system. The fate of these peptidoglycan fragments, and the outcome for the host, depends on the molecular nature of the peptidoglycan, as well the cellular profile of the recipient tissue, mechanism of cell entry, the expression of specific processing and recognition mechanisms by the cell, and the local immune context. At the target level, physiological processes modulated by peptidoglycan are extremely diverse, ranging from immune activation to small molecule metabolism, autophagy and apoptosis. In this review, we bring together a fragmented body of literature on the kinetics and dynamics of peptidoglycan interactions with the mammalian host, explaining how peptidoglycan functions as a signalling molecule in the host under physiological conditions, how it disseminates within the host, and the cellular responses to peptidoglycan.

Immune modulating effects of receptor interacting protein 2 (RIP2) in autoinflammation and immunity.

Receptor-interacting protein 2 (RIP2) is a kinase that is involved in downstream signaling of nuclear oligomerization domain (NOD)-like receptors NOD1 and 2 sensing bacterial peptidoglycans. RIP2-deficiency or targeting of RIP2 by pharmaceutical inhibitors partially ameliorates inflammatory diseases by reducing pro-inflammatory signaling in response to peptidoglycans. However, RIP2 is widely expressed and interacts with several other proteins suggesting additional functions outside the NOD-signaling pathway. In this review, we discuss the immunological functions of RIP2 and its possible role in autoinflammation and immunity.

Identification of an IRF10 gene in common carp (Cyprinus carpio L.) and analysis of its function in the antiviral and antibacterial immune response.

BACKGROUND: Interferon (IFN) regulatory factors (IRFs), as transcriptional regulatory factors, play important roles in regulating the expression of type I IFN and IFN- stimulated genes (ISGs) in innate immune responses. In addition, they participate in cell growth and development and regulate oncogenesis. RESULTS: In the present study, the cDNA sequence of IRF10 in common carp (Cyprinus carpio L.) was characterized (abbreviation, CcIRF10). The predicted protein sequence of CcIRF10 shared 52.7-89.2% identity with other teleost IRF10s and contained a DNA-binding domain (DBD), a nuclear localization signal (NLS) and an IRF-associated domain (IAD). Phylogenetic analysis showed that CcIRF10 had the closest relationship with IRF10 of Ctenopharyngodon idella. CcIRF10 transcripts were detectable in all examined tissues, with the highest expression in the gonad and the lowest expression in the head kidney. CcIRF10 expression was upregulated in the spleen, head kidney, foregut and hindgut upon polyinosinic:polycytidylic acid (poly I:C) and Aeromonas hydrophila stimulation and induced by poly I:C, lipopolysaccharide (LPS) and peptidoglycan (PGN) in peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs) of C. carpio. In addition, overexpression of CcIRF10 was able to decrease the expression of the IFN and IFN-stimulated genes PKR and ISG15. CONCLUSIONS: These results indicate that CcIRF10 participates in antiviral and antibacterial immunity and negatively regulates the IFN response, which provides new insights into the IFN system of C. carpio.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Molecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation.

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Activity of mammalian peptidoglycan-targeting immunity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most important opportunistic pathogens, whose clinical relevance is not only due to the high morbidity/mortality of the infections caused, but also to its striking capacity for antibiotic resistance development. In the current scenario of a shortage of effective antipseudomonal drugs, it is essential to have thorough knowledge of the pathogen's biology from all sides, so as to find weak points for drug development. Obviously, one of these points could be the peptidoglycan, given its essential role for cell viability. Meanwhile, immune weapons targeting this structure could constitute an excellent model to be taken advantage of in order to design new therapeutic strategies. In this context, this review gathers all the information regarding the activity of mammalian peptidoglycan-targeting innate immunity (namely lysozyme and peptidoglycan recognition proteins), specifically against P. aeruginosa. All the published studies were considered, from both in vitro and in vivo fields, including works that envisage these weapons as options not only to potentiate their innate effects within the host or for use as exogenously administered treatments, but also harnessing their inflammatory and immune regulatory capacity to finally reduce damage in the patient. Altogether, this review has the objective of anticipating and discussing whether these innate immune resources, in combination or not with other drugs attacking certain P. aeruginosa targets leading to its increased sensitization, could be valid therapeutic antipseudomonal allies.

Recombinant PAL/PilE/FlaA DNA vaccine provides protective immunity against Legionella pneumophila in BALB/c mice.

BACKGROUND: Legionella pneumophila (L.pneumophila), a Gram-negative small microorganism, causes hospital-acquired pneumonia especially in immunocompromised patients. Vaccination may be an effective method for preventing L.pneumophila infection. Therefore, it is necessary to develop a better vaccine against this disease. In this study, we developed a recombinant peptidoglycan-associated lipoprotein (PAL)/type IV pilin (PilE)/lagellin (FlaA) DNA vaccine and evaluated its immunogenicity and efficacy to protect against L.pneumophila infection. RESULTS: According to the results, the expression of PAL, PilE, FlaA proteins and PAL/PilE/FlaA fusion protein in 293 cells was confirmed. Immunization with PAL/PilE/FlaA DNA vaccine resulted in highest IgG titer and strongest cytotoxic T-lymphocyte (CTL) response. Furthermore, the histopathological changes in lung tissues of mice challenged with a lethal dose of L.pneumophila were alleviated by PAL/PilE/FlaA DNA vaccine immunization. The production of T-helper-1 (Th1) cytokines (IFNγ, TGF-α, and IL-12), and Th2 cytokines (IL-4 and IL-10) were promoted in PAL/PilE/FlaA DNA vaccine group. Finally, immunization with PAL/PilE/FlaA vaccine raised the survival rate of mice to 100% after challenging with a lethal dose of L.pneumophila for 10 consecutive days. CONCLUSIONS: Our study suggests that the newly developed PAL/PilE/FlaA DNA vaccine stimulates strong humoral and cellular immune responses and may be a potential intervention on L.pneumophila infection.

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.