ABSTRACT
The transition time during which a virus leaves its host and infects the next susceptible host is critical for virus survival. Enterovirus 71 (EV71) is stable in aqueous environments, but its molecular interactions with bacteria and their biofilms are not well-established. Helicobacter pylori is a highly successful gut bacterial pathogen, with its capacity to form biofilms being linked to its transmission. Given that both are gut-associated microbes, we hypothesized that biofilms formed by H. pylori may play a significant role in the survival of EV71 in the external environment. In this study, we examine the interactions of EV71 with the preformed biofilm of H. pylori to mimic its natural state in the environment. Immunofluorescence confocal microscopy and scanning electron microscopy revealed that EV71 particles persisted for up to 10 days when incubated with the H. pylori biofilm. Furthermore, the presence of the H. pylori biofilm significantly augmented viral viability, as verified through virus plaque assays. Interestingly, the viability of EV71 was dependent on the quantity of H. pylori biofilm formation. Thus, two H. pylori strains able to generate large amounts of biofilm could facilitate EV71 viability for up to 17 days, whereas two other H. pylori strains that produced moderate or low quantities of biofilm could not prolong virus viability. It is interesting that biofilm contains N-acetyl-glucosamine and glycosaminoglycan, and that EV71 has binding affinity to cell-surface heparan sulfate glycosaminoglycan, which acts as an EV71 attachment receptor. The synergistic ability of H. pylori biofilm to promote EV71 viability for extended periods implies that H. pylori biofilm may serve as an additional pathway of EV71 transmission.
Subject(s)
Enterovirus A, Human , Helicobacter pylori , Microbial Viability , Biofilms , GlycosaminoglycansABSTRACT
OBJECTIVE: To investigate the incidence of gastric cancer (GC) attributed to gastric intestinal metaplasia (IM), and validate the Operative Link on Gastric Intestinal Metaplasia (OLGIM) for targeted endoscopic surveillance in regions with low-intermediate incidence of GC. METHODS: A prospective, longitudinal and multicentre study was carried out in Singapore. The study participants comprised 2980 patients undergoing screening gastroscopy with standardised gastric mucosal sampling, from January 2004 and December 2010, with scheduled surveillance endoscopies at year 3 and 5. Participants were also matched against the National Registry of Diseases Office for missed diagnoses of early gastric neoplasia (EGN). RESULTS: There were 21 participants diagnosed with EGN. IM was a significant risk factor for EGN (adjusted-HR 5.36; 95% CI 1.51 to 19.0; p<0.01). The age-adjusted EGN incidence rates for patients with and without IM were 133.9 and 12.5 per 100 000 person-years. Participants with OLGIM stages III-IV were at greatest risk (adjusted-HR 20.7; 95% CI 5.04 to 85.6; p<0.01). More than half of the EGNs (n=4/7) attributed to baseline OLGIM III-IV developed within 2 years (range: 12.7-44.8 months). Serum trefoil factor 3 distinguishes (Area Under the Receiver Operating Characteristics 0.749) patients with OLGIM III-IV if they are negative for H. pylori. Participants with OLGIM II were also at significant risk of EGN (adjusted-HR 7.34; 95% CI 1.60 to 33.7; p=0.02). A significant smoking history further increases the risk of EGN among patients with OLGIM stages II-IV. CONCLUSIONS: We suggest a risk-stratified approach and recommend that high-risk patients (OLGIM III-IV) have endoscopic surveillance in 2 years, intermediate-risk patients (OLGIM II) in 5 years.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Precancerous Conditions , Stomach Neoplasms , Gastroscopy , Helicobacter Infections/complications , Helicobacter Infections/epidemiology , Humans , Metaplasia , Precancerous Conditions/epidemiology , Prospective Studies , Risk Factors , Stomach Neoplasms/diagnosis , Stomach Neoplasms/epidemiology , Stomach Neoplasms/etiologyABSTRACT
Outer inflammatory protein A (OipA) is an important virulence factor associated with gastric cancer and ulcer development; however, the results have not been well established and turned out to be controversial. This study aims to elucidate the role of OipA in Helicobacter pylori infection using clinical strains harbouring oipA "on" and "off" motifs. Proteomics analysis was performed on AGS cell pre-infection and postinfection with H. pylori oipA "on" and "off" strains, using liquid chromatography/mass spectrometry. AGS apoptosis and cell cycle assays were performed. Moreover, expression of vacuolating cytotoxin A (VacA) was screened using Western blotting. AGS proteins that have been suggested previously to play a role or associated with gastric disease were down-regulated postinfection with oipA "off" strains comparing to oipA "on" strains. Furthermore, oipA "off" and ΔoipA cause higher level of AGS cells apoptosis and G0/G1 cell-cycle arrest than oipA "on" strains. Interestingly, deletion of oipA increased bacterial VacA production. The capability of H. pylori to induce apoptosis and suppress expression of proteins having roles in human disease in the absence of oipA suggests that strains not expressing OipA may be less virulent or may even be protective against carcinogenesis compared those expressing OipA. This potentially explains the higher incidence of gastric cancer in East Asia where oipA "on" strains predominates.
Subject(s)
Apoptosis/drug effects , Bacterial Outer Membrane Proteins/metabolism , Epithelial Cells/microbiology , Epithelial Cells/physiology , Helicobacter pylori/metabolism , Bacterial Outer Membrane Proteins/genetics , Blotting, Western , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Chromatography, Liquid , Gene Deletion , Helicobacter pylori/chemistry , Humans , Mass Spectrometry , Proteome/analysis , Virulence Factors/analysisABSTRACT
For nearly five decades since its discovery, the role of natural IgG, which pre-exists in neonates and uninfected individuals, has remained unclear due to the general perception that natural antibodies lack affinity for pathogens. Here, we show for the first time that natural IgG recognizes a spectrum of bacteria through lectins like ficolin and mannose binding lectin (MBL). Infection-inflammation condition markedly increased the affinity of natural IgG for bacteria associated with ficolins. After opsonization with IgG:ficolin complex, the bacteria were phagocytosed by monocytes via FcγRI. Infection of C3(-/-) mice indicated that the natural IgG-mediated immune complex was formed independently of C3. AID(-/-) mice lacking IgG were susceptible to infection, unless reconstituted with natural IgG. Thus, we have proven that natural IgG is not quiescent; rather, it plays a vital and immediate role in immune defense. Our findings provide a fresh perspective on natural antibodies, opening new avenues to explore host-microbe interaction.
Subject(s)
Autoantibodies/immunology , Bacteria/isolation & purification , Immunity, Innate , Immunoglobulin G/immunology , Lectins/immunology , Opsins/immunology , Animals , Bacterial Infections/immunology , Complement C3/genetics , Mice , Mice, Knockout , FicolinsABSTRACT
To date, the exact route and mode of transmission of Helicobacter pylori remains elusive. The detection of H. pylori in food using molecular approaches has led us to postulate that the gastric pathogen may survive in the extragastric environment for an extended period. In this study, we show that H. pylori prolongs its survival by forming biofilm and micro-colonies on vegetables. The biofilm forming capability of H. pylori is both strain and vegetable dependent. H. pylori strains were classified into high and low biofilm formers based on their highest relative biofilm units (BU). High biofilm formers survived longer on vegetables compared to low biofilm formers. The bacteria survived better on cabbage compared to other vegetables tested. In addition, images captured on scanning electron and confocal laser scanning microscopes revealed that the bacteria were able to form biofilm and reside as micro-colonies on vegetable surfaces, strengthening the notion of possible survival of H. pylori on vegetables for an extended period of time. Taken together, the ability of H. pylori to form biofilm on vegetables (a common food source for human) potentially plays an important role in its survival, serving as a mode of transmission of H. pylori in the extragastric environment.
Subject(s)
Biofilms/growth & development , Food Microbiology , Helicobacter pylori/physiology , Vegetables/microbiology , Bacterial Adhesion/physiology , Brassica/microbiology , Humans , Microbial Viability , Plant Leaves/microbiology , Plant Stomata/microbiologyABSTRACT
Prevention of infection and enhanced osseointegration are closely related, and required for a successful orthopaedic implant, which necessitate implant designs to consider both criteria in tandem. A multi-material coating containing 1:1 ratio of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite as the top functional layer, and hydroxyapatite as the base layer, was produced via the drop-on-demand micro-dispensing technique, as a strategic approach in the fight against infection along with the promotion of bone tissue regeneration. The homogeneous distribution of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets at alternate position in silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating delayed the exponential growth of Staphylococcus aureus for up to 24 h, and gave rise to up-regulated expression of alkaline phosphatase activity, type I collagen and osteocalcin as compared to hydroxyapatite and silver-substituted hydroxyapatite coatings. Despite containing reduced amounts of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets over the coated area than silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite coatings, silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating exhibited effective antibacterial property with enhanced bioactivity. By exhibiting good controllability of distributing silicon-substituted hydroxyapatite, silver-substituted hydroxyapatite and hydroxyapatite micro-droplets, it was demonstrated that drop-on-demand micro-dispensing technique was capable in harnessing the advantages of silver-substituted hydroxyapatite, silicon-substituted hydroxyapatite and hydroxyapatite to produce a multi-material coating along with enhanced bioactivity and reduced infection.
Subject(s)
Apatites/chemistry , Coated Materials, Biocompatible/pharmacology , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects , Adipocytes/cytology , Alkaline Phosphatase/metabolism , Anti-Bacterial Agents/pharmacology , Bone Regeneration , Cell Proliferation , Collagen/chemistry , Humans , Hydroxyapatites/chemistry , Metal Nanoparticles/chemistry , Microbial Sensitivity Tests , Microscopy, Confocal , Osseointegration/drug effects , Osteocalcin/chemistry , Powders , Silicon/chemistry , Silver/chemistry , Surface PropertiesABSTRACT
Galectin-3 (Gal-3) is a ß-galactoside lectin that is upregulated and rapidly secreted by gastric epithelial cells in response to Helicobacter pylori infection. An earlier study reported the involvement of H. pylori cytotoxin-associated gene A (cagA) in the expression of intracellular Gal-3. However, the role of extracellular Gal-3 and its functional significance in H. pylori-infected cells remains uncharacterized. Data presented here demonstrate secretion of Gal-3 is an initial host response event in gastric epithelial cells during H. pylori infection and is independent of CagA. Previously, Gal-3 was shown to bind to H. pylori LPS. The present study elaborates the significance of this binding, as extracellular recombinant Gal-3 (rGal-3) was shown to inhibit the adhesion of H. pylori to the gastric epithelial cells. Interestingly, a decrease in H. pylori adhesion to host cells also resulted in a decrease in apoptosis. Furthermore, the study also demonstrated a chemoattractant role of extracellular rGal-3 in the recruitment of THP-1 monocytes. This study outlines the previously unidentified roles of extracellular Gal-3 where it acts as a negative regulator of H. pylori adhesion and apoptosis in gastric epithelial cells, and as a chemoattractant to THP-1 monocytes. Our findings could contribute to the better understanding of how Gal-3 acts as a modulator under H. pylori-induced pathological conditions.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Adhesion/physiology , Bacterial Proteins/metabolism , Chemotaxis/physiology , Epithelial Cells/microbiology , Galectin 3/metabolism , Helicobacter pylori/metabolism , Antigens, Bacterial/genetics , Apoptosis/physiology , Bacterial Proteins/genetics , Cell Line, Tumor , Helicobacter Infections/pathology , Humans , Stomach Neoplasms/pathologyABSTRACT
Galectin 3 (Gal-3) is upregulated in gastric epithelial cells as a host response to Helicobacter pylori infection. However, the significance of Gal-3 expression in H. pylori-infected cells is not well established. We analyzed Gal-3 intracellular expression, localization, and its effects in H. pylori-infected gastric epithelial cells. The predominantly nuclear confined Gal-3 was shown to be upregulated and exported out to the cytoplasm in H. pylori-infected AGS cells. The nuclear export was channeled through CRM-1 (exportin-1) protein. Interestingly, knock down of Gal-3 expression led to reduced NF-κB promoter activity and interleukin-8 (IL-8) secretion, suggesting its pro-inflammatory roles. Furthermore, Gal-3 was found to be pro-proliferative and anti-apoptotic in nature, as its knock down caused a reduction in cell proliferation and an increase in apoptosis, respectively. Taken together, our data suggest the expression and upregulation of Gal-3 as a critical endogenous event in H. pylori infection that interferes with various intracellular events, causing prolonged cell survival, which is characteristic in carcinogenesis.
Subject(s)
Galectin 3/biosynthesis , Helicobacter Infections/metabolism , Helicobacter pylori/metabolism , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/physiology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Helicobacter Infections/pathology , Humans , Interleukin-8/metabolism , Karyopherins/metabolism , NF-kappa B/metabolism , Protein Transport , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Stomach Neoplasms/pathology , Up-Regulation , Exportin 1 ProteinABSTRACT
Helicobacter pylori is a major aetiological agent in the development of various gastroduodenal diseases. Its persistence in gastric mucosa is determined by the interaction between various host, microbial and environmental factors. The bacterium colonizes the gastric epithelium and induces activation of various chemokine mediators, including NFκB, the master regulator of inflammation. H. pylori infection is also associated with an increase in expression of cell cycle regulators, thereby leading to mucosal cell hyper-proliferation. Thus, H. pylori-associated infections manifest activation of key host response events, which inadvertently could lead to the establishment of chronic infection and neoplastic progression. This article reviews and elaborates the current knowledge in H. pylori-induced activation of various host signalling pathways that could promote cancer development. Special focus is placed on the inflammatory and proliferative responses that could serve as suitable biomarkers of infection, since a sustained cell proliferation in an environment rich in inflammatory cells is characteristic in H. pylori-associated gastric malignancies. Here, the role of ERK and WNT signalling in H. pylori-induced activation of inflammatory and proliferative responses respectively is discussed in detail. An in depth analysis of the underlying signalling pathways and interacting partners causing alterations in these crucial host responses could contribute to the development of successful therapeutic strategies for the prevention, management and treatment of H. pylori infection.
Subject(s)
Helicobacter pylori/physiology , Host-Pathogen Interactions , Inflammation/microbiology , MAP Kinase Signaling System , Stomach Neoplasms/microbiology , Wnt Signaling Pathway , HumansABSTRACT
A 26-member library of novel N-hydroxyquinolinone derivatives was synthesized by a one-pot Buchwald-type palladium catalyzed amidation and condensation sequence. The design of these rare scaffolds was inspired from N-hydroxypyridones and 2-quinolinones classes of compounds which have been shown to have rich biological activities. The synthesized compounds were evaluated for their anti-plasmodial and anti-bacterial properties. In addition, these compounds were screened for their iron(II)-chelation properties. Notably, four of these compounds exhibited anti-plasmodial activities comparable to that of the natural product cordypyridone B.
Subject(s)
Amides/chemistry , Anti-Bacterial Agents/chemical synthesis , Antimalarials/chemical synthesis , Chelating Agents/chemical synthesis , Ferrous Compounds/chemistry , Quinolones/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antimalarials/chemistry , Antimalarials/pharmacology , Catalysis , Chelating Agents/chemistry , Chelating Agents/pharmacology , Cyclization , Escherichia coli/drug effects , Palladium/chemistry , Plasmodium/drug effects , Quinolones/chemical synthesis , Quinolones/pharmacology , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
Lysis of RBCs during numerous clinical settings such as severe hemolytic anemia, infection, tissue injury, or blood transfusion releases the endogenous damage-associated molecular pattern, hemoglobin (Hb), into the plasma. The redox-reactive Hb generates cytotoxic reactive oxygen species, disrupting the redox balance and impairing the immune-responsive blood cells. Therefore, it is crucial to understand how the immune system defends against the cytotoxic Hb. We identified a shortcut "capture and quench" mechanism of detoxification of Hb by the monocyte scavenger receptor CD163, independent of the well-known dominant antioxidant, haptoglobin. Our findings support a highly efficient two-pass mechanism of detoxification and clearance of Hb: 1) a direct suppression of Hb-pseudoperoxidase activity by CD163, involving an autocrine loop of CD163 shedding, sequestration of Hb, recycling, and homeostasis of CD163 in human monocytes and 2) paracrine transactivation of endothelial cells by the shedded soluble CD163 (sCD163), which further detoxifies and clears residual Hb. We showed that sCD163 and IgG interact with free Hb in the plasma and subsequently the sCD163-Hb-IgG complex is endocytosed into monocytes via FcγR. The endocytosed sCD163 is recycled to restore the homeostasis of CD163 on the monocyte membrane in an autocrine cycle, whereas the internalized Hb is catabolized. Using ex vivo coculture experiments, we demonstrated that the monocyte-derived sCD163 and IgG shuttle residual plasma Hb into the proximal endothelial cells. These findings suggest that CD163 and IgG collaborate to engage monocytes and endothelial cells in a two-pass detoxification mechanism to mount a systemic defense against Hb-induced oxidative stress.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Hemoglobins/metabolism , Hemolysis , Immunoglobulin G/metabolism , Oxidative Stress/immunology , Receptors, Cell Surface/metabolism , Anemia, Hemolytic , Apoptosis , Cell Line , Cell Survival , Endothelial Cells/metabolism , HEK293 Cells , Haptoglobins , Humans , Interleukin-10/analysis , Interleukin-8/analysis , Membrane Proteins/metabolism , Monocytes/metabolism , Oxidation-Reduction , RNA Interference , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Receptors, IgG/genetics , Receptors, IgG/metabolism , Tumor Necrosis Factor-alpha/analysisABSTRACT
The intrinsic cytotoxicity of cell-free haemoglobin (Hb) has hampered the development of reliable Hb-based blood substitutes for over seven decades. Notably, recent evidence shows that the Hb deploys this cytotoxic attack against invading microbes, albeit, through an unknown mechanism. Here, we unraveled a rapid molecular reprogramming of the Hb structure-function triggered by virulent haemolytic pathogens that feed on the haem-iron. On direct contact with the microbe, the Hb unveils its latent antimicrobial potency, where multiple antimicrobial fragments are released, each harbouring coordinated 'dual-action centres': microbe binding and pseudoperoxidase (POX) cycle activity. The activated Hb fragments anchor onto the microbe while the juxtaposed POX instantly unleashes a localized oxidative shock, killing the pathogen-in-proximity. This concurrent action conceivably restricts the diffusion of free radicals. Furthermore, the host astutely protects itself from self-cytotoxicity by simultaneously releasing endogenous antioxidants. We found that this decryption mechanism of antimicrobial potency is conserved in the ancient invertebrate respiratory protein, indicating its fundamental significance. Our definition of dual-antimicrobial centres in the Hb provides vital clues for designing a safer Hb-based oxygen carrier blood substitute.
Subject(s)
Anti-Infective Agents/pharmacology , Hemoglobins/pharmacology , Adaptation, Biological/physiology , Animals , Anti-Infective Agents/chemistry , Blood Substitutes/pharmacology , Hemoglobins/chemistry , Hemoglobins/physiology , Horseshoe Crabs , Humans , Microbial Sensitivity Tests , Models, Biological , Models, Molecular , Peroxidase/metabolism , Rabbits , Structure-Activity RelationshipABSTRACT
Although specific single Toll-like receptor (TLR) ligands are known to drive the development of Th1 or Th2 immunity, the outcome of different combinations of TLR ligands on innate immunity is not well defined. Spatiotemporal dynamics are critical in determining the specificity of the immune response, but the mechanisms underlying combinatorial TLR stimulation remain unclear. Here, we tested pairwise combinations of TLR ligands separated by different time intervals for their effect on cytokine production in macrophages. We observed that stimulation via a combination of MyD88- and TRIF-utilizing adaptors leads to a highly synergistic cytokine response. On a timescale of 4-24 h, macrophages pretreated with poly(I:C) (TLR3 ligand) are cross-primed to a second stimulation with R848 (TLR7 ligand) and vice versa, and each condition exhibits different optimal time windows of synergistic response for each cytokine. We show that the synergy resulting from combinatorial stimuli (poly(I:C) and R848 is also regulated by the order and dosage of the TLR agonists. Secondary response genes, which depend on new protein synthesis for transcription, show greater synergy than primary response genes, and such enhancement is abolished when new protein synthesis is inhibited. Synergistic cytokine production appears concordant with sustained ERK phosphorylation, suggesting that the de novo factors act via inhibition of ERK dephosphorylation, for example, by the downregulation of dual specificity phosphatase 6. Taken together, our findings illustrate a checkpoint in the innate immune system, where the synchronization of timing of both MyD88 and TRIF pathways is required for a maximal cytokine response and potential memory effect in macrophages.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Macrophages/immunology , Myeloid Differentiation Factor 88/metabolism , Animals , Cell Line , Cytokines/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Imidazoles/pharmacology , Immunity, Innate , Immunologic Memory , Membrane Glycoproteins/agonists , Mice , Mice, Inbred BALB C , Phosphorylation , Poly I-C/pharmacology , Receptor Cross-Talk , Signal Transduction , Toll-Like Receptor 3/agonists , Toll-Like Receptor 7/agonistsABSTRACT
The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM.
Subject(s)
Armadillo Domain Proteins/chemistry , Armadillo Domain Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Mitochondria/metabolism , Toll-Like Receptors/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Apoptosis/physiology , Armadillo Domain Proteins/genetics , Biological Transport, Active , Cytoskeletal Proteins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Mice , Microscopy, Immunoelectron , Mitochondria/ultrastructure , Molecular Sequence Data , Mutagenesis, Site-Directed , NIH 3T3 Cells , Protein Sorting Signals , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and ß subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbß attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.
Subject(s)
Hemoglobins/chemistry , Lipopolysaccharides/chemistry , Models, Molecular , Binding Sites , Hemoglobins/immunology , Hemoglobins/metabolism , Humans , Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Protein BindingABSTRACT
The complement system is key to innate immunity and its activation is necessary for the clearance of bacteria and apoptotic cells. However, insufficient or excessive complement activation will lead to immune-related diseases. It is so far unknown how the complement activity is up- or down- regulated and what the associated pathophysiological mechanisms are. To quantitatively understand the modulatory mechanisms of the complement system, we built a computational model involving the enhancement and suppression mechanisms that regulate complement activity. Our model consists of a large system of Ordinary Differential Equations (ODEs) accompanied by a dynamic Bayesian network as a probabilistic approximation of the ODE dynamics. Applying Bayesian inference techniques, this approximation was used to perform parameter estimation and sensitivity analysis. Our combined computational and experimental study showed that the antimicrobial response is sensitive to changes in pH and calcium levels, which determines the strength of the crosstalk between CRP and L-ficolin. Our study also revealed differential regulatory effects of C4BP. While C4BP delays but does not decrease the classical complement activation, it attenuates but does not significantly delay the lectin pathway activation. We also found that the major inhibitory role of C4BP is to facilitate the decay of C3 convertase. In summary, the present work elucidates the regulatory mechanisms of the complement system and demonstrates how the bio-pathway machinery maintains the balance between activation and inhibition. The insights we have gained could contribute to the development of therapies targeting the complement system.
Subject(s)
Complement System Proteins/physiology , Bayes Theorem , Models, TheoreticalABSTRACT
Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.
Subject(s)
C-Reactive Protein/metabolism , Immunity, Innate , Lectins/metabolism , Membrane Proteins/metabolism , Monocytes/immunology , Receptor Cross-Talk/immunology , Receptors, Cell Surface/metabolism , Signal Transduction/immunology , Acidosis/blood , Acidosis/metabolism , Animals , C-Reactive Protein/physiology , COS Cells , Cell Line , Chlorocebus aethiops , Escherichia coli Infections/blood , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Humans , Interleukin-8/antagonists & inhibitors , Interleukin-8/biosynthesis , Interleukin-8/metabolism , Lectins/deficiency , Macromolecular Substances/blood , Macromolecular Substances/metabolism , Membrane Proteins/blood , Membrane Proteins/chemistry , Monocytes/chemistry , Monocytes/metabolism , Pseudomonas Infections/blood , Pseudomonas Infections/immunology , Pseudomonas Infections/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/physiology , Salmonella Infections/blood , Salmonella Infections/immunology , Salmonella Infections/microbiology , Staphylococcal Infections/blood , Staphylococcal Infections/immunology , Staphylococcal Infections/microbiology , U937 Cells , Up-Regulation/immunology , FicolinsABSTRACT
Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human.
Subject(s)
Calcium-Binding Proteins/chemistry , Membrane Proteins/chemistry , Monosaccharide Transport Proteins/chemistry , Periplasmic Binding Proteins/chemistry , Amino Acid Sequence , Animals , Biological Evolution , Conserved Sequence , Horseshoe Crabs , Host-Pathogen Interactions , Ligands , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Pseudomonas aeruginosa/metabolism , Sequence Homology, Amino Acid , Surface Plasmon Resonance , Two-Hybrid System TechniquesABSTRACT
BACKGROUND & AIMS: gamma-Glutamyl transpeptidase (GGT) has been reported to be a virulence factor of Helicobacter pylori associated with bacterial colonization and cell apoptosis. But its mechanism of pathogenesis is not firmly established. This study aims to examine its role in H pylori-mediated infection. METHODS: Various H pylori isogenic mutants were constructed by a polymerase chain reaction (PCR) approach. H pylori native GGT protein (HP-nGGT) was purified with ion-exchange and gel-filtration chromatography. Generation of H2O2 was measured with fluorimetric analysis, whereas nuclear factor-kappaB (NF-kappaB) activation was determined by luciferase assay and Western blot. Cytokine production was examined by enzyme-linked immunoabsorbent assay and real-time PCR. DNA damage was assessed with comet assay and flow cytometry. The GGT activity of 98 H pylori isolates was analyzed by an enzymatic assay. RESULTS: Purified HP-nGGT generated H2O2 in primary gastric epithelial cells and AGS gastric cancer cells, resulting in the activation of NF-kappaB and up-regulation of interleukin-8 (IL-8) production. In addition, HP-nGGT caused an increase in the level of 8-OH-dG, indicative of oxidative DNA damage. In contrast, Deltaggt showed significantly reduced levels of H2O2 generation, IL-8 production, and DNA damage in cells compared with the wild type (P<.05). The clinical importance of GGT was indicated by significantly higher (P<.001) activity in H pylori isolates obtained from patients with peptic ulcer disease (n=54) than isolates from patients with nonulcer dyspepsia (n=44). CONCLUSION: Our findings provide evidence that GGT is a pathogenic factor associated with H pylori-induced peptic ulcer disease.
Subject(s)
Bacterial Proteins/metabolism , Gastric Mucosa/microbiology , Helicobacter Infections/microbiology , Helicobacter pylori/pathogenicity , Stomach Ulcer/microbiology , Virulence Factors/metabolism , gamma-Glutamyltransferase/metabolism , Apoptosis , Bacterial Proteins/genetics , Biopsy , Blotting, Western , Cells, Cultured , Chromatography, Gel , Chromatography, Ion Exchange , Comet Assay , DNA Damage , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorometry , Gastric Mucosa/immunology , Gastric Mucosa/pathology , Helicobacter Infections/complications , Helicobacter Infections/pathology , Helicobacter pylori/enzymology , Helicobacter pylori/genetics , Humans , Hydrogen Peroxide/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Mutation , NF-kappa B/metabolism , Oxidative Stress , Reverse Transcriptase Polymerase Chain Reaction , Stomach Ulcer/immunology , Stomach Ulcer/pathology , Time Factors , Virulence Factors/genetics , gamma-Glutamyltransferase/geneticsABSTRACT
SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.