cyclic-gmp and Staphylococcal-Infections

Topics: Chemotactic Factors; Chemotaxis, Leukocyte; Cyclic AMP; Cyclic GMP; Dermatitis Herpetiformis; Erythema; Histamine Release; Humans; Immunoglobulin E; Muramidase; Neutrophils; Skin; Skin Diseases; Staphylococcal Infections

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Adhesion; Cell Movement; Cells, Cultured; Chemokine CXCL2; Cyclic GMP; Diclofenac; Dietary Supplements; Endothelial Cells; Gene Expression; Humans; Inflammation; Intercellular Adhesion Molecule-1; Intestine, Small; Leukocyte Count; Male; Mice; Mice, Inbred C57BL; Microvessels; Mouthwashes; Neutrophil Infiltration; Nitrates; Nitrites; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Staphylococcal Infections; Staphylococcus aureus

Cyclic diguanylate (c-di-GMP) is a novel immunomodulator and immune enhancer that triggers a protective host innate immune response. The protective effect of c-di-GMP as a vaccine adjuvant against Staphylococcus aureus infection was investigated by subcutaneous (s.c.) vaccination with two different S. aureus antigens, clumping factor A (ClfA) and a nontoxic mutant staphylococcal enterotoxin C (mSEC), then intravenous (i.v.) challenge with viable methicillin-resistant S. aureus (MRSA) in a systemic infection model. Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA vaccines then challenged with MRSA produced strong antigen-specific antibody responses demonstrating immunogenicity of the vaccines. Bacterial counts in the spleen and liver of c-di-GMP plus mSEC and c-di-GMP plus ClfA-immunized mice were significantly lower than those of control mice (P<0.001). Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA showed significantly higher survival rates at day 7 (87.5%) than those of the non-immunized control mice (33.3%) (P<0.05). Furthermore, immunization of mice with c-di-GMP plus mSEC or c-di-GMP plus ClfA induced not only very high titers of immunoglobulin G1 (IgG1), but c-di-GMP plus mSEC also induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.01 and P<0.001, respectively) and c-di-GMP plus ClfA induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.001). Our results show that c-di-GMP should be developed as an adjuvant and immunotherapeutic to provide protection against systemic infection caused by S. aureus (MRSA).

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Bacterial; Antigens, Bacterial; Coagulase; Colony Count, Microbial; Cyclic GMP; Enterotoxins; Female; Immunoglobulin G; Injections, Subcutaneous; Liver; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred C57BL; Spleen; Staphylococcal Infections; Staphylococcal Vaccines

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.

Topics: Animals; Bacterial Proteins; Bombyx; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Gene Expression Regulation, Bacterial; Hemolysin Proteins; Humans; Hydrolysis; Manganese; Membrane Proteins; Metalloproteins; Mutation; Phosphates; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Recombinant Proteins; RNA; RNA-Binding Proteins; Staphylococcal Infections; Staphylococcus aureus

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-gamma, IL-8, MCP-1, IFN-gamma-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antibody Formation; Antigens, CD; Bacterial Proteins; Cells, Cultured; Coagulase; Cyclic GMP; Cytokines; Dendritic Cells; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Granulocytes; Humans; Macrophage Activation; MAP Kinase Signaling System; Mice; Monocytes; p38 Mitogen-Activated Protein Kinases; Receptors, Chemokine; Staphylococcal Infections; Staphylococcus aureus; Vaccination

The cyclic dinucleotide 3',5'-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

Topics: Animals; Anti-Bacterial Agents; Biofilms; Cattle; Cyclic GMP; Female; Mastitis, Bovine; Mice; Models, Animal; Staphylococcal Infections; Staphylococcus aureus; Virulence

Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus-induced cardiac proinflammatory mediator production and the development of cardiac dysfunction.. Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1beta, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus-induced activation of interleukin-1 receptor-associated kinase, c-Jun NH2 terminal kinase, nuclear factor-kappaB, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus-induced contractile dysfunction.. These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.

Topics: Animals; Child; Cyclic GMP; Cytokines; Heart; Humans; Interleukin-1; Mice; Mice, Knockout; Myocardial Contraction; Myocardium; Nitric Oxide; Receptors, Cell Surface; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha; Ventricular Dysfunction, Left

Experiments on rabbits with infiltrates induced by intracutaneous injection of staphylococcal culture were made to examine the content of cyclic nucleotides in the adjacent muscle tissue under the effect of administering ampiox alone or combined with guanosine triphosphate (GTP). Combined injection of ampiox and GTP to the infected animals produced a considerable elevation in the content of cAMP (more than 2 1/2-fold) and in the cAMP/cGMP ratio along with a beneficial therapeutic effect. The possible mechanisms of action of the agents administered are discussed.

Topics: Ampicillin; Animals; Chinchilla; Cyclic AMP; Cyclic GMP; Drug Combinations; Drug Therapy, Combination; Guanosine Triphosphate; Muscles; Nucleotides, Cyclic; Oxacillin; Rabbits; Staphylococcal Infections