Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs.

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-beta-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Delta4,5-anhydrogalaturonic acid (Delta4,5-GalA). Delta4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.

Staphylococcus aureus SigB activity promotes a strong fibronectin-bacterium interaction which may sustain host tissue colonization by small-colony variants isolated from cystic fibrosis patients.

Genes encoding cell-surface proteins regulated by SigB are stably expressed in Staphylococcus aureus small-colony variants (SCVs) isolated from cystic fibrosis (CF) patients. Our hypothesis is that CF-isolated SCVs are locked into a colonization state by sustaining the expression of adhesins such as fibronectin-binding proteins (FnBPs) throughout growth. Force spectroscopy was used to study the fibronectin-FnBPs interaction among strains varying for their SigB activity. The fibronectin-FnBPs interaction was described by a strength of 1000+/-400 pN (pulling rate of 2 microm s(-1)), an energetic barrier width of 0.6+/-0.1 A and an off-rate below 2 x 10(-4) s(-1). A CF-isolated SCV highly expressed fnbA throughout growth and showed a sustained capacity to bind fibronectin, whereas a prototypic strain showed a reduced frequency of fibronectin-binding during the stationary growth phase when its fnbA gene was down-regulated. Reduced expression of fnbA was observed in sigB mutants, which was associated with an overall decrease adhesion to fibronectin. These results suggest that the fibronectin-FnBPs interaction plays a role in the formation of a mechanically resistant adhesion of S. aureus to host tissues and supports the hypothesis that CF-isolated SCVs are locked into a colonization state as a result of a sustained SigB activity.

Lack of protection of mice against Staphylococcus aureus despite a significant immune response to immunization with a DNA vaccine encoding collagen-binding protein.

Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus.

Bacterial c-di-GMP is an immunostimulatory molecule.

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.

Protective immune responses to a multi-gene DNA vaccine against Staphylococcus aureus.

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-gamma and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent "Newman" strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.

DNA immunization of dairy cows with the clumping factor A of Staphylococcus aureus.

Blocking the primary stages of Staphylococcus aureus infection, specifically the bacterial adhesion to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA) is considered to be one of the most important adhesions factors of S. aureus to host cells. The present study describes the immune response of dairy cattle to a DNA vaccine against ClfA and evaluates the ability of specific genetic adjuvants, targeting sequences (granulocyte macrophage colony-stimulating factor and cytotoxic T lymphocyte antigen-4) and transporter molecules (chitosan and copolymer) to modify the immune response of cows. The results show that vaccination of cows with fibrinogen-binding region A induced a strong and specific antibody response to ClfA in comparison with a control group injected with the pCI vector alone. Although the co-expression of both genetic adjuvants and the addition copolymer transporter did not augment the overall antibody response, these approaches decreased the number of non-responsive cows. Chitosan was the only factor that did not enhance the immune response. Three months after the last DNA immunization, three cows from each of the pGM-CSF, internal ribosomal entry site (IRES), pCTLA and pCI groups were injected with 200 microg of recombinant ClfA protein in incomplete Freund's adjuvant. A strong humoral response was observed in all groups following this protein boost, with the response occurring slightly earlier in DNA-primed protein boost cows. Sera and milk samples taken from cows after the second DNA injection or after the protein boost (sera only) were analyzed for their ability to block adherence and increase phagocytosis. Pre-incubation of S. aureus with sera or milk from vaccinated cows significantly reduced the pathogen's ability to adhere to MAC-T cells relative to the sera and milk samples from the pCI-injected control cows. Similarly, pools of sera and milk from vaccinated cows increased phagocytosis of S. aureus by neutrophils. After the protein boost, sera were more efficient promoters of phagocytosis, reflecting the higher anti-ClfA antibody level of these sera. DNA-prime/protein boost regimes combined with molecular adjuvants appeared to be effective in generating a strong immune response to S. aureus antigens in cattle.

Synthesis and characterization of new permanently charged poly(amidoammonium) salts and evaluation of their DNA complexes for gene transport.

A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected.

Inflammatory cell infiltration as an indicator of Staphylococcus aureus infection and therapeutic efficacy in experimental mouse mastitis.

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.

Immune responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows.

The fibronectin binding protein (FnBP) and clumping factor A (ClfA) of Staphylococcus aureus are important proteins involved in the pathogenesis of staphylococcal bovine mastitis. These antigens were the targets of a DNA and protein vaccination strategy against S. aureus induced mastitis in dairy cows. The DNA vaccine comprised the bicistronic plasmid (pCI-D(1)D(3)-IRES-ClfA) that encoded the fusion of two sequences, (D1(21-34); D3(20-33)) from the fibronectin-binding motifs of FnBP and a fragment from ClfA (aa 221-550) of S. aureus 8325-4 separated by an Internal Ribosomal Entry Site (IRES) sequence. In addition, the vaccine contained the plasmid encoding the bovine granulocyte-macrophage-colony stimulatory factor gene (pCI-bGM-CSF). Four, 7-month pregnant heifers were immunized twice with the DNA vaccine and boosted once with recombinant D(1)D(3) and ClfA proteins while four others were not immunized. The immunization induced lymphoproliferative responses and functional antibodies against D(1)D(3) and ClfA antigens. Three weeks after calving, three mammary quarters of each vaccinated and non-vaccinated cow were challenged with 900 CFU/each of S. aureus Newbould 305. The fourth quarter received saline only. Serum haptoglobin levels, cardiac rhythm and the body temperature of vaccinated cows during the 24-72 h post-challenge were lower than in non-vaccinated animals. At 21 days post-challenge, bacteria were present in 5 of the vaccinated and 11 of the control challenged quarters. The bacteria averaged 1.4 and 3.3 log(10) CFU/ml of milk from vaccinated and control cows respectively. In summary, DNA-protein vaccination against FnBP and ClfA of S. aureus caused both lymphoproliferative and humoral immune responses that provided partial protection of mammary gland from staphylococcal mastitis and better post-challenge conditions in vaccinated cows.

Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms of Staphylococcus aureus.

Despite the general in vitro susceptibility of Staphylococcus aureus isolates that cause infectious bovine mastitis, the pathogen remains difficult to eradicate with the available antibiotics. The capacity to survive intracellularly has been proposed as a factor contributing to the persistence of S. aureus in the bovine mammary gland. The costs associated with the use of cows or goats to assess the in vivo efficacy of new antibacterial compounds constitute a major drawback. Therefore, in the present study, a mouse model of intramammary infection has been characterized for in vivo testing of new experimental drugs. An inoculum of 100 CFU of S. aureus per gland caused an important level of infection with minimal tissue damage as observed at 24 h post-inoculation. By microscopy, polymorphonuclear neutrophil cell infiltration of the infected mammary glands was observed to increase over time. At 12-24 h of infection, the pathogen was primarily found alive and dividing in neutrophils and occasionally within mammary epithelial cells. Intramuscular or intravenous injections of cephapirin at t = 0 and 10 h reduced the number of CFU/g of gland in a dose-dependent manner. In conclusion, the mouse model of infectious mastitis proposed here is suitable for primary evaluation of experimental drugs after parenteral treatment of intramammary infection with a pathogen such as S. aureus that presents both intracellular and extracellular phases of growth.

Lactoferrin against Staphylococcus aureus Mastitis. Lactoferrin alone or in combination with penicillin G on bovine polymorphonuclear function and mammary epithelial cells colonisation by Staphylococcus aureus.

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

In vivo and in vitro demonstration that Staphylococcus aureus is an intracellular pathogen in the presence or absence of fibronectin-binding proteins.

Staphylococcus aureus is the most significant bacterial pathogen associated with bovine mastitis. However, the relevance of intracellular infection to mastitis pathogenesis is poorly understood. We used in vitro assays and a mouse model of mastitis to demonstrate the intracellular component of the infection and to identify the importance of fibronectin-binding proteins in the processes of colonization and internalization. In vitro, a mutant strain, lacking fibronectin-binding protein (FnBPs(-)), had a reduced ability to bind fibronectin and to infect epithelial cells when compared to its parental wild type strain. After 2 h of infection, the internalization of the mutant bacteria into epithelial cell cultures was reduced by 60% compared with the wild type. After in vivo infection, microscopic examination using the FnBPs(-) strain revealed that production of a high density of live bacteria within the mammary gland epithelial cells was delayed. Both parental and mutant strains were identified within neutrophils, macrophages and epithelial cells suggesting a close similarity between the mouse mastitis model and bovine mastitis. These results demonstrate that S. aureus was able to cause an intracellular infection in the mouse model of mastitis and that the elimination of one adhesion protein delayed, but did not prevent, infection.

Optimization of DNA vaccination immune responses in dairy cows: effect of injection site and the targeting efficacy of antigen-bCTLA-4 complex.

The objectives of this study were to evaluate the effects of immunization site and antigen presenting cell targeting on cattle immune responses to DNA immunization. Cows were vaccinated with the plasmid expression vector pCI alone, pCI encoding the bacterial antigen beta-galactosidase (pCI-beta-gal) or pCI encoding bCTLA 4 fused to beta-gal (pCI-bCTLA-hIgG-beta-gal). The plasmids were delivered by intramuscular, intradermal, intramammary gland, or intra supramammary lymph node needle-injection. Both vaccines induced significant humoral and cellular immune responses. pCI-beta-gal elicited a higher IgG response than immunization with pCI-bCTLA-hIgG-beta-gal. Cows injected intramuscularly and intramammary had higher IgG and IgG-1 humoral responses than cows immunized intradermaly or in the lymph nodes. The injection site did not significantly affect the magnitude of the IgG2 and IgM antibody responses, although a trend similar to the IgG results was observed. The lymphocyte proliferation index was higher with pCI-beta-gal but was not affected by the injection site. These results suggest that in bovine, the injection site can affect immune responses but they do not provide evidence that bCTLA-4-hIgG-antigen targeting is effective in cattle.

The fibronectin-binding proteins of Staphylococcus aureus may promote mammary gland colonization in a lactating mouse model of mastitis.

The fibronectin-binding proteins (FnBPs) of Staphylococcus aureus are believed to be implicated in the pathogen's adherence to and colonization of bovine mammary glands, thus leading to infectious mastitis. In vitro studies have shown that FnBPs help the adhesion of the pathogen to bovine mammary epithelial cells. However, the importance of FnBPs for the infection of mammary glands has never been directly established in vivo. In this study with a mouse model of mastitis, the presence of FnBPs on the surface of S. aureus increased the capacity of the bacterium to colonize mammary glands under suckling pressure compared to that of a mutant lacking FnBPs.

DNA immunization against the clumping factor A (ClfA) of Staphylococcus aureus.

Adhesins are considered the most important virulence factors during early phases Staphylococcus aureus infection. Antibodies induced by vaccination toward an adhesin should reduce the adherence of the pathogen and augment its phagocytosis. The present report describes the immune response of mice to a DNA vaccine directed against one of these adhesins, clumping factor A (ClfA). Injection of plasmids expressing the fibrinogen-binding region A of ClfA induced a strong and specific antibody response to ClfA in mice. In addition, splenocyte proliferation was provoked by in vitro stimulation with recombinant ClfA, thus, indicating direct implication of these cells in the immune response. Pre-incubation of S. aureus with sera of vaccinated mice reduced the pathogen's ability to bind fibrinogen by up to 92%. These pre-incubated bacteria were phagocytosed by macrophages at an increased level in vitro and were less virulent in vivo in a mouse mastitis model. However, DNA-immunized mice were not protected against an intraperitoneal challenge. Overall, the results suggest that DNA immunization against adhesins represents a new and valuable approach to combat S. aureus infections.