Adília Ribeiro

Streptococcus agalactiae GAPDH Is a Virulence-Associated Immunomodulatory Protein

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.

Protection against systemic candidiasis in mice immunized with secreted aspartic proteinase 2

Secreted aspartic proteinases (Sap) have been described as virulence factors implicated in the mechanisms of host colonization by the yeast Candida albicans in different types of candidiasis. Intraperitoneal inoculation of C. albicans into BALB/c mice rapidly leads to systemic candidiasis, with significant colonization of the kidneys measurable in the following week. In this study we assessed the potential of vaccination with C. albicans secreted aspartic proteinase 2 (Sap2) in preventing systemic candidiasis in BALB/c mice. Intradermal injection of highly purified native Sap2 protein incorporated in alum adjuvant provided efficient immune protection, as indicated by a 20‐fold decrease in the colonization of kidneys. The protective effect of Sap2 immunization with alum adjuvant was also observed in mice infected with a lethal inoculum of C. albicans. Immunization with the native Sap2 alone, as well as with a denatured recombinant form of the protein, also conferred protection, albeit to a lesser level. In all cases, protection correlated with an increase in serum antibodies to Sap2. Moreover, passive transfer of anti‐Sap2 immunoglobulin G (IgG) significantly decreased the yeast burden in kidneys of C. albicans‐infected mice. This result shows that immune protection against systemic candidiasis in mice immunized with Sap2 is antibody‐mediated. Taken together, these analyses demonstrate that Sap2 can be successfully used as a vaccination target in systemic candidiasis and reveals the potential immunomodulatory role of Sap2 on C. albicans infection.

Staphylococcus epidermidis biofilms with higher proportions of dormant bacteria induce a lower activation of murine macrophages

Staphylococcus epidermidis is an opportunistic pathogen and, due to its ability to establish biofilms, is a leading causative agent of indwelling medical device-associated infection. The presence of high amounts of dormant bacteria is a hallmark of biofilms, making them more tolerant to antimicrobials and to the host immune response. We observed that S. epidermidis biofilms grown in excess glucose accumulated high amounts of viable but non-culturable (VBNC) bacteria, as assessed by their low ratio of culturable bacteria over the number of viable bacteria. This effect, which was a consequence of the accumulation of acidic compounds due to glucose metabolism, was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VBNC bacteria within S. epidermidis biofilms. Using bacterial inocula obtained from biofilms with high and low proportions of VBNC bacteria, their stimulatory effect on murine macrophages was evaluated in vitro and in vivo. The inoculum enriched in VBNC bacteria induced in vitro a lower production of tumour necrosis factor alpha, interleukin-1 and interleukin-6 by bone-marrow-derived murine macrophages and, in vivo, a lower stimulatory effect on peritoneal macrophages, assessed by increased surface expression of Gr1 and major histocompatibility complex class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can induce dormancy in S. epidermidis biofilms. Moreover, they show that bacterial suspensions enriched in dormant cells are less inflammatory, suggesting that dormancy can contribute to the immune evasion of biofilms.

TLR2-Induced IL-10 Production Impairs Neutrophil Recruitment to Infected Tissues during Neonatal Bacterial Sepsis

Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti–IL-10R mAb treatment or by using IL-10–deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis.

Oral Therapeutic Vaccination with Streptococcus sobrinus Recombinant Enolase Confers Protection against Dental Caries in Rats

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites.

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite‐evasion immune‐mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69‐expressing peritoneal and splenic B cells. This early B‐cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin‐secreting splenic B cells and a rise in serum levels of N. caninum‐specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum‐infected mice. The B‐cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with γ‐irradiated parasites. Contrasting with the peripheral B‐cell expansion, a depletion of B‐lineage cells was observed in the bone‐marrow of the N. caninum‐infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite‐specific antibodies nevertheless impaired interferon‐γ (IFN‐γ) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non‐lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti‐N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite‐specific, non‐polyclonal, B‐cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN‐γ in the host protective immune mechanisms against this parasite.

Group B Streptococcus Hijacks the Host Plasminogen System to Promote Brain Endothelial Cell Invasion

Group B Streptococcus (GBS) is the leading cause of meningitis in neonates. We have previously shown that plasminogen, once recruited to the GBS cell surface and converted into plasmin by host-derived activators, leads to an enhancement of bacterial virulence. Here, we investigated whether plasmin(ogen) bound at the GBS surface contributes to blood-brain barrier penetration and invasion of the central nervous system. For that purpose, GBS strain NEM316 preincubated with or without plasminogen plus tissue type plasminogen activator was analyzed for the capacity to adhere to, invade and transmigrate the human brain microvascular endothelial cell (hBMEC) monolayer, and to penetrate the central nervous system using a neonatal mouse model. At earlier times of infection, plasmin(ogen)-treated GBS exhibited a significant increase in adherence to and invasion of hBMECs. Later, injury of hBMECs were observed with plasmin(ogen)-treated GBS that displayed a plasmin-like activity. The same results were obtained when hBMECs were incubated with whole human plasma and infected with untreated GBS. To confirm that the observed effects were due to the recruitment and activation of plasminogen on GBS surface, the bacteria were first incubated with epsilon-aminocaproic acid (εACA), an inhibitor of plasminogen binding, and thereafter with plasmin(ogen). A significant decrease in the hBMECs injury that was correlated with a decrease of the GBS surface proteolytic activity was observed. Furthermore, plasmin(ogen)-treated GBS infected more efficiently the brain of neonatal mice than the untreated bacteria, indicating that plasmin(ogen) bound to GBS surface may facilitate the traversal of the blood-brain barrier. A higher survival rate was observed in offspring born from εACA-treated mothers, compared to untreated mice, and no brain infection was detected in these neonates. Our findings suggest that capture of the host plasmin(ogen) by the GBS surface promotes the crossing of the blood-brain barrier and contributes to the establishment of meningitis.

Analysis of the immune response to Neospora caninum in a model of intragastric infection in mice.

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll‐like Receptor 4‐ and functional IL‐12Rβ2 chain‐deficient C57BL/10 ScCr mice were challenged intragastrically with 5 × 106 N. caninum tachyzoites. All parasite‐inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 × 107 N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B‐ and T‐cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN‐γ and IL‐10 mRNA were detected in both groups of infected BALB/c mice compared with non‐infected controls, whereas in the Peyers patches only IFN‐γ mRNA levels were found to be increased. Parasite‐specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum‐specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.

Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth

Staphylococcus epidermidis is the most commonly isolated aetiological agent of nosocomial infections, mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark of the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of the icaA gene, as well as higher susceptibility to vancomycin and penicillin. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR green I staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics, suggesting these cells may contribute to the recalcitrant nature of biofilm infections.

Therapeutic Vaccine against Streptococcus sobrinus-induced Caries

Streptococcus sobrinus produces a virulence-associated immunomodulatory protein (VIP) which suppresses the host-specific immune response and induces the early production of IL-10. In this study, we evaluated the effects of therapeutic immunization with this VIP on the incidence of caries in S. sobrinus-infected rats. Groups of Wistar rats were orally infected with S. sobrinus and fed with sucrose-sweetened drinking water ad libitum. Five days later, rats were immunized intranasally with active or heat-inactivated VIP plus alum as adjuvant or PBS plus adjuvant (sham-immunized). After 3 wks, all rats were re-immunized as above. Evaluation of dental caries showed that VIP-immunized animals had significantly fewer enamel sulcal and proximal caries lesions than did the sham-immunized animals (p < 0.001). The protective effects following therapeutic VIP immunization were attributed to the induced salivary immunoglobulin A specific to the VIP. These results offer a promising and safe strategy for the development of a vaccine against dental caries.