Development of a bioluminescent reporter system to monitor neonatal Group B Streptococcal infection

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

Staphylococcus epidermidis an opportunistic pathogen due to its ability to establish biofilms on indwelling medical devices. 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 TNF-α, 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 MHC class II molecules. Overall, these results show that environmental conditions, such as pH and extracellular levels of calcium and magnesium, can account to 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.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) PTDC/BIAMIC/113450/2009 and FEDER FCOMP-01-0124-FEDER-014679. FC, AF and EBA were respectively supported by FCT fellowships SFRH/BD/27638/ 2006, SFRH/BD/62359/2009 and SFRH/BD/38380/200

Group B Streptococcus GAPDH Is Released upon Cell Lysis, Associates with Bacterial Surface, and Induces Apoptosis in Murine Macrophages

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages

Inhibition of IL-10 Production by Maternal Antibodies against Group B Streptococcus GAPDH Confers Immunity to Offspring by Favoring Neutrophil Recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10−/−) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen

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

Plasmin(ogen)-coated GBS displays enhanced abilities to adhere to and invade hBMECs <i>in vitro</i>.

<p>(A) GBS cells were incubated with FITC-labeled human plasminogen (hPLG) (grey filled histogram) or PBS (white dotted histogram). Plasminogen binding was measured by a FACScan cytometer as the increase in FITC mean fluorescent intensity (MFI). Each histogram shows cell number as a function of relative fluorescence obtained for 10,000 events per population. Results are shown for 10, 20, and 50 µg of FITC-conjugated hPLG. (B) Representative growth curves of GBS preincubated without (untreated GBS) or with (PLG-treated GBS) plasminogen plus tPA in complete hBMEC growth medium. Data are from a experiment performed in triplicate that is representative of three independent experiments. Each point is the mean of three samples ± SEM. (C and D) HBMEC monolayers were infected with 10⁶ cells of GBS preincubated without (untreated GBS) or with (PLG-treated GBS) plasminogen plus tPA (MOI of 10 bacteria per cell). (C) HBMECs surface adherent GBS cells and (D) intracellular bacteria were isolated and enumerated after 30, 60, and 90 min of infection. The percentages of hBMECs surface adherent GBS and intracellular bacteria are expressed relative to the initial inoculums. Data are the mean + SEM of three independent experiments. Statistical differences (P values) are indicated; ND – not detected.</p

Plasmin(ogen)-coated GBS induces hBMECs detachment and injury.

<p>GBS cells were preincubated without (untreated GBS) or with (PLG-treated GBS) plasminogen plus tPA, or pre-treated with εACA prior to plasminogen plus tPA incubation (εACA-treated GBS). HBMECs monolayers were infected with 10⁶ CFU (MOI of 10) of untreated GBS, PLG-treated GBS or εACA-treated GBS, for 120 min at 37°C; uninfected cells were used as negative controls. (A) <i>Upper panel</i>: representative microscopic photos of the average cell density were taken at ×100 magnification for visualization purposes. <i>Lower left panel</i>: percentage of viable cells, determined by the neutral red assay, expressed relative to the number of viable cells observed in uninfected control. <i>Lower right panel</i>: Cell viability determined by measuring the LDH release. Data represents mean the values normalized to the mean 100%-death control + SEM from an experiment performed in triplicate that is representative of three independent experiments. (B) Plasmin-like activity in bacterial cell surface. The plasmin activity in GBS surface was assessed following incubation with its specific chromogenic substrate S-2251 and determination of the absorbance at 405 nm. Data represents mean + SEM from an experiment performed in triplicate that is representative of three independent experiments.</p

Transmigration of GBS across hBMECs.

<p>(A) Confluent hBMECs monolayers grown in the upper chamber of Transwell inserts were infected for a 2 h period with 10⁶ GBS cells preincubated without (untreated GBS) or with (PLG-treated GBS) plasminogen plus tPA. The total lower chamber medium was collected at the indicated time points and total GBS CFU were enumerated. (B) The integrity of the HBMEC monolayers infected with 10⁶ GBS CFU previously incubated with plasminogen plus tPA (PLG-treated GBS), untreated (untreated GBS) or pre-treated with εACA prior to plasminogen plus tPA incubation (εACA-treated GBS) was monitored by measuring the change in TEER. Data are the mean values + SEM of at least two experiments. Statistical differences (P values) are indicated.</p