A phosphoramidon-sensitive metalloprotease induces ... - Springer Link

Antonie van Leeuwenhoek (2013) 104:1125–1133 DOI 10.1007/s10482-013-0034-y

ORIGINAL PAPER

A phosphoramidon-sensitive metalloprotease induces apoptosis of human endothelial cells by Group B Streptococcus Michelle Hanthequeste Bittencourt dos Santos • Andreía Ferreira Eduardo da Costa Beatriz Jandre Ferreira • Simone Lima Souza • Pamella da Silva Lannes • Gabriela Silva Santos • Ana Luiza Mattos-Guaraldi • Prescilla Emy Nagao

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Received: 5 June 2013 / Accepted: 6 September 2013 / Published online: 20 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract We explored Group B Streptococcus (GBS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and the role of phosphoramidon, a zinc metalloprotease inhibitor, in this process. GBS 90186 strain (serotype V, a blood isolate) and concentrated supernatant (CS) were used to investigate the viability and morphological alterations in HUVEC by Trypan blue uptake, electrophoresis in 2 % agarose gel and scanning electron microscopy assays. Apoptosis before and after phosphoramidon-

M. H. B. dos Santos A. F. E. da Costa B. J. Ferreira P. da Silva Lannes G. S. Santos P. E. Nagao (&) Departamento de Biologia Celular, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, R. Saõ Francisco Xavier, 524 – PHLC, 5° andar, sala 501B, Maracana˜, Rio de Janeiro CEP 20.550-013, Brazil e-mail: [email protected] S. L. Souza Departamento de Patologia Geral e Laborato´rios, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil e-mail: [email protected] A. L. Mattos-Guaraldi Disciplina de Microbiologia e Imunologia, Faculdade de Cieˆncias Me´dicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil e-mail: [email protected]

treatment were verified by flow cytometry using annexin V-FITC labeling. Differences were considered significant when P \ 0.05 using unpaired Student’s t test. GBS and CS induced HUVEC death by apoptosis (76.5 and 32 %, respectively) with an increasing pro-apoptotic Bax expression and decreasing anti-apoptotic Bcl-2 expression. Caspase-3 was activated during GBS-induced endothelial apoptosis. Phosphoramidon reduced 89.3 and 100 % of GBS and CS cell death by apoptosis, respectively. Some GBS strains may induce cell death by apoptosis with involvement of metalloproteases and signaling through the intrinsic pathway of apoptosis, which may contribute to GBS survival during sepsis of adults and neonates. Keywords Group B Streptococcus Endothelial cells Metalloprotease Phosphoramidon Apoptosis

Introduction Group B Streptococcus (GBS; Streptococcus agalactiae) emerged in the 1960s as a major cause of neonatal morbidity and mortality in the United States and Europe. Three decades later, GBS arose as a pathogen responsible for various infections in non pregnant adults, particularly in elderly subjects with

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underlying conditions (Farley 1993). Primary bacteremia is the most serious clinical syndrome reported in adults and meningitis is associated with very high mortality rates (27–34 %) (Salloum et al. 2011). Disruption of the collagen fibrils of the amniotic membrane has been shown in the presence of a GBS isolate from the placenta of a septic neonate born after premature rupture of membranes (Jackson et al. 1994; Winram et al. 1998). Considering that the amniotic membrane consists largely of several layers of collagen fibrils, collagenolytic activity by GBS may be an important spreading factor for these bacteria that facilitates bacterial passage from the blood stream to other tissues (Rubens et al. 1991). We previously demonstrated that peptidases produced by GBS belong to the metalloprotease class capable of cleaving matrix extracellular proteins and that two serotype III GBS strains (80340 and 90356) isolated from vagina and central nervous system produced similar metalloprotease profiles under the same growth conditions (Soares et al. 2008). Following bacteremia, the immune response can be insufficient, presumably due to immature innate immunity of the neonate or adults with significant underlying conditions (Farley 2001). GBS are able to penetrate the blood–brain barrier by targeting human brain microvascular endothelial cells and endothelial dysfunction is a major component in the pathophysiology of septicemic GBS infection (Lembo et al. 2010). Therefore, analysis of the interaction between GBS and endothelial cells is of major interest, especially when considering the septic and hemorrhagic complications in humans. In this work, the induction of apoptosis in human umbilical vein endothelial cells (HUVEC) by an invasive GBS strain and the role of phosphoramidon-sensitive metalloprotease in this process were investigated.

Materials and methods Bacterial strain and growth conditions The GBS 90186 strain (serotype V, a blood isolate) was used in this study. The strain was identified as GBS and serotyped as previously described (Lancefield 1934). The microorganisms were stored after lyophilization and recovered in Brain Heart Infusion broth (BHI; Difco Laboratories, Detroit, MI). The GBS isolate was cultured on Blood Agar base (Oxoid)

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Antonie van Leeuwenhoek (2013) 104:1125–1133

plates containing 5 % sheep defibrinated blood (BAB/ blood) for 24 h at 37 °C and then grown in BHI at 37 °C until optical density reached 0.4 (*108 CFU/ml). In preliminary experiments, bacteria were previously incubated at 37 °C for 1 h, in the absence or presence of the metalloprotease inhibitor at different concentrations (5, 10, 25, 30 lM). A concentration of 25 lM phosphoramidon effectively inhibited the metalloprotease activity and was used in all assays (data not shown). Human umbilical vein endothelial cell (HUVEC) culture Primary HUVEC were obtained by treating umbilical veins with 0.1 % collagenase IV solution (Sigma Chemical Co., St. Louis, MO, USA) as previously described (Jaffe et al. 1973) and maintained in 199 medium (M199)/HEPES modification (Sigma) supplemented with antibiotics (penicillin 100 U/ml, streptomycin 100 lg/ml and anphotericin-B 2.5 lg/ml), glutamine 2 mM and 20 % fetal bovine serum (FBS, Cultilab, Campinas, Brazil) at 37 °C in a humidified 5 % CO2 atmosphere until they reached confluence. Cells were used during first or second passages only, and subcultures were obtained by treating the confluent cultures with 0.025 % trypsin/ 0.2 % EDTA solution in phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.2). Cell-free culture supernatants All media and solutions were prepared with sterile apyrogenic deionized water. Bacterial cells grown in broth medium were centrifuged and the supernatants were filtered through a 0.22-lm membrane (Millipore). Cell-free culture supernatants (CS) were concentrated 100-fold using a 10,000 molecular weight cut-off Amicon micropartition system (Beverly, MA, USA) and immediately used in zymography assays (Soares et al. 2008). Protein concentration was determined as cited below. HUVEC viability and morphology analysis Confluent HUVEC on glass coverslips (13 mm diameter) coated with porcine skin gelatin 1 % in 24-welltissue culture plates were incubated with GBS at a


multiplicity of infection (MOI) of 1:100 HUVEC/ bacteria or CS (60 lg), in M199 media with 3 % fetal calf serum (FCS) for 2, 4, 16 and 24 h in 5 % CO2 at 37 °C. After each incubation period, stimulated cells were rinsed with M199 and stained with 0.4 % Trypan blue solution diluted at a ratio of 10:1 in M199. Total cells (residual adherent cells plus detached floating cells) were evaluated. The viability of cells was determined as follow: [viable cells 9 100/total cells]. A minimum of 100 cells were counted for each treatment. For the examination of nuclear morphology, HUVEC were grown on 12 mm-diameter glass coverslips coated with porcine skin gelatin 1 % and incubated with GBS (MOI, 1:100 HUVEC/bacteria) or CS for 4 h. For metalloprotease inhibition, GBS (GBS?P) or CS (CS?P) were pre-incubated with phosphoramidon (25 lM) in M199 media with 3 % FCS for 1 h at 37 °C. HUVEC in M199 media with plus 3 % FCS (C) or treated with phosphoramidon (25 lM) in M199 media with 3 % FCS (C?P) were used as negative controls. HUVEC treated with LPS (1,000 ng/ml) or LPS?P (plus 25 lM phosphoramidon) was used as positive controls. HUVEC were washed with PBS containing 1 % bovine serum albumin (PBS-BSA), fixed in 3.7 % formaldeyde in PBS for 10 min and permeabilized with 0.1 % Triton X-100 in PBS for 6 min. Then, cells were stained with 1 lg/ml DAPI (40 ,6-diamidino-2-phenylindole; Sigma) for 5 min and residual adherent cells were examined by fluorescence microscope. Apoptotic nuclei were identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. DNA samples from total cells (residual adherent cells plus detached floating cells) were also analyzed by electrophoresis in 2 % agarose gel with ethidium bromide. The total cells were evaluated after incubation with GBS, GBS?P, CS, CS?P, LPS and LPS?P (Fettucciari et al. 2000). In scanning electron microscopy assays, HUVEC were grown onto 12 mm diameter glass coverslips coated with porcine skin gelatin 1 %, infected with GBS or CS for 24 h at 37 °C and incubated overnight at 4 °C in a solution of 3 % paraformaldehyde and 2.5 % glutaraldehyde. Following treatments, the cells were post-fixed with 1 % OsO4 in 0.1 M cacodylate buffer supplemented with 0.8 M potassium ferrocyanide and 10 mM CaCl2. The cells were dehydrated in a graded series of ethanol, and then subjected to critical

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point drying with CO2. Samples were covered with a gold film and examined with a Zeiss scanning electron microscopy. Analysis of HUVEC by flow cytometry for apoptosis and necrosis For identification of apoptosis in HUVEC stimulated with GBS or CS in M199 media with 3 % FCS during 24 h at 37 °C, the binding of annexin V and the uptake of propidium iodide (PI) were measured by flow cytometry with a FACScan flow cytometer (Becton– Dickinson Immunocytometry Systems Europe, Erembodegem, Belgium) equipped with an air-cooled 15 mW argon-ion laser operating at 488 nm. GBS (GBS?P) or CS (CS?P) were pre-incubated with phosphoramidon (25 lM) in M199 media with 3 % FCS for 1 h at 37 °C and phosphoramidon was present throughout the incubation time (24 h). HUVEC in M199 media with plus 3 % FCS (C) or treated with phosphoramidon (25 lM) in M199 media with 3 % FCS (C?P) were used as negative controls. HUVEC treated with LPS (1,000 ng/ml) or LPS?P (plus 25 lM phosphoramidon) were used as positive controls. The experiments were performed using the apoptosis and necrosis detection kit TACS Annexin V-FITC (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Flow cytometric analysis was performed using the cellQuest software program (Becton–Dickinson). Logarithmic fluorescence intensity of annexin V-FITC was plotted versus the fluorescence intensity of PI in a dot plot. Total cells (residual adherent cells plus detached floating cells) were evaluated. Data from 10,000 endothelial cells were analyzed for each plot (Costa et al. 2011). Immunoblot analysis Cellular proteins (80 lg per slot) were separated by SDS-PAGE (15 % acrylamide gels) and proteins were electro-transferred to polyvinylidene fluoride membranes (Immobilion-P 45 lm, Millipore, Bedford, USA) for 1 h at 4 °C. Membranes were incubated with primary antibodies (Bcl-2, Bax or caspase-3; 1:1,000, rabbit; Santa Cruz Biotechnology) overnight at 4 °C for 48 h. Membranes were washed, incubated with horseradish-peroxidase-conjugated anti-rabbit IgG (goat anti-rabbit HRP, 1:1,500, Bio-Rad, Munich,

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Germany) and the immunoreactivity was detected using an ECL-plus detection kit (Amersham Pharmacia, Freiburg, Germany). The protein bands on X-ray films were analyzed using NIH Image software (version 1.61). A loading control was performed with b-actin (1:100, rabbit, Santa Cruz). The density of Bcl2, Bax and caspase-3 protein expression was expressed in arbitrary units. Bacterial cellular extracts All media and solutions were prepared with sterile apyrogenic deionized water. GBS (three bacterial colonies) were grown overnight in M199 at 37 °C. GBS cultures were centrifuged (4,0009g, 10 min, 4 °C) and washed in PBS. Bacteria were then suspended in 500 ll of PBS supplemented with 0.1 % Triton X-100. An equivalent volume of glass beads (0.3 mm in diameter) was then added to the suspensions and cells were broken in a cell homogenizer (Braun Biotech International, Melsungen, Germany) by alternating 2-min shaking periods and 2-min cooling intervals (5 cycles). After removal of the glass beads, the suspensions were centrifuged at 4,0009g for 10 min at 4 °C, and the supernatants were used as cellular extract (CE). Viable bacteria were not observed in CE preparations. Protein concentration was determined by the method described previously (Lowry et al. 1951) using BSA as standard. Zymography Peptidases in CE and CS, treated and untreated with phosphoramidon (25 lM) for 1 h at 37 °C, were analyzed by electrophoresis on 10 % acrylamide SDSPAGE with 0.1 % co-polymerized gelatin as substrate. Gels were loaded with 20 lg of protein per slot. After electrophoresis, at a constant voltage of 120 V at 4 °C, SDS was removed by incubation with 10 volumes of 1 % Triton X-100 for 1 h at room temperature under constant agitation. In order to promote proteolysis, the gels were incubated for 48 h at 37 °C in 20 mM glycine-NaOH (pH 10.0). The gels were stained for 2 h with 0.2 % Coomassie Brilliant Blue R-250 in methanol:acetic acid:water (50:10:40) and discolored overnight in a solution containing methanol:acetic acid:water (5:10:85), to intensify the digestion halos. The molecular masses of the peptidases were calculated by comparison with the mobility of low

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molecular mass standards. The gels were dried, scanned and digitally processed (Soares et al. 2008). Statistical analysis Statistical differences between groups were determined with the unpaired Student’s t test and by analysis of variance followed by Tukey’s test. All experiments were replicated 3–4 times. P values of \0.05 were considered statistically significant.

Results HUVEC viability analysis The capacity of GBS or CS to impair HUVEC cell viability was revealed by Trypan blue (Fig. 1a, b). The HUVEC death post-stimulation was time-dependent with GBS (4–24 h incubation; P \ 0.001) and CS (16–24 h incubation; P \ 0.01). Blebs at the HUVEC surface plasma membrane were seen by scanning electron microscope after exposure to GBS or CS for 24 h (Fig. 1c). Nuclear morphological changes and assessment of apoptosis by double staining propidium iodide (PI)/ annexin V (AV) assay Figure 2a shows data from experiments using flow cytometry to give information about the numbers of viable cells (PI-/AV-) versus apoptotic (PI-/AV?) cells and that concurrently provided the number of late apoptotic or secondary necrotic cells (PI?/AV?). Compared to untreated HUVEC (C; control), significant proportions of early (PI-/AV?) and late (PI?/ AV?) apoptotic cells appeared only at 24 h postexposure of HUVEC to GBS (55.6 %; P \ 0.001 and 20.9 %; P \ 0.02, respectively) and CS (20.8 %; P \ 0.02 and 11.2 %; P \ 0.05, respectively). Higher apoptotic activity was observed for GBS (76.5 %) when compared with CS (32 %; P \ 0.02). Phosphoramidon inhibited cell death by apoptosis induced by GBS?P (P \ 0.002; Fig. 2a) and CS?P (P \ 0.0002; Fig. 2a) by 89.3 and 100 %, respectively. Represented in Fig. 2b are the results of nuclear morphological changes by DAPI staining of HUVEC treated with GBS or CS (4 h) with aggregation and condensation of DNA (arrowheads) as compared HUVEC in M199 media with plus 3 % FCS (C) or treated with phosphoramidon (25 lM) in M199 media with 3 %


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Fig. 1 Alterations in human umbilical endothelial cells (HUVEC) by group B Streptococcus 90186 strain (GBS) or concentrated supernatant (CS). HUVEC viability at 2, 4, 16 and 24 h with culture medium as negative control (C) or postexposure to GBS or CS analyzed by Trypan blue exclusion assay (a). The viability of cells was determined as viable cells 9 100/total cells. A minimum of 100 cells were counted for each treatment (b). Scanning electron microscopy of HUVEC infected by GBS (asterisks) or CS showed blebs (black square) at the HUVEC surface plasma membrane. Scale bars 1, 20 and 50 lm (c). Data were expressed as mean ± SD of four experiments. *P \ 0.05

FCS (C?P). Nuclear morphological changes were reduced by phosphoramidon in HUVEC treated with GBS (GBS?P) or CS (CS?P) but not in HUVEC treated with LPS (LPS?P). Apoptosis induced by

GBS and CS were also evaluated by 2 % agarose gel electrophoresis of DNA. DNA fragmentation was detected in extracts from HUVEC after GBS and CS exposure during 24 h (Fig. 2c). DNA fragmentation

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Fig. 2 Flow cytometric analysis showing HUVEC apoptosis inhibition by phosphoramidon. The HUVEC were incubated with: culture medium (C), group B Streptococcus 90186 strain (GBS), concentrated supernatant (CS), phosphoramidon (C?P), GBS plus phosphoramidon (GBS?P), CS plus phosphoramidon (CS?P), LPS and LPS?P as positive controls for 24 h at 37 °C. The viable cell population in the lower left quadrant (annexin V-/PI-), the early apoptotic cells in the lower right quadrant (annexin V?/PI-), late apoptotic cells in the upper right

quadrant (annexin V?/PI?) and necrosis cells in the upper left quadrant (a). Detection of apoptosis by DAPI stain in HUVEC after exposure to the same conditions of flow cytometric analysis. Arrowheads represent the condensed or fragmented nuclei of cells. Magnification 1,0009 (b). Agarose gel (2 %) showing DNA fragmentation of HUVEC incubated with the same conditions of flow cytometric analysis (c). Data were expressed as mean ± SD of four experiments. *P \ 0.05

caused by GBS or CS was partially inhibited by phosphoramidon (GBS?P and CS?P, respectively) but not in DNA extract from HUVEC treated with LPS or LPS?P. The experiments were performed three times and results of a representative experiment are shown.

Bax and Bcl-2 regulation during apoptosis by GBS

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Bax upregulation was observed after HUVEC exposure to GBS (P \ 0.0001) and CS (P \ 0.0001), while Bcl-2 downregulation was observed with GBS (P \ 0.0002) and CS (P \ 0.0002) (Fig. 3a, b).


Fig. 3 Expression of apoptotic regulators Bax, Bcl-2 and caspase-3 in HUVEC after incubated with C, group B Streptococcus 90186 strain (GBS) CS, phosphoramidon (C?P), GBS plus phosphoramidon (GBS?P), CS plus phosphoramidon (CS?P), LPS and LPS?P as positive controls for 24 h at 37 °C. Representative immunoblot analysis of Bax upregulation, Bcl-2 downregulation, and pro-caspase-3 activation detected by caspase-3 subunits (17 and 12 kDa). Actin was used as a control for protein loading (a). Data were expressed as mean ± SD of three experiments (b). *P \ 0.05

Activation of caspase-3 in HUVEC during apoptosis by GBS Procaspase-3 downregulation was detected along with caspase-3 subunits (17 and 12 kDa) after incubation of HUVEC with GBS (P \ 0.0001), CS (P \ 0.001), LPS (P \ 0.002) and LPS?P (P \ 0.01) (Fig. 3a, b). GBS proteolytic activity SDS-PAGE analysis of CS showed a peptidase of 200 kDa (Fig. 4a). The proteolytic activity of the 200 kDa protein was demonstrated by zymography (Fig. 4b). Both cellular extract (CE) and CS proteolytic activities were reduced by phosphoramidon by 82.2 % (CEP; P \ 0.0002) and 92.5 % (CSP;

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Fig. 4 Proteolytic profiles of group B Streptococcus 90186 strain (GBS) was analyzed by SDS-PAGE on a 10 % acrylamide gel (a). Cellular extract (CE) and concentrated supernatants (20 lg protein; CS) were analyzed by zymography assay. Data showed a reduction in peptidase activity of CE and CS after phosphoramidon treatment (CE?P and CS?P, respectively) (b). Data were analysed by densitometry analysis (c) as mean ± SD of three experiments. *P \ 0.05

P \ 0.0001) of control values, respectively (Fig. 4b, c). Similar data are also found with GBS serotype III strain 90356 cell supernatant (data not shown).

Discussion GBS strains have been found to be capable of causing devastating invasive disease in neonates and adults. While it has been previously established that GBS is able to invade epithelial and endothelial cells (Soares et al. 2008; Costa et al. 2011), the mechanisms of cellular injury by GBS remain uncharacterized. The interaction of GBS strain or CS with HUVEC demonstrated in the present study suggests that one of the consequences of GBS adhesion/invasion and/or metallopeptidases production by the bacteria is injury to the endothelial cells and cell death by apoptosis. In response to bacterial infection, programmed cell death, such as apoptosis, is induced as a host innate immune response. Cell death can also benefit

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pathogens: one prominent strategy of many bacterial pathogens is to induce the demise of infected host cells, which allows the bacteria to efficiently exit the host cell, spread to neighbouring cells, evade immune cells, and/or gain nutrients. Many bacterial pathogens, especially those capable of invading within host cells, use multiple mechanisms to manipulate host cell death and survival pathways in order to maintain their replicative compartment (Ashida et al. 2011; Wu and Chen 2011). For GBS, endothelial cell death by apoptosis may contribute to blood and tissue dissemination of infection in human adults or neonates. The balance of pro-apoptotic to anti-apoptotic factors is important in the regulation of the mitochondrial pathway of apoptosis. Microbial infection affects this balance both by triggering the activation of proapoptotic factors and by inducing expression of antiapoptotic proteins (Tait and Green 2010). For many non-pathogenic bacteria, these two events are balanced and apoptosis is prevented. The added stress on cells infected with pathogenic microbes, however, will typically result in apoptosis unless the pathogen has the ability to alter the function of proteins involved in regulation of cell death (Faherty and Maurelli 2008). Among them, the Bcl-2 family plays a central role in the activation of caspases and dominates the regulation of apoptosis. Some Bcl-2 family members can promote cell death, such as Bax, Bad, Bid, Bcl-xS while others promote cell survival, like Bcl-2 and BclxL. Bax are critical mediators of mitochondrial outer membrane permeabilization (Tait and Green 2010). Phosphoramidon markedly inhibited the apoptosis of HUVEC induced by the invasive GBS strain tested as evidenced by flow cytometry analysis, DAPI staining and DNA fragmentation. Infection of HUVEC with GBS induced apoptosis cell death with downregulation of Bcl-2 and significantly upregulated the expression of Bax, suggesting the involvement of intrinsic pathway of apoptosis. In addition, we showed procaspase-3 fragmentation by GBS and CS, which was inhibited after phosphoramidon treatment (GBS?P and CS?P). After apoptotic injury, the 32 kDa caspase-3 pro-enzyme was cleaved to 17 and 12 kDa fragments, which are likely able to catalyse the specific cleavage of many key cellular proteins. A previous study demonstrated that GBS induced macrophages apoptosis by a caspase-3 and caspase-1 independent pathway (Fettucciari et al. 2000). However, other authors showed that GBS-induced

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macrophage apoptosis was caspase-3-dependent and also involved Bax (Ulett et al. 2005), suggesting the involvement of the intrinsic mitochondrial pathway of programmed cell death in GBS-mediated apoptosis. GBS b-hemolysin also contributes to the apoptotic response triggered by GBS (Fettucciari et al. 2000), although additional study showed that serotype III GBS–induced apoptosis in macrophages was induced by a factor that is co-regulated with b-hemolysin by glucose, rather than by b-hemolysin alone (Ulett et al. 2003). The potential of use in endothelial cells of these mechanisms known in macrophages is a possibility. Further investigations are in progress to clarify this process in HUVEC, including using other GBS strains from important serotypes Ia and III. Our data also suggested that GBS cell surface components and/or secreted molecules, including metalloproteases, may contribute to the apoptotic effect in human endothelial cells. Metalloproteases occupy a number of roles in their ability to function as virulence factors for microorganisms. In some instances, they directly damage the tissue during the infection or inactivate endogenous factors that normally are involved in the regulation of the host response to the infection (Supuran et al. 2002). In a previous study, we demonstrated that a 200-kDa metallopeptidase in culture supernatants of GBS is capable of cleaving fibronectin, laminin, type IV collagen, fibrinogen and albumin (Soares et al. 2008) in GBS serotypes III and V. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, in this present work, the activity of the 200-kDa metallopeptidase synthesized by GBS serotype V invasive isolate 90186 was reduced by phosphoramidon, a zinc metalloprotease inhibitor. Other authors demonstrated that incubation of surfactant protein-A (SP-A) with several clinical isolates of Pseudomonas aeruginosa resulted in concentration- and temperature-dependent degradation of SP-A that was inhibited by phosphoramidon (Mariencheck et al. 2003). Gentlyase, an extracellular metalloprotease of Paenibacillus polymyxa that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases was also inhibited in the presence of phosphoramidon (Ruf et al. 2013). Pathologically evolving vascular sites are indeed impregnated with proteases, which are capable of detaching vascular cells, such as human endothelial


cells, from their extracellular matrix and then inducing apoptotic cell death (Mosnier et al. 2003). Pathogenic bacteria, including GBS, express proteinases that can also alter host tissue extracellular matrix and cell viability. Although apoptosis can be beneficial for the animal host by playing an important role in the host defense mechanism (Beaufort et al. 2011), apoptosis can play dual roles in the infected animals, depending on the situation. Similar to other pathogens, if GBS infection induces massive apoptosis in a broad area of tissue or in essential organs, induction of apoptosis would bring serious results in the infected host. Thus, apoptosis can have a primary role in the pathogenesis of bacteria. In conclusion, some GBS strains may induce cell death by apoptosis, with the involvement of proteases and intrinsic signaling, that may contribute to GBS invasive growth and survival during sepsis of adults and neonates. Acknowledgments This work was supported by Fundaçaõ de Amparo a` Pesquisa do Estado do Rio de Janeiro—FAPERJ, Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico—CNPq, Universidade do Estado do Rio de Janeiro (SR-2/UERJ), Programa de Apoio aos Nućleos de Exceleˆncia—PRONEX.

References Ashida H, Mimuro H, Ogawa M, Kobayashi T, Sanada T, Sasakawa C (2011) Cell death and infection: a double-edged sword for host and pathogen survival. J Cell Biol 195:931–942 Beaufort N, Corvazier E, Hervieu A, Choqueux C, Dussiot M, Louedec L, Cady A, de Bentzmann S, Michel JB, Pidard D (2011) The thermolysin-like metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells. Cell Microbiol 13:1149–1167 Costa AFE, Pereira CS, Santos GS, Carvalho TM, Hirata RJR (2011) Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells. Int J Mol Med 27:739–744 Faherty CS, Maurelli AT (2008) Staying alive: bacterial inhibition of apoptosis during infection. Trends Microbiol 16:173–180 Farley MM (2001) Group B streptococcal disease in nonpregnant adults. Clin Infect Dis 15:556–561 Farley MM, Harvey RC, Stull T, Smith JD, Schuchat A, Wenger JD, Stephens DS (1993) A population-based assessment of invasive disease due to group B Streptococcus in nonpregnant adults. N Engl J Med 328:1807–1811 Fettucciari K, Rosati E, Scaringi L, Cornacchione P, Migliorati G, Sabatini R, Fetriconi I, Rossi R, Marconi P (2000)

1133 Group B Streptococcus induces apoptosis in macrophages. Am Assoc Immunol 22:1767–1778 Jackson RJ, Dao ML, Lim DV (1994) Cell-associated collagenolytic activity by group B streptococci. Infect Immun 62:5647–5651 Jaffe EA, Nacchman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells of derivate from umbilical cords veins. Identification on by morphologic and immunologic criteria. J Clin Invest 52:2745–2756 Lancefield RC (1934) A serological differentiation of specific types of bovine hemolytic streptococci (group B). J Exp Med 59:441–458 Lembo A, Gurney MA, Burnside K, Banerjee A, de Los reyes M, Connelly JE, Lin WJ, Jewell KA, Vo A, Renken CW, Doran KS, Rajagopal L (2010) Regulation of CovR expression in Group B Streptococcus impacts blood-brain barrier penetration. Mol Microbiol 77:431–443 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275 Mariencheck WI, Alcorn JF, Palmer SM, Wright JR (2003) Pseudomonas aeruginosa elastase degrades surfactant proteins A and D. Am J Respir Cell Mol Biol 28:528–537 Mosnier LO, Griffin JH (2003) Inhibition of staurosporineinduced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor. Biochem J 373:65–70 Rubens CE, Raff HV, Jackson JC, Chi EY, Bielitzki JT, Hillier SL (1991) Pathophysiology and histopathology of group B streptococcal sepsis in Macaca nemestrina primates induced after intraamniotic inoculation: evidence for bacterial cellular invasion. J Infect Dis 164:320–330 Ruf A, Stihle M, Benz J, Schmidt M, Sobek H (2013) Structure of gentlyase, the neutral metalloprotease of Paenibacillus polymyxa. Acta Crystallogr D Biol Crystallogr 69:24–31 Salloum M, van der Mee-Marquet N, Valentin-Domelier AS, Quentin R (2011) Diversity of prophage DNA regions of Streptococcus agalactiae clonal lineages from adults and neonates with invasive infectious disease. PLoS One 6:e20256 Soares GC, Santos MHB, Costa AF, Santos AL, Morandi V, Nagao PE (2008) Metallopeptidases produced by group B Streptococcus: influence of proteolytic inhibitors on growth and on interaction with human cell lineages. Int J Mol Med 22:119–125 Supuran CT, Scozzafava A, Clare BW (2002) Bacterial protease inhibitors. Med Res Rev 22(4):329–372 Tait SWG, Green DR (2010) Mitochondria and cell death: outer membrane permeabilizationand beyond. Nature 11:621–632 Ulett GC, Bohnsack JF, Armstrong J, Adderson EE (2003) bHemolysin-independent induction of apoptosis of macrophages infected with serotype III group B Streptococcus. J Infect Dis 188:1049–1053 Ulett GC, Maclean KH, Nekkalapu S, Cleveland JL, Adderson EE (2005) Mechanisms of Group B Streptococcal-induced apoptosis of murine macrophages. J Immunol 175:2555–2562 Winram SB, Jonas M, Chi E, Rubens CE (1998) Characterization of group B streptococcal invasion of human chorion and amnion epithelial cells in vitro. Infect Immun 66:4932–4941 Wu JW, Chen XL (2011) Extracellular metalloproteases from bacteria. Appl Microbiol Biotechnol 92:253–262

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