A phosphoramidon-sensitive metalloprotease induces apoptosis of human endothelial cells by Group B Streptococcus.

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-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.

Metallopeptidases produced by group B Streptococcus: influence of proteolytic inhibitors on growth and on interaction with human cell lineages.

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.

Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells.

Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.

Effect of chelating agents on the growth, surface polypeptide synthesis and interaction of Streptococcus agalactiae with human epithelial cells.

In the present study, the influence of the chelating agents of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (ß-aminoethyl ether) (EGTA) and 1,10-phenanthroline (PHEN) on growth rate, cytoadherence ability and surface protein expression was examined in a clinical strain of Streptococcus agalactiae isolated from a blood sample. EDTA and EGTA at 10 mM did not inhibit the growth of S. agalactiae, while PHEN significantly arrested bacterial proliferation in a concentration range of 10-0.01 mM. The in vitro interaction between S. agalactiae and A549 cells was a time-dependent process; adherence and bacterial intracellular viability were more pronounced after 3 h of contact. The pre-treatment of bacterial cells with EDTA adversely influenced the adhesive properties of S. agalactiae to A549 cells after 2 and 3 h, whereas EGTA only blocked this process after 3 h. Viable intracellular bacteria were just detected after 3 h of interaction, and EDTA and EGTA inhibited intracellular viability in a similar fashion. Conversely, PHEN inhibited neither the adherence nor the intracellular viability of the microorganism. Furthermore, EDTA robustly suppressed surface polypeptide synthesis, suggesting a decline in the possible bacterial ligands responsible for S. agalactiae adhesive properties.