Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

Abstract

Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by S. gordonii CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of S. gordonii, S. sanguis, S. mutans, S. mitis, and S. oralis but only weakly by S. salivarius. Comparison of isogenic pairs of S. gordonii revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.Viridans group streptococci comprise a large proportion of the commensal bacteria that colonize oral surfaces (20,24,25). These bacteria frequently enter the bloodstream following trauma to oral tissues (12,17,41,58) and can then adhere to surfaces of abnormal or previously damaged heart valves (15,21,29,47) or become implanted in arterial atherosclerotic plaques (11). Streptococci growing on heart valve surfaces (causing infective endocarditis) become encased in a matrix of fibrin and platelets, which form macroscopic verrucous lesions and can lead to valve perforation, abnormalities in cardiac conduction, valve ring abscesses, pericarditis, aneurysm of the sinus of Valsalva, and release of peripheral emboli (21, 56). Viridans group streptococci are the most common cause of native valve endocarditis in humans, accounting for 45 to 80% of cases (5, 55). A variety of virulence factors have been implicated in the initial colonization of bacteria to cardiac valve surfaces (1, 32, 49, 50, 54), but those responsible for the ultimate destruction of underlying tissues are not well unders...

Streptococcus Mutans and Streptococcus Sobrinus are Able to Adhere and Invade Human Gingival Fibroblast Cell Line

“…Invasion of host cells represents a bacterial mechanism to evade innate and adaptative immunity and to persist in host cells and tissues (21)(22). The invasion ability of human oral cells has been described for oral bacterial species as viridans streptococci (23)(24), Porphyromonas gingiva/is and Aggregatibacter actnomycetemcomitans, causative agents of periodontitis (25)(26). Concerning S. mutans, it is able to gain access to the bloodstream, leading to transient bacteraemia (27)(28).…”

Use of Phylogenetic and Phenotypic Analyses To Identify Nonhemolytic Streptococci Isolated from Bacteremic Patients

“…It has been suggested that the glucosyltransferase enzyme is an important virulence factor in systemic infections due to its ability to synthesize capsule-like extracellular polysaccharide (25) and to enhance adhesion to and invasion and killing of endothelial cells in vitro (46,52). In this study, 39% of the 107 isolates from blood possessed the gtf gene.…”

“…The shortcoming of this method is that it is unable to identify other species. However, it has been suggested that the glucosyltransferase enzyme (GTF) is an important virulence factor in systemic infections, being responsible for biosynthesis of the capsule-like extracellular polysaccharide (25) and for adhesion to and invasion and killing of cultured human umbilical endothelial cells (46,52). For this reason, it was interesting to investigate the proportion of streptococci from cases of endocarditis and septicemia that carry the gtf gene.…”

Integrin-mediated Adhesion and Soluble Ligand Binding Stabilize COX-2 Protein Levels in Endothelial Cells by Inducing Expression and Preventing Degradation

“…COX-2 Degradation Studies-Cells cultured for 12 h on gelatincoated plates were washed twice with PBS, detached by trypsin treatment, and immediately exposed to different inhibitors: protease inhibitor mixture at 1:1000 dilution (33) Cell Adhesion Assays-Maxisorp II Nunc enzyme-linked immunosorbent assay plates (Roskilde, Denmark) were coated with 10 g/ml laminin, collagen I, fibronectin, or vitronectin overnight at 4°C and blocked with BSA for 2 h at room temperature. Assays were done as previously described (32).…”