Bacterial adherence to human endothelial cells in vitro

Abstract

Differences in the ability of bacteria to adhere to normal valvular endothelium may account for the predominance of particular species as pathogens in acute endocarditis. An in vitro adherence assay was developed to simulate the host surface encountered in acute bacterial endocarditis by using confluent monolayers of human endothelial cells. Adherence of 32 gram-positive and -negative blood culture isolates to this surface was compared. All five Staphylococcus aureus strains tested were highly adherent to endothelial cells, as was one gram-negative strain (Serratia marcescens). The remaining gram-positive and -negative isolates, including four viridans streptococci, were relatively nonadherent. Transmission electron microscopy demonstrated attachment of Staphylococcus aureus and invagination of the underlying endothelial cell membrane at 1 h followed by engulfment of large numbers of bacteria after 3 h. The intracellular bacteria appeared to be contained within vacuoles. Preferential attachment of some strains of bacteria, in particular Staphylococcus aureus, to human endothelial cells occurred in vitro, suggesting that adherence is an important determinant of bacterial pathogenicity in acute endocarditis. Active uptake of bacteria by endothelial cells may help account for the virulence of Staphylococcus aureus in endovascular infections and for the ability of this organism to establish multiple metastatic foci of infection.

Staphylococcus aureus α-toxin induces apoptosis in endothelial cells

“…On the other hand, in a less localized staphylococcal infection or when the bacterial density is low such as with bacteremia or during the initial stage of localized infection, su¤ciently low amounts of K-toxin may be elaborated for induction of apoptosis in a¡ected tissues. Moreover, if the laboratory-observed phenomenon of S. aureus internalization within non-professional mammalian cells [2,3,6] is relevant in vivo, then cytotoxic e¡ects may be manifest in apoptotic cell death. Denudement of the endothelial cell lining would disrupt homeostasis in many critical tissues, such as lung and kidney, allowing tissue invasion by extracellular staphylococci.…”

“…Direct interaction of S. aureus with the vascular endothelium has been demonstrated in the laboratory by the avid adherence of the staphylococci to cultured endothelial cells via adhesin^receptor interactions that occur between the bacterium and the endothelial cell surface. Following adherence, S. aureus can be ingested or internalized by endothelial cells in vitro [2,3]. The organisms are internalized within the vacuoles of endothelial cells and the resulting phagosome may fuse with lysosomes [4].…”

Prevention of endothelial cell cytokine induction by a Staphylococcus aureus lipoprotein

“…Bacteria were collected by centrifugation, washed and resuspended in medium 199 (M199, Gibco, Grand Island, NY, USA). The bacterial concentration was measured spectrophotometrically at a wavelength of 620 nm and the bacterial inoculum con¢rmed by colony counts [8].…”

“…Endothelial cells were harvested from human umbilical veins using 0.1% collagenase (Sigma, St. Louis, MO, USA) and maintained in tissue culture at 37³C in 5.5% CO 2 as previously described [8,9]. Cells between the third and sixth passage were grown to con£uence in 100-mm tissue culture plates (Falcon, Cockeysville, MD, USA) and passaged every four days in endothelial cell growth media consisting of M199, supplemented with 20% newborn calf serum (Gibco), 5% human AB serum, 1.6 mM glutamine (Gibco), 15 mM HEPES bu¡er (Calbiochem-Behring, La Jolla, CA, USA), 50 Wg ml 31 of porcine heparin (Sigma), 50 Wg ml 31 ascorbate (Fisher, Fairlawn, NJ, USA), partially puri¢ed acidic ¢broblast growth factor (10 Wg ml 31 ), penicillin and streptomycin [9,10].…”

“…Infection assays were performed to determine the number of adherent and internalized bacteria as previously described [5,8]. Suspensions of staphylococci were added to monolayers of endothelial cells (24-well tissue culture plates) in the presence of equal volumes of 8325-4, Wb supernatant or THB control.…”