INFECTION AND IMMUNITY,0019-9567/98/$04.0010

Dec. 1998, p. 5994–5998 Vol. 66, No. 12

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Internalization of Staphylococcus aureus by Endothelial CellsInduces Apoptosis

BARBARA E. MENZIES1,2* AND IORDANKA KOURTEVA1

Division of Infectious Diseases and Department of Medicine, Vanderbilt University School of Medicine,Nashville, Tennessee 37232,1 and Department of Veterans Affairs Medical Center,

Nashville, Tennessee 372122

Received 22 July 1998/Returned for modification 11 August 1998/Accepted 24 August 1998

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute toits propensity to cause persistent endovascular infection with endothelial destruction. In the present study, weshow that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organismsremain viable over a 72-h period and, as determined by transmission electron microscopic examination, thatthe bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial celldeath following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation andchanges in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelialcells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosisinhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to beinternalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factorassociated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosisof endothelial cells and that internalized staphylococci can exist free within the cytoplasm.

Staphylococcus aureus is a common cause of acute endocar-ditis which is associated with fulminant valvular destructionand metastatic infection (13, 16). The propensity of S. aureus toinfect endovascular tissue and to cause disseminated infectionis incompletely understood. It has been suggested that staph-ylococcal adherence to the endothelium can occur by at leasttwo different mechanisms (7). S. aureus can adhere to damagedendocardium by binding to matrix proteins such as fibrinogenand to platelets. Alternatively, S. aureus can bind to normal orundamaged endothelium via specific receptors that have yet tobe fully characterized. Since most cases of S. aureus endocar-ditis involve heart valves that exhibit normal gross morphology(17), elucidation of the interaction of this bacterial pathogenwith normal endothelial cells should provide insight into thepathogenesis of this infection.

Following adherence of S. aureus to endothelial cell mono-layers, invasion may occur via endothelial cell ingestion. Anumber of studies have reported that endothelial cells functionas nonprofessional phagocytes and thus are able to ingest S.aureus organisms (5, 6, 12). Transmission electron micrographsof bovine aortic endothelial cell monolayers infected with S.aureus have demonstrated phagocytosis following a sequenceof events: (i) adherence of S. aureus to the endothelial cell, (ii)formation of cup-like processes on the endothelial cell surfaceunderlying the adherent bacteria, and (iii) elongation of thecup and engulfment of the bacteria within a phagosome (10).Despite lysosomal fusion, the endothelial cell is unable to killthe ingested S. aureus. Viable staphylococci can be isolatedafter treatment of infected endothelial cell monolayers withlysostaphin, which lyses extracellular staphylococci only.Uptake of S. aureus by endothelial cells offers a unique expla-nation for the frequency and persistence of invasive staphylo-

coccal infections involving endothelialized surfaces such asheart valves, prosthetic vascular grafts, and catheterized ves-sels.

The consequences of intracellular invasion of endothelialcells by S. aureus have only recently been investigated. Vannand Proctor showed that ingestion of a high-alpha-toxin-pro-ducing S. aureus strain by bovine aortic valve endothelial cellsresulted in cytotoxic damage to cell monolayers (14). In con-trast, cytotoxicity was not induced with an alpha-toxin-negativederivative strain, and furthermore, cytotoxicity was reduced byanti-alpha-toxin antiserum, suggesting that alpha-toxin, a pore-forming extracellular toxin, mediates the cytotoxic effect. In-vestigations by Yao et al. demonstrated that internalization ofS. aureus by human umbilical vein endothelial cells induced theexpression of the proinflammatory cytokines interleukin-1 andinterleukin-6 (18). Other evidence of potentially deleteriouseffects of internalization in endothelial cells has been the find-ings of stimulation of adhesion molecule expression and hy-peradhesiveness for human monocytes and granulocytes in in-fected endothelial cells (3).

One consequence of invasion by an intracellular pathogen isapoptosis or programmed cell death of the host cell, which isusually observed in professional phagocytic cells such as mono-cytes and macrophages (19). Although S. aureus is not consid-ered a classic intracellular pathogen as are microorganismssuch as Listeria monocytogenes and Mycobacterium tuberculosis,its intracellular residence in endothelial cells could potentiallyelicit this type of cell death response. Recent evidence tosupport this hypothesis has been the finding of apoptosis inhuman monocytes and in bovine mammary epithelial cells thathave phagocytized S. aureus (1, 2). We thus sought to deter-mine whether apoptosis contributes to S. aureus-induced en-dothelial cell death. We show for the first time that cytotoxicityin human umbilical vein endothelial cells infected with S. au-reus occurs via apoptosis and that internalization of metabol-ically active S. aureus organisms is necessary for apoptosis tooccur.

* Corresponding author. Mailing address: Division of InfectiousDiseases, A-3310 Medical Center North, Vanderbilt University, Nash-ville, TN 37232-2605. Phone: (615) 327-4751, ext. 5495. Fax: (615)343-6160. E-mail: menziebe@ctrvax.vanderbilt.edu.

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MATERIALS AND METHODS

Media and chemicals. Medium 199 (M199), Hanks’ balanced salt solution,penicillin G, streptomycin, amphotericin B, and trypsin-EDTA were purchasedfrom Life Technologies GIBCO BRL (Gaithersburg, Md.). Fetal calf serum waspurchased from Hyclone (Logan, Utah). Heparin sodium salt, lysostaphin, andcytochalasin D were obtained from Sigma Chemical Co. (St. Louis, Mo.). En-dothelial cell mitogen was purchased from Biomedical Technologies, Inc.(Stoughton, Mass.).

S. aureus strains and cultivation. S. aureus 8325-4 is a prototypic laboratorystrain cured of prophages that produces hemolysins and is used for staphylococ-cal genetic studies (11). DK2076 is a penicillin-sensitive clinical S. aureus isolatethat is virulent in animal models of infection (9). Aliquots from overnight brothcultures were grown in brain heart infusion broth at 37°C until cultures hadreached an optical density at 600 nm of 0.30 (.1.5 to 2 h), corresponding toapproximately 108 organisms/ml. The staphylococci were harvested by centrifu-gation at 7,000 3 g for 10 min, washed twice with phosphate-buffered saline(PBS), and resuspended in M199. Bacterial concentrations were estimated spec-trophotometrically and confirmed by plating serial dilutions on agar plates andcounting CFU. For UV-treated S. aureus, a logarithmic-phase growth culture wassuspended in M199 and UV irradiated for 1 h at a wavelength of 254 nm.

Cultivation of human umbilical vein endothelial cells. Human umbilical veinendothelial cells were purchased from Clonetics (San Diego, Calif.). Cells weregrown at 37°C in 5% CO2 in medium consisting of M199 supplemented with 15%fetal bovine serum, penicillin, streptomycin, amphotericin B, endothelial cellmitogen (50 mg/ml), and heparin (10 U/ml) on gelatin-coated 100-mm-diametertissue culture plates (Falcon, Cockeysville, Md.) or 24-well plates (Corning,Corning, N.Y.). All experiments were performed with second- to fourth-passagecells that had reached confluence.

Invasion assays. The S. aureus inoculum was suspended in complete mediumwithout antimicrobials and adjusted by optical density to 108 CFU/ml, and 0.4 mlwas added to washed endothelial cell monolayers that had been subcultured toconfluence in 24-well tissue culture plates. Human AB serum at 5% was addedimmediately afterwards. Following incubation of the bacteria with endothelialcells, the wells were washed three times with M199 to remove nonadherentbacteria, and then the endothelial cells were lifted with trypsin, hypotonicallylysed in sterile water, and serially diluted for agar plating to determine colonycounts for total numbers of adherent and internalized bacteria. For determiningS. aureus invasion, the endothelial cells were treated with lysostaphin (10 mg/ml)for 20 min to lyse extracellular bacteria prior to lifting with trypsin. Assays wereperformed in triplicate.

Apoptosis assays. Human umbilical vein endothelial cells were cultivated in100-mm-diameter tissue culture plates to confluence and infected with S. aureusfor at least 1 h. Lysostaphin was added to kill extracellular staphylococci. Theendothelial cells were harvested for either DNA isolation or nuclear staining.

(i) DNA isolation and gel electrophoresis. Detached endothelial cells werecentrifuged at 1,000 3 g for 5 min at 4°C, and the pellet was washed with ice-coldPBS and resuspended in lysis buffer. Adherent cells were lysed directly in thedish. DNA was isolated from both lysates with the Genzyme TACS ApoptoticDNA Laddering Kit according to the manufacturer’s guidelines and analyzed onan ethidium bromide-stained 1.5% TreviGel (Genzyme, Cambridge, Mass.).

(ii) Terminal deoxynucleotide transferase-mediated dUTP-biotin nick endlabeling of DNA (TUNEL assay). Detached cells were combined with adherentcells after lifting with trypsin-EDTA, centrifuged at 1,000 3 g for 5 min at 4°C,and washed with ice-cold PBS. The cell pellet was resuspended in 4% methanol-free formaldehyde in PBS, fixed for 10 min, and stored in 70% ethanol at 220°C.The Genzyme TACS in situ Apoptosis Detection Kit-Blue Label was used tolabel apoptotic cells. Briefly, the cells were permeabilized with proteinase K, andthe 39-hydroxyl ends of DNA fragments were labeled with biotinylated dUTP byterminal deoxynucleotidyl transferase. Detection was performed with a strepta-vidin-horseradish peroxidase conjugate and TACS Blue Label substrate, gener-ating a blue insoluble product. Finally, the cells were counterstained with RedCounterstain B provided by the manufacturer. Analysis was performed by oilimmersion light microscopy at magnifications of 31,000 and 32,000.

Electron microscopy. Endothelial cell monolayers in 24-well plates were inoc-ulated with S. aureus and treated identically to those used in the invasion assaysdescribed above. Cells were fixed for 24 h in 2% glutaraldehyde in 0.1 M PBS(pH 7.4). Cells were then washed in 7.5% sucrose–PBS buffer and dehydratedthrough a graded ethanol series. The cells were then embedded in Spurrs resin,thin sectioned, stained with uranyl acetate and lead citrate, and examined with amodel 300 transmission electron microscope (Philips Electronic Instruments,Mahwah, N.J.).

RESULTS

Internalization of S. aureus by endothelial cells. To study theconsequences of S. aureus invasion of endothelial cells, weexamined the ability of two S. aureus isolates, 8325-4 andDK2076, to invade human umbilical vein endothelial cells intissue culture. The invasion assay is based upon the principlethat intracellular staphylococci are protected from the lytic

action of lysostaphin which is added to the medium to lyseextracellular or adherent staphylococci. Both S. aureus strainswere internalized within 30 min to 1 h after infection in endo-thelial cells in a dose-dependent fashion up to a multiplicity ofinfection of 10 (data not shown). To determine survival ofingested S. aureus over time, endothelial cells harboring S.aureus were incubated in the continuous presence of lyso-staphin until cells were harvested at selected time points overa 72-h period for enumeration of CFU. The numbers of inter-nalized staphylococci decreased slightly, but a significant pro-portion of each strain remained viable (Fig. 1). Negative con-trols with uninfected cells were also performed to excludecontamination during the 72-h incubation period.

Transmission electron microscopy of endothelial cells afterthe invasion assay in which lysostaphin was used to removeextracellular bacteria confirmed that S. aureus organisms wereinternalized. Staphylococci were evident within vacuoles, asdescribed by others (6, 12); however, it was observed that overa 4-h period an increased number appeared to be free withinthe cytoplasm (Fig. 2).

Infection by S. aureus induces apoptosis. To determinewhether endothelial cells undergo apoptosis after infectionwith S. aureus, internucleosomal DNA fragmentation, a hall-mark of apoptotic cells, was assessed by examining for thecharacteristic 180-bp laddering pattern on agarose gels. DNAwas isolated from endothelial cells which had been infectedwith S. aureus 8325-4 and DK2076 in M199 for 4 h and treatedwith lysostaphin for 1 h. DNA laddering occurred in S. aureus-infected endothelial cells but not in uninfected endothelialcells also treated with lysostaphin (Fig. 3). In addition, S. au-reus-infected endothelial cells stained positively when assayedby the TUNEL method, which is based on the specific stainingof 39-hydroxyl ends of the fragmented DNA within apoptoticcells (Fig. 4). In addition, by the TUNEL technique, lightmicroscopy of 8325- and DK2076-infected endothelial cells

FIG. 1. Change in mean number of S. aureus CFU in human umbilical veinendothelial cell cultures with time. Cultures were incubated with S. aureus at aninfection ratio of 100:1 for 1 h, washed three times with medium, and thenexposed to lysostaphin continuously until the endothelial cells were harvested fordetermination of intracellular CFU by colony plate counting at 1, 24, 48, and72 h. Bars indicate the standard errors of the means for three different experiments.

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showed nuclear segmentation and chromatin condensationcharacteristic of apoptosis.

Internalization of S. aureus by endothelial cells is requiredfor apoptosis. To establish whether S. aureus has to be inter-nalized for apoptosis to be induced, we compared apoptosisinduced by 8325-4 and DK2076 in the presence or absence ofcytochalasin D, which inhibits actin polymerization and hencephagocytosis. Cytochalasin D at 1 mg/ml inhibited internaliza-

tion in 99.9% of untreated controls by the invasion assay. Thiscorrelated with a reduced number of apoptotic cells in cytocha-lasin D-treated, S. aureus-infected endothelial cells (Fig. 4D).

Effect of UV-killed S. aureus on apoptosis. To determinewhether apoptosis occurs as a result of metabolically active S.aureus, the bacteria were killed by UV irradiation and endo-thelial cell cultures were incubated with the killed bacteria.Apoptosis was not detectable by the TUNEL assay in endo-thelial cells exposed to UV-killed bacteria (data not shown).Transmission electron microscopy confirmed that UV-killedbacteria retained the capacity to be internalized by endothelialcells.

DISCUSSION

Although primarily known as an extracellular pathogen, S.aureus has been shown to invade and survive within endothelialcells. We confirm this finding and show that intracellular staph-ylococci remain viable up to at least 72 h without evidence ofintracellular multiplication. Furthermore, we show not onlythat S. aureus is engulfed within vacuoles but that it exists freewithin the cytoplasm, implying that the bacterium must be ableto escape from the phagolysosome.

The fate of the host cell in the presence of viable internal-ized S. aureus was investigated. Previously, Vann and Proctorhave shown that ingestion of S. aureus results in damage toendothelial cell monolayers by loss of membrane integrity (14).The effect was dependent on the time after exposure to S.aureus and on the inoculum size. In the present study, we showfor the first time that endothelial cell toxicity mediated by S.aureus occurs through apoptosis as determined by both hostcell DNA fragmentation and characteristic nuclear morpho-logical changes. The use of the phagocytosis inhibitor cytocha-lasin D demonstrated that S. aureus adherence to endothelialcells does not induce apoptosis and that when internalization

FIG. 2. Transmission electron micrographs of an endothelial cell infected with S. aureus DK2076 for 4 h in the presence of lysostaphin. (A) S. aureus organisms(arrows) appear to be free within the cytoplasm. Magnification, 314,000. (B) Higher magnification shows S. aureus free in the cytoplasm (arrows) as well as in vacuoles(asterisk). Magnification, 317,125.

FIG. 3. Agarose gel (1.5%) electrophoretic separation of DNA extractedfrom human umbilical vein endothelial cells infected with S. aureus for 4 h. Lanea, uninfected endothelial cells treated with lysostaphin (negative control); lane b,endothelial cells infected with S. aureus 8325-4; lane c, endothelial cells infectedwith S. aureus DK2076; lane d, 200-bp DNA ladder. Data are from a represen-tative experiment repeated three times.

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of adherent bacteria is inhibited, no apoptosis occurs. Thisindicates a requirement for intracellular residence of S. aureusfor induction of apoptosis in endothelial cells and is consistentwith the study results of Baran et al., who noted that apoptosiswas observed predominantly in mononuclear cells that hadingested S. aureus (1). The propensity for intracellular patho-gens to cause apoptosis in the host cell is well known. Otherbacterial pathogens, such as Shigella flexneri, need to be withinthe cytoplasm to cause apoptosis in murine macrophages (19).

It is unlikely for several reasons that a staphylococcal prod-uct acting extracellularly is responsible for the apoptotic pro-cess observed in this study. The S. aureus inoculum was washedcarefully to remove extracellular toxins and enzymes. In addi-

tion, the absence of apoptosis with the use of cytochalasin Dduring infection of endothelial cells also excludes the contri-bution of extracellular staphylococcal products that may beexpressed during the infection or incubation period.

The mechanisms of induction of apoptosis in endothelialcells which have internalized S. aureus are likely mediated byone or more bacterially secreted staphylococcal products andnot a surface component, since UV-killed S. aureus, althoughretaining the capacity to become internalized, was not capableof inducing apoptosis. Further studies employing isogenic S.aureus strains will be necessary for this determination.

Little is known about the nature of S. aureus gene expressionor secreted gene products that would contribute to cytotoxicity

FIG. 4. Evaluation of DNA fragmentation in human umbilical vein endothelial cells by the TUNEL method. Endothelial cells were grown in the absence or presenceof S. aureus for 1 h prior to treatment with lysostaphin. The incorporation of biotinylated dUTP at the free 39-hydroxyl ends of fragmented DNA is shown by the bluecolor. (A) Uninfected endothelial cells; (B) endothelial cells infected with S. aureus 8325-4; (C) endothelial cells infected with S. aureus DK2076; (D) endothelial cellspreincubated with cytochalasin D for 30 min prior to infection with DK2076. Magnifications, 32,000 (A and B) and 31,000 (C and D).

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during intracellular residence in the endothelial cell. Frombroth medium cultures, it is known that the majority of theputative virulence factors that are expressed by S. aureus areregulated by at least two distinct global regulatory loci, calledagr and sar (4, 11). However, there may be altered expressionof staphylococcal genes from S. aureus organisms in contactwith cultured mammalian cells compared to that of genes fromS. aureus cultivated in vitro within broth suspensions. Findingsby Proctor and colleagues support altered S. aureus physiologyduring intracellular residence within endothelial cells. Theycite evidence to support that the intracellular milieu of endo-thelial cells which have phagocytized S. aureus may induce atbetween 24 and 48 h the emergence of a subpopulation ofmetabolically hypoactive small-colony variants of S. aureus(15). Given that in the present study apoptosis was observed asearly as 1 h after infection of endothelial cells, one may spec-ulate that the small-colony variants of S. aureus as observed inother studies may arise in response to a nutritionally depletedand dying host cell.

The membrane-damaging S. aureus alpha-toxin has beenimplicated in triggering apoptosis in human T lymphocytes.Evidence for apoptosis was found in lymphocytes treated withlow (nanomolar) concentrations of alpha-toxin and not inthose treated with high toxin doses (8). On the basis of per-meability studies, those authors associate the induction of ap-optosis with the formation of very small alpha-toxin poreswithin the plasma membrane of the lymphocyte. A similar typeof mechanism may be operative in endothelial cells that haveingested S. aureus. Bovine aortic endothelial cells that haveingested S. aureus show cytotoxicity as early as 3.5 h, and thisappears to be attributable to intracellular production of S.aureus alpha-toxin (14). It is conceivable that very small quan-tities of alpha-toxin expressed by intracellular S. aureus mayinduce apoptosis of the endothelial cell. Certainly, furtherwork needs to be done to investigate the role of alpha-toxin inthe triggering of endothelial cell apoptosis during infectionwith S. aureus.

The ability of S. aureus to survive intracellularly in endothe-lial cells would benefit the bacterium, as it would be protectedagainst host immune control mechanisms and most antimicro-bials. Does, however, apoptosis of the endothelial cell result inany advantage for ingested S. aureus organisms? Since endo-thelial cells have no known significant bactericidal functionslike that of macrophages, death of the endothelial cell wouldnot compromise immune clearance of the organism. However,death of endothelial cells which have phagocytized S. aureusand detached from the endothelial surface would provide aroute for dissemination of S. aureus to distant sites where fociof metastatic infection could be established.

ACKNOWLEDGMENTS

This work was funded by Minority Medical Faculty DevelopmentProgram of the Robert Wood Johnson Foundation grant no. 030802

(to B.E.M.) and by grant-in-aid TN97G13 from the Southeast Affiliateof the American Heart Association (to B.E.M.).

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Editor: E. I. Tuomanen

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