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Microbial Pathogenesis 57 (2013) 62e69

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Microbial Pathogenesis

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Enterococcus faecalis internalization in human umbilical vein endothelial cells(HUVEC)

Diana Millán a, Carlos Chiriboga b, Manuel A. Patarroyo c,d, Marta R. Fontanilla a,*

a Tissue Engineering Group, Universidad Nacional de Colombia, Carrera 45#26-85, Bogotá, Colombiab School of Medicine, Universidad de los Andes, Carrera 1#18A-12, Bogotá, ColombiacMolecular Biology Department, Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50#26-20, Bogotá, Colombiad School of Medicine and Health Sciences, Universidad del Rosario, Calle 63D#24-31, Bogotá, Colombia

a r t i c l e i n f o

Article history:Received 5 May 2012Received in revised form6 November 2012Accepted 8 November 2012Available online 19 November 2012

Keywords:Enterococcus faecalisNosocomial diseaseHuman umbilical vein endothelial cell

* Corresponding author. Tel.: þ57 1 3165000x14663165000x16959.

E-mail addresses: mrfontanillad@unal.edu.co(M.R. Fontanilla).

0882-4010/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.micpath.2012.11.007

a b s t r a c t

Initial Enterococcus faecaliseendothelial cell molecular interactions which lead to enterococci associatingin the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models.Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positivebacteriaecell interactions; however, few studies have been aimed at establishing the relationship ofE. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents anE. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence.The Asc10 protein (a member of this family) promotes bacteriumebacterium aggregation and bacteriumehost cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shownthat this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structuralcomponents, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thusrelevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role inE. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins inE. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction byenterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeletoncomponents were partially involved in E. faecalis entry to endothelial cells, thereby suggesting thatE. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Enterococci are Gram-positive bacteria which are usually foundas innocuous inhabitants of human gastro-intestinal flora, the oralcavity and the female genital tract [1]. These opportunistic micro-organisms are known for being multi-resistant pathogens and forcausing nosocomial bacteremia, endocarditis and surgical andurinary infection [1,2]. Risk factors for acquiring Enterococcus faecalisnosocomial infections include long hospitalization, being exposed tocontaminated medical equipment, proximity to infected patientsand receivingorganor tissue transplants [3,4];E. faecalis accounts forw60% of clinical enterococcal isolates [4]. The emergence ofE. faecalis strains suchashigh level aminoglycoside resistance (HLAR)and vancomycin-resistant enterococci (VRE) [3,5,6] has limited the

4, þ51 1 3116960; fax: þ57 1

, m.fontanilla.d@gmail.com

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success of antibiotic treatment and highlighted the importance ofstudying such bacteria’s interaction with their target tissues.

Endocarditis by E. faecalis is characterized by lesions known asvegetations which are biofilms formed on the endothelial surface[7,8]. Efforts at studying the mechanism by which this bacteriumforms vegetations have mainly been carried out in experimentalmodels of endocarditis [9e11]. An ex vivo porcine heart valveadherence model has been developed for studying initial interac-tions between bacteria and valve tissue [12]; however, few in vitrostudies have assessed E. faecalis-cultured endothelial cell interac-tions. Our group has successfully infected HUVEC primary cultureswith an E. faecalis clinical isolate, intracellular bacteria being recov-ered 4 h post-infection in a gentamicin survival assay; this indicatedthat the bacterium was internalized by endothelial cells [13]. Thissuggested that such model could be used to study initial bacteriaeendothelial cell molecular interactions which lead enterococci toassociate, colonize and proliferate in host endothelial tissue.

The aggregation substance (AS) family of adhesins representsE. faecalis virulence factors which are involved in promoting

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e69 63

effective endothelial cell adherence and colonization; these surfaceproteins have been implicated in endocarditis severity and bacterialpersistence [10,11] and are encoded by genes present in conjugativeplasmids [14]. A member of this family, the Asc10 protein, promotesbacteriumebacteriumaggregationandbacteriumehost cell binding[15e19], being encoded by conjugative plasmids, such as pCF10, andcan be expressed in vivo [20,21]. The evaluation of Asc10 role inbacterial internalization bycultured enterocytes has shown that thisadhesin facilitates E. faecalis endocytosis by HT-29 cells [22,23]. Itthus seemed relevant to determine whether Asc10 played a role inE. faecalis internalization by cultured endothelial cells. Some studieshave analyzed the role played by cell components, such as cyto-skeletal proteins, in E. faecalis entry to cell-lines [24]; however, thishas not been done with endothelial cells to date. The role of Asc10andcytoskeletonproteins inE. faecalis ability toenterHUVEC, aswellas the induction of cell apoptosis by enterococcal internalization byHUVEC was determined in the present study.

2. Results

2.1. Characterizing clinical isolates

Colony PCR, RT-PCR and Western blot analysis were carried outwith E. faecalis 9031 and E. faecalis 1441 clinical isolates, kindlyprovided by Dr. Martha Garzón (Hospital El Tunal, Colombia) andDr. Jinnethe C. Reyes (Bacterial Molecular Genetics Unit,

Fig. 1. Detection and expression of ASC10 in E. faecalis clinical isolates and strains. A. Colonyand the OG1-SSpP11 strain; a 441 bp band was observed. B. RT-PCR analysis of prgB gene traband was observed in OG1-SSpP11 and 9031. Control: RNA extracted from the samples butOG1-SSpP11 and 9031 bacterial lysate (w137 kDa) as detected by Western blot. D. Clumpisolate. An additional assay (1441/9031 culture medium) to the right of the panel shows thaMWM: molecular weight markers; Hyperladder II (Bioline) in Panels A and B, and PageRul

Universidad El Bosque, Colombia) to qualitatively assess Asc10 genepresence, transcription and expression. OG1-SSpP11 (carryingpCF11, a mutant of pCF10) and OG1-SSp (plasmid-free) strains,kindly donated by Dr. Gary Dunny from the University of Minnesotaand previously characterized as being Asc10þ and Asc10� respec-tively [25e27], were included as positive and negative controls.

Fig. 1A shows colony PCR products. An expected 441 bp ampli-fication fragment was observed in the E. faecalis OG1-SSpP11 strain(positive control) as well as in isolates 9031 and 1441. RT-PCR andWestern blot experiments indicated that Asc10 was not transcribedor expressed in isolate 1441 while this did occur in E. faecalis OG1-SSpP11 and isolate 9031 (Fig. 1B, Panel 1C). Clumping experimentsshowed that only positive control and isolate 9031 culturesaggregated in the presence of cCF10 pheromone, thereby con-firming this finding (Fig. 1, Panel D). A clumping experimentinvolving adding filteredmedium from an E. faecalis 9031 culture toan E. faecalis 1441 culture was carried out to evaluate whetherAsc10 expression in isolate 1441 required conditions that were notpresent in the culture medium used for its growth. The resultsindicated that growing the 1441 isolate bacterial inoculum in thismedium did not induce clumping (Fig. 1, Panel D).

2.2. Asc10 involvement in enterococci internalization by HUVEC

Three gentamicin survival assays carried out on different daysindicated enterococcal entry to cultured HUVEC; each represented

PCR analysis of the Asc10-encoding prgB gene in E. faecalis clinical isolates 9031, 1441nscription in E. faecalis clinical isolates 9031, 1441 and the OG1-SSpP11 strain; a 441 bpwithout Superscript III treatment was used for cDNA synthesis. C. Asc10 expression ining experiments showing aggregation in the OG1-SSpP11 strain and the 9031 clinicalt 1441 did not clump when grown in filtered medium from an E. faecalis 9031 culture.er TM (Fermentas) in Panel C.

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e6964

the average of triplicate infected cultures. Fig. 2 shows mean values(�SD) of log10 colony forming units (CFU) recovered from 50,000infected cells after beingwashed and lysed. All E. faecalis strains andisolates were able to enter cultured HUVEC. Mean log10 CFU valuesfrom the OG1-SSpP11 (Asc10þ) and OG1-SSp (Asc10�) strains andclinical isolates 9031 (Asc10þ) and 1441 (Asc10�) were similar(p > 0.05). As expected, HUVEC invasion by positive control(Staphylococcus aureus) and no invasion by negative control(Bacillus subtilis) were observed. Transmission electron microscopydata agreed with the above-mentioned antibiotic protection assayresults. Cultured HUVEC had cell junctions and junction structurescharacteristic of a monolayer; although no bacteria was observed inmost HUVEC, some had one to four enterococci inside them (Fig. 3,Panels AeB). Unusually, few HUVEC having more than 30 entero-cocci inside themwere observed (Fig. 3, Panel C). Many enterococciwere also seen close to HUVEC membrane protrusions, resemblinglamellipodia and filopodia (Fig. 3, Panel D). Some invaded HUVECshowed apoptotic changes, such as condensed chromatin, apoptoticbodies and nuclear fragmentation (Fig. 3, Panels C and EeF).

2.3. Apoptosis assessment

HUVEC apoptosis after E. faecalis infection and the role ofmultiplicity of infection (MOI) were determined. Flow cytometryusing an FITC Annexin V apoptosis kit was carried out to determinewhether cultured HUVEC underwent apoptosis after infection(Fig. 4). The data indicated that enterococcus strains and isolatesinduced HUVEC apoptosis and that apoptosis increased as MOIbecame greater. No significant differences between apoptosispercentages were observed when OG1-SSpP11 and OG1-SSp strainMOI (500:1 and 1000:1) were evaluated (p ¼ 0.136 and p ¼ 0.092,respectively). All apoptosis percentages for enterococcus-infectedHUVEC were significantly higher than those observed in unin-fected HUVEC (base apoptosis). As expected, staurosporine-treatedHUVEC (positive control) had the highest percentage of apoptosis.

2.4. Cytoskeleton role in enterococci internalization by HUVEC

Antibiotic protection assays in the presence of eukaryotic cyto-skeleton inhibitors were carried out to determine the role that thecytoskeleton might play in bacterial entry. The E. faecalis isolatehaving the highest intracellular log10 CFU (9031), and OG1-SSpP11and OG1-SSp strains were used for these experiments. Cells werepre-incubated with different cytochalasin D and colchicineconcentrations which inhibit actin polymerization andmicrotubule

Fig. 2. Enterococcal internalization by HUVEC Log10 CFU counts � SD showing that despiteisolate 1441), bacteria were similarly internalized by HUVEC. S. aureus and B. subtilis were

assembly, respectively [28]. The data showed a decrease in log10CFU values when adding increasing concentrations of each inhib-itor. Fig. 5A shows the results obtained when 1.0, 2.0 and 4.0 mg/mLcytochalasin D were added. Experiments performed in the pres-ence of the inhibitor were compared with their respective controls(without the inhibitor). The statistical analysis showed that log10CFU for S. aureus 6538, E. faecalis OG1-SSpP11 and the E. faecalis9031 clinical isolate were significantly lower (p < 0.05) than theircontrol values at all concentrations tested. The E. faecalis OG1-SSplog10 CFU was significantly lower (p < 0.05) when only 2.0 and4.0 mg/mL cytochalasin D were used. 100% invasion inhibition wasseen when assays with E. faecalis OG1-SSpP11 and 9031 were donein the presence of the highest cytochalasin D concentration tested.The lowest OG1-SSp and S. aureus ATCC 6538 invasion values wereobserved at the same concentration; however, complete invasioninhibition was not detected. Fig. 5B shows data obtained when 10,20 and 40 mg/mL of colchicine were added to HUVEC cultures; allinhibitor concentrations tested significantly reduced bacterialinternalization (p < 0.05). However, 100% invasion inhibition wasonly reached in E. faecalis isolate 9031-infected cultures.

3. Discussion

Enterococcal bacteremia and endocarditis are difficult to treatand can be fatal. The mechanisms by which E. faecalis causesendocarditis are poorly understood and studies have mainly beenfocused on investigating bacterial factors involved in infection andthe bacterial pathways involved in such bacteria moving from thegastro-intestinal tract into the blood stream and tissues in animalmodels [9e12,29e31], as well as assessing E. faecalis entry intocultured epithelial cell-lines, neutrophils and macrophages in vitro[16,19,23,24]. It has already been proven that regardless of beingconsidered an extra-cellular pathogen, E. faecalis can invadeeukaryotic cells [23,24,32] by means of its binding to cell surface[32e36] through adhesins that interact with host cell receptors[14,16,32,36,37]; bacterial adhesion thus activates host signaltransduction cascades and endocytic pathways promotingE. faecalis internalization [24,37,38]. Although E. faecalis role inbacteremias and endocarditis has been established, little is knownabout its interaction with cultured endothelial cells.

Our group has infected HUVEC primary cultures with a clinicalE. faecalis isolate in a previous study; viable intracellular bacteriawere recovered 4 h post-infection in an antibiotic protection assay.It was observed that bacteria adhered to HUVEC and that they hadintracellular localization inside spacious vacuoles. E. faecalis strains

Asc10 presence (OG1-SSpP11 strain and isolate 9031) or absence (OG1-SSp strain andused as positive and negative controls, respectively.

Fig. 3. Transmission electron microscopy (TEM) of infected HUVEC. A. Presence of internalized bacteria in cultured HUVEC; non-invaded cells are also shown to the left-hand andright-hand sides. B. A cell containing a few bacteria. C. An endothelial cell with numerous intracellular bacteria showing chromatin condensation and membrane blebbing. D.Bacteria in close proximity to philopodia-like membrane protrusions and membrane-attached bacteria (arrows). E. and F. Infected HUVEC exhibiting apoptotic changes indicated byarrows (masses of condensed chromatin, apoptotic bodies and nuclear fragmentation). Bars 0.6 mm.

Fig. 4. Apoptosis assessment by flow cytometry. Infected HUVEC were double-labeled with FITC-conjugated annexin V and propidium iodide (PI) to assess bacterial-inducedapoptosis/necrosis.

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e69 65

Fig. 5. Cytoskeleton role in enterococci internalization by HUVEC. A. Log10 CFU counts of infected cells at various cytochalasin D concentrations (1, 2, and 4 mg/mL). B. Log10 CFUcounts of infected cells at various colchicine concentrations (10, 20, and 40 mg/mL). A and B: untreated cells were used as control (C). The graph shows the mean of log10 CFUcounts � SD (p < 0.05) of three independent experiments performed in triplicate.

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e6966

and clinical isolates’ ability to enter cultured HUVEC was investi-gated using positive and negative invasion controls (S. aureus ATCC6538 and B. subtilis). The results showed that E. faecalis was able toenter and survive within HUVEC cells in a similar way to that ofS. aureus and viridian group streptococci [39,40].

Earlier studies have shown that E. faecalis aggregation substanceAsc10 expression facilitates such bacteria’s internalization incultured enterocytes [15,23,41]. Although Asc10� E. faecalis isolate1441 log10 CFU was significantly lower than that displayed byS. aureus 6538 (p ¼ 0.000), OG1-SSpP11 (p ¼ 0.004), OG1-SSp(p ¼ 0.005) and isolate 9031 (p ¼ 0.02) in the present study, bothE. faecalis Asc10þ (OG1-SSpP11 strain (pCF11) and isolate 9031) andE. faecalis Asc10� (OG1-SSp strain and isolate 1441) were able toenter cultured HUVEC. This data thus suggested that Asc10 was nota critical factor for E. faecalis entry into cultured HUVEC. Theaforementioned results were consistent with those previously ob-tained in endocarditis animal models, showing that despite thisprotein contributing to bacterial virulence, enterococcal adherenceto host cells and increased severity of the vegetations so formed, itwas not required for causing the disease [9e11].

The RT-PCR andWestern blot results shown here indicated that,despite E. faecalis 1441 having the prgB gene, it was not transcribed

or expressed in the presence of cCF10. Interestingly, internalizationinto HUVEC was significantly lower for this isolate than for theother Asc10� and Asc10þ strains studied here. This observationmaysuggest that despite Asc10 not being required for E. faecalis entryinto HUVEC, pCF10-related mechanisms involved in the negativeregulation of prgB may play a role in decreasing E. faecalis 1441entry into HUVEC. Data from clumping experiments carried out todetermine whether Asc10 expression in isolate 1441 requiredconditions that were not present in the culture medium used for itsgrowth supported such hypothesis. Transcriptional and post-transcriptional regulatory regions of prgB expression in the pres-ence of pheromone cCF10 have been described [27,42e44]; theimproper functioning of any of them may thus have been pre-venting Asc10 expression.

The role of eukaryotic cytoskeleton elements in bacterial entryto host cells was evaluated once E. faecalis ability to enter culturedHUVEC had been established. A partial (but statistically significant)reduction of intracellular log10 CFU in the presence of both inhibi-tors was found, thereby indicating that E. faecalis internalizationpartially relied on endothelial cell cytoskeleton components.Similar results have been found when studying E. faecalis inter-nalization in cell-lines (HeLa, HT-29, Caco-2), peritoneal

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e69 67

macrophages and neutrophils [16,19,23,24,37,38], suggesting thatthe underlying mechanism might be common for non-phagocyticand phagocytic cells. The experiments shown here did not lead toascertaining whether cytoskeleton rearrangements were beinginduced by E. faecalis isolates or directed by endothelial cells;however, finding that this bacterium could enter non-phagocyticcells led us to suggest that this microorganism might be able tocross endothelial barriers.

Various studies have shown that cultured endothelial cellsundergo apoptosis after being invaded by bacteria (i.e. S. aureus,Streptococcus sp., Pseudomonas aeruginosa, and Porphyromonasgingivalis) [39,45e48]. Studies using non-endothelial cell-lines(macrophages, HeLa, U937, J774A.1, Jurkat J16, thymocytes) havealso shown that E. faecalis and Enterococcus faecium strains caninduce cell death without invading cells [49,50]; however, this isthe first report to describe enterococcus-induced apoptosis inprimary HUVEC. Transmission electron microscopy (TEM) imagesclearly displayed cells having characteristic apoptotic features.

Evaluating the effect of MOI on HUVEC apoptosis showed thatthis increased as MOI increased; internalized bacteria were onlyobserved when the highest MOI (1000:1) was tested. No or lowapoptosis was seenwhen using the lowest MOI. The data suggestedthat some bacteria studied here (OG1-SSpP11 and OG1-SSp) couldinduce HUVEC apoptosis without entering cells.

Overall, the data indicated that E. faecalis entered endothelialcells, cell apoptosis increased and that cytoskeleton componentswere partially involved. The data also suggested that Asc10 was nota critical factor for E. faecalis entry into cultured HUVEC.

4. Materials and methods

4.1. Bacterial strains

E. faecalis clinical isolates 9031, 1441 and OG1-SSpP11 and OG1-SSp strains (Asc10þ and Asc10�, respectively) were used; S. aureus(ATCC#6538) and B. subtilis (ATCC#8037) strains were included aspositive and negative invasion controls, respectively. All strainswere grown in brain heart infusion (BHI) broth and agar andincubated at 37 �C.

4.2. Gene evaluation

Colony PCR detected the Asc10-encoding prgB gene in E. faecalisclinical isolates; E. faecalis OG1-SSpP11 and E. faecalis OG1-SSpstrains were included as positive and negative prgB gene amplifi-cation controls, respectively. The pCF10 (prgB) nucleotide sequence(GenBank ID: NC_006827.2) was used as template for designinga set of primers for gene amplification; primers were designed withGene Runner v3.05 software. prgB-forward 50-CTTTGGTTCAGGTG-TAGGTCT-30 and prgB-reverse 50-CTTGCGTCTGCTTGATAAACT-30

primers were used for amplifying a 441 bp region. The amplifica-tion was carried out using BIOLASE DNA polymerase enzyme(Bioline, London, UK) in a 10 mL final reaction, according to themanufacturer’s instructions. Amplification conditions were asfollows: a 5-min cycle at 95 �C, followed by 35 cycles of 1 min at60 �C, 1 min at 72 �C and 1 min at 95 �C, followed by a final 10-minextension step at 72 �C. Products were visualized on 1% agarose gel.

4.3. Evaluating Asc10 transcription and expression by RT-PCR andWestern blot

Overnight cultures (OD600 z 0.1) of E. faecalis clinical isolates9031, 1441 and OG1-SSpP11, OG1-SSp strains were diluted (1:4) inBHI broth; pheromone (25 ng/mL of cCF10) was added to stimulateAsc10 expression. Cultures were incubated at 37 �C for 3 h with

continuous shaking (150 rpm) and then centrifuged (180� g,20 min). The resulting pellet was washed with PBS (pH: 7.4) andused for RT-PCR and Western blot.

Total RNA was extracted by the Trizol method for the RT-PCRassay and then treated with RNase-free DNase for 30 min at 37 �C(Epicentre Biotechnologies, Madison, WI, USA). One microgram ofRNA was used as cDNA synthesis template using Superscript IIIenzyme (Invitrogen, Carlsbad CA) and prgB primers in a 5-min cycleat 65 �C, followed by 60 min at 50 �C, followed by a final 15-mincycle at 70 �C. RNA extracted from the samples but not treatedwith Superscript III was used as control for cDNA synthesis. The RT-PCR conditions used were the same as those described for thecolony PCR.

Western blot involved the pellet being suspended in extractionbuffer (6e8 M urea, 15 mM imidazole, 10 mM TriseHCl, 100 mMNaH2PO4, 1 mg/mL lysozyme) and lysed by sonication. Thesuspension was centrifuged at 12,000� g for 10 min, the superna-tant was collected, the total amount of protein was measured (BCAassay) and samples were stored at �20 �C for Western blot.

Samples were separated by 8% SDS-PAGE and electro-transferred to nitrocellulose membrane for Western blot analysis.The membrane was blocked with 5% skimmed milk in PBS-0.05%Tween 20 and washed thrice with PBS-0.05% Tween 20 for 5 min.A polyclonal anti-Asc10 antibody (1:5000), kindly provided by Dr.Gary M. Dunny, was used to detect the Asc10 adhesin. After incu-bating for 2 h, the membrane was washed twice with PBS-0.05%Tween 20, and incubated with phosphatase-coupled anti-rabbitIgG (Pierce, Rockford, IL, USA) (1:5000 dilution) for 2 h. A BCIP/NBTkit (Promega) was used as developing solution, according to themanufacturer’s instructions.

4.4. Clumping experiments

Overnight cultures (OD600 z 0.1) of E. faecalis clinical isolates9031 and 1441, as well as OG1-SSpP11 and OG1-SSp strains werediluted (1:4) in BHI broth and 25 ng/mL of cCF10 pheromone wasadded to stimulate Asc10 expression. Cultures were incubated at37 �C for 3 h with continuous shaking (150 rpm) and then centri-fuged (180� g, 20 min). The 9031 supernatant was filtered usinga 0.2 mm-pore membrane, diluted 1:2 in BHI broth and added toa 1441 inoculum to assess whether bacteria clumped as a result ofASC10 expression due to certain conditions present in the mediumculture inwhich the 9031 (ASC10þ) clinical isolate had been grown.Bacteria were grown for 4 h and clumping was then assessed.

4.5. HUVEC culture

Human umbilical vein endothelial cells (HUVEC) (Gibco/Invi-trogen, Carlsbad, CA, USA) were used for establishing endothelialcell cultures, following the supplier’s indications. Cells werecultured in T75 culture dishes (Falcon, Corning Costar, Bodenheim,Germany) with 200PRF medium (Gibco/Invitrogen, Carlsbad, CA,USA) supplemented with LSGS (Gibco/Invitrogen, Carlsbad, CA,USA) and incubated (5% CO2 at 37 �C) until reaching 80% conflu-ence. A trypsin/EDTA solution (Gibco/Invitrogen, Carlsbad, CA, USA)and a trypsin neutralizer solution (Gibco/Invitrogen, Carlsbad, CA,USA) were added to sub-confluent cultures for sub-culturing,following manufacturer’s instructions. Cell viability was deter-mined with trypan blue staining.

4.6. Assessing Asc10 role in E. faecalis entry to HUVEC (antibioticprotection assays)

Invasion assays were carried out following previously describedprotocols [51], with a few modifications. Briefly, 1 mL of an O/N

D. Millán et al. / Microbial Pathogenesis 57 (2013) 62e6968

bacterial culture was added to 9 mL of RPMI 1640 medium andincubated until the logarithmic growth phase was reached. HUVECcells were seeded at 2.5 � 104/well density into 12-well cultureplates (BD-Biosciences) and incubated with 200PRF-500 medium(5% CO2 at 37 �C) for 36 h to double the cell population. Cell numberwas assessed using a vital stain (trypan blue). The culture mediumwas then replaced with antibiotic-free RPMI 1640 supplementedwith 10% fetal bovine serum (FBS) and cultures were incubated foran hour prior to the invasion assay. The enterococci concentrationwas spectrophotometrically adjusted to 1.5 � 108 CFU/mL at600 nm and confirmed by dilution plating. Thereafter, bacteriawere suspended in RPMI 1640 mediumwithout antibiotics and theresulting suspensionwas sonicated at 20W for 10 s using a Bransondigital sonifier (Branson, Los Angeles, USA) to avoid bacterialclumping [41]. Cultured HUVEC were incubated using a 1000:1(bacteria:host cell) multiplicity of infection (MOI). This value waschosen since preliminary assays at different MOI (100:1, 500:1,1000:1) showed internalized bacteria when using it. After co-culturing for 2.5 h in 5% CO2 at 37 �C, infected cultures werewashed with PBS and incubated for 2 h with RPMI medium sup-plemented with gentamicin (500 mg/mL) to kill extra-cellularbacteria for clinical isolates, while 250 mg/mL ampicillin was usedto kill OG1-SSpP11 and OG1-SSp strains. Supernatant aliquots wereremoved and plated to confirm extra-cellular bacterial death. Afterwashing with PBS, cells were lysed with 1% Triton X-100 bypipetting several times. Serial dilutions of 100 mL of lysate fromeach well were plated onto BHI agar and incubated (24 h at 37 �C).Intracellular bacteria were calculated by counting CFU. Similarexperiments were carried out using non-invasive B. subtilis andinvasive S. aureus. The assays were performed in triplicate andrepeated independently at least three times.

4.7. Cytoskeleton role in enterococci internalization by HUVEC

After establishing that cytoskeleton inhibitors were harmless forHUVEC, cells were sub-cultured into 12-well culture plates andincubated for 24 h (37 �C, 5% CO2). Thereafter, the medium wasreplaced with RPMI 1640 supplemented with cytochalasin D (1, 2,and 4 mg/mL, SigmaeAldrich) or colchicine concentrations (1, 10, 20and 40 mg/mL, SigmaeAldrich); the cultures were incubated (1 h,37 �C) to inhibit microfilament and microtubule polymerization.Cells were washed with PBS 1� and infected with E. faecalis strainsand isolates as described. After antibiotic treatment and washingwith PBS, cells were lysed with 1% Triton X-100 and repeatedpipetting. Serial dilutions of 100 mL of lysate from each well wereplated onto BHI agar and incubated (24 h at 37 �C). Intracellularbacteria were calculated by counting CFU.

Cells were cultured in medium supplemented with the sameconcentrations used in the invasion assays and cell viability wasassessed with a vital stain (trypan blue) to ascertain that cyto-skeleton inhibitors were harmless to HUVEC.

4.8. Transmission electron microscopy (TEM) of infected HUVEC

Infected confluent HUVEC werewashed with PBS and processedfor TEM. Briefly, HUVEC incubated with the enterococcal isolatesbeing studied and strains were washed with PBS and incubatedwith trypsin/EDTA (Gibco Invitrogen, Carlsbad, CA, USA) followingthe supplier’s indications. The cell suspension was collected andcentrifuged (180� g); the resulting pellet was fixed with 3%glutaraldehyde in PBS (pH: 7.4) at 4 �C and post-fixed in 1% osmiumtetroxide for 1 h at 4 �C. Fixed cells were washed and dehydrated ina graded ethanol series and embedded in LR-White resin (SPIsupplies, PA, USA). Sections were cut with a glass knife on anultramicrotome (LKB-Ultrome), stained with saturated aqueous

uranyl acetate and Reynold’s lead citrate and then observed bytransmission electron microscope (JEOL JEM-1200 EX TEM).

4.9. Apoptosis assessment (flow cytometry analysis)

E. faecalis-infected HUVEC apoptosis was assessed by flowcytometry. HUVEC cultures infected at different MOI (100:1, 500:1,1000:1) were assayed to determine whether cells underwentapoptosis after E. faecalis infection and the effect of bacterialnumber on HUVEC viability was compared. Infected cells weredetached with trypsin/EDTA (Gibco Invitrogen, Carlsbad, CA, USA)and a trypsin neutralizer solution (Gibco/Invitrogen, Carlsbad, CA,USA) was then added, following the supplier’s instructions. HUVECincubated with bacterial strains and clinical isolates were thenlabeled with a FITC Annexin V Apoptosis Detection Kit I (BectonDickinson), according to the manufacturer’s instructions. HUVECwith staurosporine (2 mM final concentration) were incubated for3 h (37 �C in 5% CO2) and used as a positive apoptosis control. Atleast 20,000 cells were counted in each sample. A FACScan flowcytometer was used for reading the samples (Becton Dickinson,Mountain View, CA, USA).

4.10. Statistical analysis

All total CFU numbers were converted to log10 values beforecarrying out the statistical analysis. Data from E. faecalis entry tocultured HUVEC were reported as the mean (�SD) of three log10CFU counts obtained in at least three independent experiments. Aone-way ANOVA with Scheffe’s post-hoc adjustments was per-formed using STATA software to statistically determine significantdifferences between CFU data from the antibiotic protection assayscarried out with the bacterial strains and clinical isolates evaluatedhere. Data from experiments using bacteria tested in the presenceor absence of invasion inhibitors (colchicine and cytochalasin D)were compared using a one-tailed Student’s t-test. In all casesa p < 0.05 value was considered statistically significant.

Acknowledgments

We would like to thank Dr. Ricardo Sanchez for his assistancewith the statistical analysis, Dr. Jinnethe Reyes and Dr. MarthaGarzón for providing us with clinical isolates and Dr. Gary Dunnyand Dr. Dawn A. Manias for providing OG1-SSpP11 and OG1-SSpstrains, Dr. Diego Garzón and Dr. Andrea Castillo for their assis-tance with experimental assays and Jason Garry for reviewingEnglish style and expression. This study was partly financed byColciencias (contract 13080412635). We would like to dedicate thisarticle to the lovingmemory of Doctor Carlos Chiriboga, a long-timefriend who passed away recently.

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