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Istituto Superiore di Sanità, Rome, Italy
Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Enterococcus faecalis is responsible for a large variety of nosocomial infections. The intestinal barrier is thought to be one of the preferential portals of entry of enterococci, and the ability of E. faecalis to survive within peritoneal macrophages may contribute to spreading to distant sites. We examined the ability of a polysaccharide-expressing (biofilm-positive) E. faecalis strain and an isogenic biofilm-negative mutant to enter and survive within professional and nonprofessional phagocytes. Biofilm-positive bacteria survived longer in all cell systems than did biofilm-negative bacteria, through a process of receptor-mediated endocytosis that is dependent on functional reorganization of microtubules and polymerization of microfilament and on activation of protein kinases but not ATPases or protein phosphatases. We suggest that glycosaminoglycansspecifically heparin, heparan sulfate, and chondroitin sulfate Aare the host receptors for enterococci on professional and, possibly, nonprofessional phagocytes, allowing entry of enterococci into cell compartments where killing mechanisms are inhibited.
c Enterococci are important opportunistic pathogens and one of the leading causes of endocarditis and nosocomial bacteremia in the United States [13] and northern Europe [4]. The mechanisms involved in the pathogenicity of these microorganisms are not well understood, although some putative virulence factors have been described. Besides several surface proteins, enzymes, and capsular polysaccharides possibly involved in virulence, the ability of Enterococcus faecalis to survive inside polymorphonuclear leukocytes (PMNLs) [5] and macrophages [68] has been observed. Survival of E. faecalis within peritoneal macrophages may contribute to pathogenesis by facilitating spread to distant sites [9] after translocation through the intestinal barrier.
In recent studies [7, 8], we hypothesized that biofilm-forming E. faecalis was more resistant to killing by rat peritoneal macrophages than were biofilm-negative organisms. These data were obtained by growing strains in different media that either supported or inhibited biofilm formation. In the present study, we used a previously described biofilm-forming E. faecalis strain and its biofilm-negative transposon mutant derivative [10]. The term "biofilm formation" was used in the original article to discriminate between mutants; strain 10D5, used here, was transposon-inserted into a 4-gene locus named "biofilm on plastic surfaces" (bop), which showed a polar effect on the downstream regulatory gene bopD. On this basis, the isogenic pair used here will be referred to as biofilm-positive and biofilm-negative, for the sole purpose of strain identification. We examined the ability of these strains to enter and survive within rat and human macrophages and dendritic cells. Inhibitors of various cell structures or specific functions were also used to identify the pathway of entry and the cell receptors involved.
MATERIALS AND METHODS
Bacterial strains.
An isogenic pair of E. faecalis strains, type 9 and 10D5, created previously by Hufnagel et al. [10], were used to evaluate the role that extracellular polysaccharides play in enterococcal survival within macrophages. The wild-type (wt) E. faecalis strain, initially characterized by Maekawa et al. [11], produces high levels of biofilm. The biofilm-negative mutant (10D5), created by transposon insertion mutagenesis, is still able to form biofilm, but at levels 5-fold less than the parent strain. Bacteria were grown at 37°C without agitation, in either plain trypticase soy broth (TSB) or TSB supplemented with 1% glucose (TSBG). E. faecalis 10D5 was grown in the same media, supplemented with erythromycin at 10 g/mL.
Chemicals and antibodies.
Chemicals and antibodies used in the present study were purchased from Sigma, except where specified, and are presented in the following list:
Chemicals: cytochalasin D, colchicine, sodium ortho-vanadate, amiloride, ammonium chloride, monodansylcadaverine (MDC), wortmannin, and staurosporine;
Sugars: mannose, laminarin, mannan, fucoidan, and -mannose 6-P;
Antibodies: -CD11b, -CD206 (PharMingen), -CD14, -CD62L (Chemicon), and -CD62L (Sigma); and
Glycosaminoglycans (GAGs): heparin, heparan sulfate, mucin, and chondroitin sulfate A, B, and C.
Reagents were added to the cell culture medium at different concentrations; possible effects on cell and bacterial viability were tested by trypan blue dye exclusion test and colony-forming unit counting, respectively.
Cells.
HeLa cells (American Type Culture Collection) were cultivated in MEM supplemented with 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 100 IU/mL penicillin, 100 g/mL streptomycin, and 10% fetal calf serum (FCS). Peritoneal macrophages were harvested from adult female rats as described elsewhere [7]. Briefly, a peritoneal lavage was performed with 2 applications of 5 mL of Hanks' balanced salt solution. Viability of recovered cells was determined by trypan blue dye exclusion test. Cell suspensions were normalized to 2.5 × 105 cells/mL in RPMI 1640 medium supplemented with 1% FCS, dispensed into 24-well plates, and incubated for 30 min at 37°C in an atmosphere of 5% CO2. Unattached cells were removed by washing, and macrophages were incubated for an additional 24 h at 37°C before being used in experiments.
Human monocytic cells were isolated from heparinized venous blood of healthy volunteers by Ficoll-Hypaque density-gradient centrifugation and hypotonic lysis of residual erythrocytes. Mouse fetal skinderived dendritic cells (FSDCs) [12] were grown in Dulbecco's modified Eagle medium supplemented with 10% FCS, 1% pyruvate, 1% glutamine, 1% nonessential amino acids, 0.002% -mercaptoethanol, 100 IU/mL penicillin, and 100 g/mL streptomycin.
THP-1, a myelomonocytic human cell line expressing Fc and C3b receptors [13], was maintained in RPMI 1640 medium supplemented with 10% FCS and 2 mmol/L glutamine in an atmosphere of 5% CO2. Cells were differentiated by incubation with phorbol miristate acetate (0.16 mol/L; Sigma) for 24 h at 37°C in 24- or 96-well plates, as described by Scorneaux et al. [14].
Adherence/invasion assay.
Cells were prepared as described and seeded in 24-well plates at different concentrations. Bacterial cells grown overnight in TSB or TSBG were washed and suspended in the cell medium appropriate to give a bacteria-to-cell ratio of 100 : 1. Epithelial cells were infected for 2 h at 37°C, washed, and further incubated for 3 h in medium supplemented with 10% FCS and 250 g/mL gentamicin. Macrophages were infected for 1 h at 37°C, thoroughly washed with PBS, and further incubated, for 3, 24, and 48 h, in medium supplemented with 10% FCS and 250 g/mL gentamicin. At the different time points, duplicate wells were washed and lysed with 0.1% Triton X-100 in PBS for 5 min. Lysates were diluted in PBS and plated on TSB agar plates to quantitate viable bacteria. Throughout the experiment, viability of cells was confirmed by trypan blue dye exclusion test and counting with a hemocytometer. Except for the treatment with NH4Cl and amiloride, which is described below, inhibitors were added to cells 30 min before infection and remained in the medium throughout the experiment. Antibodies were added 15 min before infection and remained in the medium for the first hour (adherence stage) of infection.
Treatment with NH4Cl and amiloride.
Cytosol acidification was performed in accordance with the method of Reynolds et al. [15]. Briefly, monolayers were pretreated with NH4Cl (Merck) (80 mmol/L in MEM) for 35 min and washed, and then the medium was replaced with MEM containing 3 mmol/L amiloride (Sigma-Aldrich). After 5 min of incubation, cells were washed, infected with E. faecalis, and processed as described for the invasion assay.
Biofilm formation.
Stability of the mutant strain was evaluated throughout the study by testing for biofilm formation. We used a quantitative adherence assay described elsewhere [7]; the optical density of the biofilm formed on the bottom of polystyrene plates and stained with Hucker's crystal violet was measured at 570 nm in an automatic spectrophotometer (Bio-Rad Laboratories).
Polymerase chain reaction (PCR).
Genes encoding aggregation substance (AS), cytolysin (cylA), gelatinase (gelE), and enterococcal surface protein (esp) were analyzed by PCR, as described elsewhere [16].
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
Infected cells were fixed, dehydrated, and embedded, as described elsewhere [8]. In some experiments, macrophages were pulsed with cationized ferritin (Sigma) in RPMI 1640 medium, at a final concentration of 0.2 mg/mL, for 3 h at 37°C before infection [17]. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by use of an EM 208 Philips electron microscope. For SEM, fixed samples were critical-point dried after ethanol dehydration, gold sputtered, and examined by use of a Cambridge SE360 scanning electron microscope.
Statistical analysis.
Data were analyzed for statistical significance by the t test, by use of PRISM (version 3; GraphPad software).
RESULTS
Survival of E. faecalis inside rat peritoneal macrophages or human monocytes.
We have previously reported [7, 8] that E. faecalis strains were able to survive within rat peritoneal macrophages for up to 48 h. In our model, survival seemed to be related to the ability of bacteria to synthesize extracellular polysaccharide (i.e., to form biofilm) induced by the presence of an additional carbohydrate source in the medium. To confirm the role of extracellular polysaccharides, an isogenic pair of strains was used [10]. The parent strain was able to form high-density biofilm. The biofilm-negative mutant expressed 5-fold less biofilm than did the parent strain. Characteristics of the strains used in the present study are listed in table 1. We also found that the parent strain expressed large amounts of biofilm in the presence of additional glucose in the medium (TSBG), whereas, in unsubstituted TSB (containing 0.25% glucose), biofilm formation was comparable to that of the mutant strain (table 2). Quantitative biofilm evaluation by standard plate test was performed to ensure the maintenance of the phenotype.
Survival of E. faecalis type 9 and its biofilm-negative mutant was tested in rat peritoneal macrophages, a mouse dendritic cell line, human monocytes, and a myelomonocytic human cell line. In all cell systems, the biofilm-positive strain survived longer than its biofilm-negative mutantin rat peritoneal macrophages (figure 1A), biofilm-positive and biofilm-negative cells showed a decrease of 2 and 4 log units, respectively, at 3 h after infection; biofilm-negative cells were rapidly killed thereafter, with complete clearance of the bacteria at 24 h. Biofilm-positive cells persisted up to 48 h, which was the last time point examined, because after that time viability of both infected and noninfected macrophages started to decline. Notably, E. faecalis type 9 grown in TSB, thus not producing extracellular polysaccharides, exhibited behavior comparable to that of the biofilm-negative mutant (figure 1A). In human monocytes, survival was studied up to 24 h (figure 1B), and behavior similar to that in rat macrophages was observed. In THP-1 cells, a decrease of 1 and 2.5 log units of E. faecalis type 9 and 10D5, respectively, was observed at 3 h after infection (figure 1C). Clearance of biofilm-negative cells was complete by 48 h.
Survival of the isogenic pair in FSDCs was tested only at 1 h. E. faecalis type 9 invaded and survived 3 times longer than did 10D5 (figure 2). In preliminary long-term invasion experiments, bacteria were cultivable for 48 h, with declining numbers. By TEM, intact intracellular bacteria were still detectable at 120 h after infection. Long-term invasion experiments were also conducted with mouse bone marrowderived dendritic cells (harvested fresh from the femurs and tibias of female C57BL mice), confirming the faster clearance of 10D5, compared with that of E. faecalis type 9 (not shown).
In parallel, we performed experiments with HeLa cells in which bacteria were allowed to adhere for 1 h before washing and addition of antibiotics. Under these conditions, no differences were observed in the adhesion/entry rates of E. faecalis type 9 and 10D5 (figure 3). However, by prolonging the adhesion step for an additional hour, the process appeared to be more specific, and surviving biofilm-positive cells could be recovered at 3 h after addition of antibiotics, although biofilm-negative cells could not (figure 3).
Entry into human macrophages via receptor-mediated endocytosis.
The THP-1 system was chosen for further characterization of the mechanism of entry of the polysaccharide-positive enterococcal strain into macrophages. Different inhibitors of cellular structures (e.g., microtubules) and functions (e.g., kinases) were tested. All inhibitors were added 30 min before infection and were maintained in the medium throughout the experiment, except where indicated otherwise. Bacteria were incubated with the cells for 1 h at 37°C. After removal of unbound bacteria by washing, gentamicin was added to kill extracellular bacteria, and incubation was prolonged for 3 h.
Cytochalasin D, which inhibits the polymerization of filamentous actin, reduced the number of viable intracellular bacteria in a concentration-dependent manner, up to 80% (figure 4A). When, in control experiments, phalloidin was used to stain the actin cytoskeleton of macrophages, typical actin cups could be observed at the sites of attachment of both E. faecalis type 9 and 10D5 (data not shown). Notably, the number of actin cups observed per macrophage was similar with biofilm-positive and biofilm-negative bacteria, indicating that the presence of extracellular polysaccharide did not prevent signaling to actin polymerization.
Colchicine, which blocks the elongation of microtubules, reduced entry of E. faecalis type 9 into THP-1 cells by 58% at the highest concentration used (figure 4B) and of biofilm-negative cells by 62%. MDC, an inhibitor of receptor-mediated endocytosis, dramatically reduced the number of viable intracellular biofilm-positive bacteria, by 90% (figure 4C); 10D5 cells were affected to a significantly lesser extent (27%) (figure 4C).
Staurosporine (figure 4D) and wortmannin (figure 4E), inhibitors of different classes of protein kinases and of phosphatidylinositol-triphosphate kinase (P13K), respectively, strongly inhibited entry of biofilm-positive bacteria. Sodium ortho-vanadate, which inhibits tyrosine phosphatases and ATPases, did not affect entry of enterococci at any concentration tested (30, 100, and 300 mol/L) (data not shown).
The role that cytosol acidification plays in the invasion process was determined by prepulsing of cell monolayers with NH4Cl and amiloride. Neither pretreatment of cells with 80 mmol/L NH4Cl (to lower the cytoplasmic pH) nor amiloride treatment alone appeared to interfere with entry of enterococci (figure 4F). On the other hand, pretreatment of cell monolayers with both drugs markedly decreased entry of enterococci into cells (figure 4F).
In an attempt to characterize the putative receptor involved in entry of biofilm-positive enterococci into macrophages, mannose, mannose 6-P, laminarin, and mannan were used as antagonists of -glucan and macrophage mannose receptors (MMRs). A partial effect (50% inhibition) was observed for all sugars tested (figure 5), suggesting that either CR3 or MMR might be involved. Thus, antibodies to CD11 and CD206 were used to block bacterial adherence to macrophages, but no effect was observed at any of the concentrations tested (up to 30 g/mL). We also followed the recommendations of Vanek et al. [18], who reported that L-selectin possibly contributes to enterococcal adhesion to PMNLs. By use of monoclonal antibodies (MAbs) against L-selectin (Chemicon epitope unknown; Sigma-lectinbinding domain specific), no reduction in adherence or entry was observed.
Electron microscopy showed that numerous cells could adhere to macrophagesthe entry process started with the formation of small ruffles closely encircling bacterial cells (figure 7A and 7B). Well-preserved E. faecalis type 9 were present inside macrophage vacuoles at 24 h after infection, as either single or multiple cells contained within vacuoles (figure 7C). Intact biofilm-negative bacteria were more difficult to trace, even at 3 h after infection. In both cases, results obtained with ferritin-pulsed macrophages indicated that lysosome-phagosome fusion did occur (figure 7D).
DISCUSSION
E. faecalis is an important pathogen of nosocomial infections that causes a substantial percentage of community-acquired urinary tract and wound infections, bacteremia, and endocarditis [13]. Enterococcal translocation from the intestine has been suggested as one of the primary sources of such infections [9], and this hypothesis is also supported by the recent reports of the ability of enterococci to survive within macrophages [68] and PMNLs [5]. Sussmuth et al. [19] showed that the RGD sequences present in the pAD1-encoded AS were responsible for binding to integrins, fast uptake by macrophages, and resistance to intracellular killing during the first 3 h after infection. In 2 recent studies [7, 8], we hypothesized that extracellular polysaccharides could be responsible for adhesion to macrophages and intracellular survival. We observed that killing rates remained different at later time points for biofilm-positive and biofilm-negative enterococci, suggesting that the survival ability could be attained through a route different from the one proposed by Sussmuth et al. [19]. In the present study, using a strong biofilm-positive wt strain and its isogenic biofilm-negative mutant, we found further evidence that the ability to express extracellular polysaccharides appears to be associated with delayed killing by macrophages. Four different cell-culture models of professional phagocytes were used: in all systems, E. faecalis type 9 survived longer than did its biofilm-negative mutant. Furthermore, E. faecalis type 9 grown in TSB without additional glucose (i.e., not forming biofilm) was comparable to the biofilm-negative mutant. Interestingly, similar behavior was observed with epithelial, nonprofessional phagocytes.
THP-1 cells were chosen for further characterization of entry of enterococci and the survival process because they are easy to handle and reproduce. When E. faecalis type 9infected macrophages were examined by SEM at different time points, almost all cells showed adhering/entering bacterial cells, mainly in aggregates. However, to detect macrophages phagocytosing 10D5 cells, >500 cells had to be examined. In these cases, again, small aggregates were associated with amorphous material, which suggests that the mutant cells are more readily phagocytosed, possibly because they present themselves as single entities, whereas cell aggregates, such as those of the wt strain or the few cells of the mutant strain still able to produce aggregating extracellular material, take longer to be ingested.
A variety of bacteria bind to phagocytes through the CR3 or the mannose receptor [1921], since this opsonin-independent binding allows bacteria to bypass the bactericidal process in macrophages and contributes to intracellular survival rather than killing. We could rule out that binding to macrophages of biofilm-positive E. faecalis was mediated by CR3. Partial inhibition by laminarin, a (1-3)(1-6) glucan, initially suggested that CR3 or dectin-1 [22] was involved in bacterial adherence. On the other hand, mannan is known to inhibit the MMR receptor but to have only a limited effect on nonopsonic phagocytosis mediated by dectin-1. In any case, neither MAbs to CD11b nor MAbs to CD206 (MMR) had any effect on enterococcal adherence. For other pathogens, such as group B streptococci, it has been observed that capsular polysaccharide does not influence adherence to epithelial cells but does reduce internalization [23]. Therefore, we tested all the above-mentioned antagonists to determine their effect on the adherence or entry step, but neither seemed to be affected. As well, neither of 2 MAbs to different epitopes of L-selectin affected binding/entry of either of our strains to macrophages. Since information regarding the biological activities of these antibodies is scarce, we also attempted to measure the reactivity of THP-1 cells to the different antibodies, before and after bacterial infection, by flow cytometry. If one of these specific antigens was indeed the receptor for enterococci, the percentage of THP-1 cells reacting to a given antibody would be expected to differ between infected and noninfected cells. However, no differences were observed with either antibody used (data not shown), indicating the involvement of receptors different from those tested.
Numerous sugars appeared to interfere, to various degrees, with attachment of the E. faecalis type 9 isolate to macrophages, particularly fucoidan. Further testing revealed that chondroitin sulfate A and heparin/heparan sulfate mediate a specific interaction of E. faecalis with macrophages. Adhesion of E. faecalis type 9 was inhibited by almost 90% by these molecules, whereas the effect of the biofilm-negative mutant was much lower. It is well known that GAG-binding surface molecules are used by different pathogens [2426] that thereby gain entry into macrophages while bypassing the bactericidal response. Recently, Baron et al. [27] found that Streptococcus agalactiae adheres to glycoconjugates present on vaginal epithelial cells through -C protein. A protein structurally similar to -C protein (Esp) was also expressed by the enterococci used in the present study; Toledo-Arana et al. [28] and Tendolkar et al. [29] have suggested that esp is involved in biofilm formation of enterococcal strains that are not very strong producers of biofilm [28]. However, biofilm has also been shown to be formed by mechanisms independent of esp [7, 16, 30]. Preliminary observations [31] indicate that the transcriptional regulator bopD asserts pleiotropic effects that up-regulate (and down-regulate) several genes involved in exopolysaccharide biosynthesis and possibly other metabolic pathways. It may even be that the observed effects on internalization and survival of enterococci within macrophages are due to extra production of polysaccharides, without bacteria ever going through the biofilm state. As well, surface molecules other than extracellular polysaccharides may contribute to the adherence and intracellular survival of enterococci observed in the present study.
We investigated whether the binding of biofilm-positive enterococci to the cell membrane started a signal cascade mediated by kinases. Staurosporine is a potent inhibitor of different classes of phosphokinases, including protein kinase C, some phosphotyrosinekinases, and cAMP-dependent protein kinase. Wortmannin blocks P13K, and ortho-vanadate inhibits tyrosine phosphatases and ATPases. Binding of E. faecalis type 9 was reduced by both wortmannin and staurosporine (the latter reduced binding by a slightly lesser extent), and no effect was observed with ortho-vanadate. Together with the observed effect of cytochalasin D and colchicine and the effects of cytosol acidification, the data obtained indicate that entry of the biofilm-positive strain is mediated by a process of receptor-mediated endocytosis that is dependent on functional reorganization of microtubules and polymerization of microfilament and on activation of protein kinases but not ATPases or protein phosphatases. We recently demonstrated the involvement of a receptor-mediated endocytosis mechanism for invasion of HeLa cells by clinical isolates of E. faecalis [32], a finding that is confirmed by the observed behavior of E. faecalis type 9 and 10D5 with both phagocytic and nonphagocytic cells.
bopD-controlled genes appear to enable enterococci to establish a successful infection in multiple ways: first, by supporting colonization of inert surfacessuch as prosthetic valves, catheters, and biliary stentsthrough synthesis of extracellular polysaccharides and biofilm formation; and, second, by allowing bacteria to penetrate inside both professional and nonprofessional phagocytes, facilitating translocation through the intestinal barrier and/or dissemination to distant, sterile sites of the host. Further studies are needed to evaluate which specific process, ultimately controlled by the transcriptional regulator bopD, stimulates enterococcal endocytosis through a route that allows survival inside different cell types.
Acknowledgment
We wish to thank D. L. Hasty for helpful discussion.
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