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1Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 2Department of Food Sciences, ARO, the Volcani Center, POB 6, Bet Dagan, 50250 Israel
Correspondence Shlomo Sela shlomos{at}volcani.agri.gov.il
Received March 20, 2003
Accepted September 8, 2003
The fate of GAS and epithelial cells following internalization was determined in this study. HEp-2 cells harbouring intracellular bacteria were treated with antibiotics to kill extracellular adherent bacteria, washed, and the fate of bacteria and epithelial cells was assessed up to 24 h post-infection. In the absence of antibiotics, massive bacterial growth was apparent in the cell medium, accompanied by extensive cell death, suggesting that intracellular bacteria had multiplied and damaged the monolayer. Addition of the internalization inhibitor, cytochalasin D, either pre- or post-internalization prevented bacterial growth and cell injury; post-internalization treatment with chloramphenicol had the same effect. Analysis of three apoptotic markers in HEp-2 cells – chromatin condensation, DNA laddering and translocation of phosphatidylserine onto the cell-surface membrane – indicated that HEp-2 cells underwent apoptosis. Taken together, the data presented here support a model in which intenalized bacteria can induce their own externalization into the medium by a process that requires both an intact host-cell cytoskeleton and de novo synthesis of bacterial proteins. Concomitantly, intracellular and, apparently, extracellular free bacteria induce apoptosis through their cytotoxic activity, and release essential nutrients required for their growth.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Much of the knowledge regarding GAS internalization and intracellular survival has been gained from studies that employed the conventional antibiotic-protection assay, in which cultured epithelial cells are infected with bacteria for a predetermined time. The infected cells are then washed to remove unattached bacteria and antibiotics that cannot enter the cells are added to the culture medium. In this way, extracellular adherent bacteria are killed, while intracellular protected bacteria remain viable (LaPenta et al., 1994). By means of the conventional antibiotic-protection assay, earlier studies demonstrated that the number of intracellular bacteria gradually decreases with time until no viable bacteria are recovered. Various GAS strains were shown to survive intracellularly from 24 h up to 7 days (LaPenta et al., 1994; Greco et al., 1995; Österlund & Engstrand, 1995). Furthermore, when an infected monolayer, incubated for 7 days with antibiotic-containing medium, was washed and incubated with fresh antibiotic-free medium, bacteria reappeared in the fluid, which suggested that GAS is capable of cell externalization (Österlund & Engstrand, 1995). It was hypothesized that an increased internalization rate and prolonged intracellular survival are associated with persistent GAS carriage and recurrent infections (Österlund & Engstrand, 1995; Österlund et al., 1997; Neeman et al., 1998; Sela & Barzilai, 1999; Sela et al., 2000). Although the antibiotic-protection assay seems to be a valuable test for measuring internalization, it might suffer from limitations in the study of intracellular survival during long periods following internalization. Since, the majority of survival studies used media that contained antibiotics, it is possible that differences among strains in their survival rates were actually related to differences in their capacity to escape from the cell into the extracellular medium, rather than to bona fide intracellular viability.
To gain better understanding of the interaction between GAS and epithelial cells, the fate of the bacterium and the HEp-2 cell following internalization was examined in an antibiotic-free medium.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Internalization assay.
The antibiotic-resistance assay was used to determine entry of GAS into the HEp-2 cells, essentially according to Jadoun et al. (1998). Briefly, HEp-2 cells were seeded in 24-well tissue culture plate, and grown for 24 h in RPMI 1640 supplemented with 10 % of fetal calf serum (FCS) without antibiotics. Each monolayer was infected with 106 bacteria suspended in RPMI 1640, to obtain a multiplicity of infection (m.o.i.). of 10. Infected monolayers were incubated at 37 °C for 2 h in an atmosphere containing 5 % CO2 and at 95 % relative humidity. The monolayers were then washed three times in order to remove unattached bacteria, and antibiotic solution containing gentamicin at 100 µg ml-1 and penicillin at 5 µg ml-1 was added to kill extracellular attached bacteria. Control experiments using bacteria alone in RPMI 1640 or in RPMI 1640 containing lysate of HEp-2 cells verified that these concentrations of antibiotics killed 108 c.f.u. ml-1 under the assay conditions. Following incubation for 2 h, the monolayers were thoroughly washed and fresh RPMI 1640 (devoid of serum and antibiotics) was added to each well. At the indicated times, the monolayers were washed and lysed. Tenfold dilutions of cell lysates were plated on TSA and incubated at 37 °C for 24 h, for enumeration of intracellular bacteria. In some experiments, cytochalasin D (1 µg ml-1) was added to the monolayer for 1 h prior to or after the internalization assay (at 4 h post-infection). The concentration of cytochalasin D was constant throughout the assay. In other experiments, chloramphenicol (20 µg ml-1) was added to each well following the 2 h killing period. The number of viable extracellular bacteria in the tissue culture fluid was determined at different times following infection. The fluid from each well was removed, centrifuged at 250 g for 1 min to remove any detached epithelial cells, and tenfold dilutions of the medium were plated on TSA and incubated, as described above.
Analysis of internucleosomal fragmentation.
HEp-2 cells (105) were infected with GAS at an m.o.i. of 10 as described for the internalization assay. At the indicated time points, cells were washed, trypsinized, and treated for 10 min at room temperature with lysis buffer containing 20 mM Tris/HCl (pH 7.4), 10 mM EDTA (pH 8.0) and 0.2 % Triton X-100. The lysate was centrifuged, and the supernatant, which contained the DNA, was collected. The DNA was treated with Proteinase K (100 µg ml-1) at 50 °C for 1 h, and then with RNase (0.5 µg ml-1) at 50 °C for 1 h. The DNA was extracted twice with an equal volume of phenol/chloroform (1 : 1, v/v) and once with an equal volume of chloroform-isoamyl alcohol mixture (24 : 1, v/v). The DNA was then precipitated with 0.3 M sodium acetate (pH 5.2) and 2 vols ethanol for 3 h at -70 °C. After centrifugation, the DNA pellet was washed once with 70 % ethanol and resuspended in TE buffer containing 10 mM Tris/HCl and 1 mM EDTA, pH 8.0. Electrophoresis was carried out on 1.5 % agarose gel, and the DNA was stained with ethidium bromide. DNA from non-infected cells served as a control.
Detection of phosphatidylserine.
Translocation of phosphatidylserine to the outer layer of the cell membrane is an early feature of apoptosis. The presence of exposed phosphatidylserine on epithelial cell surfaces was detected with Annexin-V-FLOUS (Roche Diagnostics). Briefly, HEp-2 cells grown in a 96-well plate were infected with GAS at an m.o.i. of 10 for 2 h. The culture medium was then replaced with fresh medium supplemented with penicillin and gentamicin, and incubation proceeded at 37 °C. At various time intervals, cells were washed with PBS and resuspended with labelling solution containing Annexin-V-FLOUS. Incubation buffer, containing 10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 5 mM CaCl2, was added to each sample and the phosphatidylserine was detected with a FACScan flow cytometer (Becton Dickinson).
Trypan blue exclusion assay.
Infected and non-infected (control) HEp-2 cells were washed, trypsinized and mixed with an equal volume of trypan blue (0.5 %, w/v, in PBS). Ten microlitres of the mixture were placed on a Neubauer chamber, and the numbers of total and stained cells were determined under a light microscope. The percentage of dead cells was calculated by dividing the mean number of dead (stained) cells by the total number of cells in 50 microscopic fields, and multiplying by 100.
Statistical analysis.
All assays were performed in triplicate, and repeated at least three times on different days. Student's t-test was applied to the results, which were considered significant if P < 0.05.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Since, GAS were found unable to multiply in serum-free RPMI 1640 (unpublished data), several explanations could account for this observation: (i) intracellular bacteria multiplied within the cells and escaped into the medium, where they were killed in the presence of antibiotics, or remain viable in their absence; (ii) intracellular bacteria lysed the cells (rather than multiplying intracellularly) and then, in the absence of antibiotics, grew extracellularly by feeding on nutrients available in the cell lysate; (iii) there was a combination of these two possibilities, i.e. intracellular bacteria first multiplied and then lysed the cells, and the released bacteria continued to multiply extracellularly by feeding on the cell lysate.
Recent studies which demonstrated that some GAS strains are capable of intracellular multiplication (Molinari et al., 2000, 2001) support the first and last hypotheses.
Epithelial cells are injured following GAS internalization
It is likely that in order to escape from epithelial cells, intracellular GAS need to lyse the cell membrane. To extend our study of the fate of HEp-2 cells after internalization, cell death was quantified. At various time points HEp-2 cells were tested for viability by determining their capacity to exclude trypan blue dye from their cytoplasm. The advantage of this assay is that it can detect the minimal cell damage that is inflicted in the early stages of attack by pore-forming toxins, such as streptolysin O and streptolysin S. As shown in Fig. 2, cell injury occurred whether or not antibiotics were present in the medium, but it was much more prominent in their absence. It is worth noting that during 12 h post-infection, cell injury increased to 18 % (Fig. 2), whereas there was only a small increase in the number of external bacteria (Fig. 1). These findings suggest that cell injury preceded bacterial escape and extracellular multiplication. Furthermore, it is obvious from these results that the sharp decrease in the number of intracellular bacteria (at 12 h) in the standard internalization assay (Table 1) is not associated with bacterial release from the cells, but rather is related to cell injury and penetration of antibiotics into epithelial cells, as was proposed by Molinari et al. (2001). Cell injury increased rapidly, and at 18 and 24 h post-infection the injury rates reached about 50 and 78 %, respectively. In agreement with these findings, light microscopy showed no significant morphological damage in the presence of antibiotics (Fig. 3a), whereas there was extensive cellular destruction in their absence (Fig. 3b).
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GAS internalization and de novo protein synthesis are necessary for cell damage induction
It could be hypothesized that a small number of extracellular adherent bacteria were not killed by the antibiotics during the internalization assay; they would then have resumed their metabolic activity when the antibiotics were removed from the medium, and induced external cell injury. To test this unlikely possibility, cytochalasin D, an inhibitor of GAS internalization (Greco et al., 1995) was added to epithelial cells prior to the internalization assay. Treated cells were incubated with bacteria in the presence of antibiotic-free medium, and cell cytotoxicity was determined by trypan blue staining. After 24 h only a slight increase (up to 25 %) in the number of dead cells was observed among the cytochalasin D-treated cells, compared with about 80 % among the untreated cells (Fig. 2). Concomitantly, only a few hundred free-living bacteria were recovered at 24 h post-infection from the cell culture fluid of treated cells, compared with > 106 from that of the untreated control cells (Fig. 1). Although we observed that in the presence of antibiotics the number of intracellular bacteria declined with time (Table 1), we found that the level of cell injury increased (Fig. 2). A possible explanation could be that the progressive increase in the cell injury level resulted from continuing activity of preformed enzymes (such as SpeB) which had been secreted by intracellular bacteria before they were killed.
These results support the notion that intracellular, rather than extracellular bacteria were responsible for the observed cell injury and the re-emergence of free-living bacteria into the external medium. To determine whether the host cell cytoskeleton is also involved in GAS externalization, cytochalasin D was added to the culture medium following internalization (4 h post-infection). Interestingly, this treatment also resulted in a small number of extracellular bacteria and inhibition of epithelial cell injury, which suggests that the cytoskeleton was involved in the processes leading to bacterial release and cell death.
In order to determine whether de novo protein synthesis in GAS is required for inducing cytotoxicity and bacterial release, cell injuries and the numbers of c.f.u. in the external medium were examined in the presence of chloramphenicol, a bacteriostatic antibiotic that inhibits prokaryotic protein synthesis. When chloramphenicol was added at 4 h post-infection (after internalization), cell injury was significantly reduced (Fig. 2), as was the number of extracellular free-living bacteria (Fig. 1).
Taken together, these data could indicate that cell injury and bacterial externalization require both GAS internalization and de novo protein synthesis. Our results agree well with recent findings that, following internalization, GAS escape from endocytic vacuoles into the cytosol, where they can multiply (Molinari et al., 2000).
HEp-2 cell damage is caused by programmed cell death
Many bacterial pathogens are capable of inducing cell death in the host via apoptosis (reviewed by Zychlinsky & Sansonetti, 1997; Weinrauch & Zychlinsky, 1999; Grassme et al., 2001). Apoptosis – programmed cell death – is a fundamental cellular mechanism which ensures homeostasis of the organism. It is characterized initially by a series of stereotypic morphological changes, such as DNA fragmentation, nuclear condensation and fragmentation, and membrane blebbing. GAS express a variety of cytotoxic enzymes and toxins, which might be responsible for epithelial cell injury, therefore, the cell deaths that are observed following GAS internalization could be a result of bacterial activity alone, and/or induced apoptosis. Recent studies have revealed that GAS are capable of inducing apoptosis in epithelial cells and in monocyte-like cells. Streptococcal pyrogenic exotoxin B (SpeB), SpeA, streptolysin O and streptolysin S have all been implicated in this process (Tsai et al., 1999; Molinari et al., 2001; Kuo et al., 1999; Bricker et al., 2002). To determine whether HEp-2 cells undergo apoptosis after internalization, we looked for several indicators of apoptosis at various time points. Infected HEp-2 cells were washed, fixed and stained with DAPI. Microscopic examination of uninfected (control) cells showed uniform nuclear morphology, whereas infected cells showed a highly condensed nucleus (Fig. 4a), characteristic of apoptotic cells. Another general hallmark of apoptotic cells is internucleosomal DNA fragmentation. DNA isolated from infected and uninfected cells was assessed by looking for presence of a 180-bp laddering pattern on agarose gels. Gel electrophoresis of chromosomal DNA revealed a characteristic fragmentation of DNA derived from infected but not of that derived from control cells at 24 h post-infection, with partial fragmentation appearing as early as 12 h (Fig. 4b).
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An earlier indicator of apoptosis is the detection of phosphatidylserine on the outer leaflet of the plasma membrane bi-layer (Raffray & Cohen, 1997). During the early stages of apoptosis, the asymmetry of plasma membrane phospholipids is lost, which leads to the external exposure of phosphatidylserine. To obtain further confirmation of the nature of the cell death, the appearance of phosphatidylserine on the surface of HEp-2 cell membrane was determined at 8 and 12 h post-infection. Annexin-V-FLOUS, a specific and sensitive phosphatidylserine binding protein, was used, and showed that at 8 h a significantly higher proportion of the infected cells than of the control cells expressed phosphatidylserine on their surface: about 35 and 5 %, respectively. At 12 h post-infection, the percentage of infected cells expressing phosphatidylserine on their surface increased to 60 %, which was significantly higher than the 7 % of the control cells that did so (data not shown). These findings support the notion that cell death following GAS internalization is mainly caused by programmed cell death.
GAS internalization has previously been shown to be necessary and sufficient for inducing apoptosis in mammalian cells (Tsai et al., 1999; Nakagawa et al., 2001). Similarly, internalization has been reported to be a prerequisite for inducing apoptosis in cells infected with Staphylococcus aureus (Nuzzo et al., 2000; Kahl et al., 2000; Menzies & Kourteva, 1998; Bayles et al., 1998), suggesting that the induction of programmed cell death by facultative intracellular bacteria is a common occurrence in host cell–bacterium interactions. Tsai et al. (1999) reported that 11–13 % of HEp-2 cells showed indications of apoptosis at 20 h following GAS infection, and that about 40 % of the cells exhibited membrane injury. The relatively low percentage of injured cells, compared with our present data, might be a reflection of the differing experimental conditions used in the two studies. Tsai et al. (1999) measured cytotoxicity in cells overlaid with antibiotic-supplemented medium, whereas no antibiotics were present in the culture medium in our study. Hence, it is conceivable that apoptosis was enhanced by the presence of extracellular multiplying bacteria. The recent finding that extracellular GAS are also capable of inducing apoptosis by insertion of NAD+-glycohydrolase into the cytoplasm of infected keratinocytes (Bricker et al., 2002), supports such a notion.
It has been suggested that apoptosis of intestinal epithelial cells following pathogen infection may be a means of eliminating infected and damaged epithelial cells, and restoring epithelial cell growth regulation and cell integrity (Kim et al., 1998). Likewise, apoptosis may lead to eradication of engulfed streptococci via desquamation of infected cells and/or ingestion by resident macrophages. Phagocytosis of apoptotic cells does not elicit bactericidal activities and therefore provides a protective intracellular niche, shielded against exogenous host immune defences, as well as against extracellular antibiotics. It is noteworthy that electron microscopy has revealed that eight out of 11 tonsils removed from asymptomatic GAS carriers contained intracellular GAS residing in macrophage-like cells (Österlund et al., 1997), an observation that supports the notion that some GAS strains may have developed a unique survival strategy to subvert the host defence mechanism and to persist within the human host. It is yet to be determined whether GAS strains derived from other sources interact in the same way with epithelial cells.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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