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Previous Article | Next Article 
Infection and Immunity, May 2003, p. 2318-2325, Vol. 71, No. 5
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.5.2318-2325.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Clinical Microbiology, The Hebrew University—Hadassah Medical School, Jerusalem 91120,1 Department of Poultry and Fish Diseases, The Kimron Veterinary Institute, Bet Dagan 50250,3 Dan Fish Farms, Kibbutz Dan, Upper Galilee 12245, Israel,4 Unité de Virologie et Immunologie Moleculaires, Institut National de la Recherche Agronomique, Jouy en Josas 78352, France,2 Department of Fish Pathology, IZS—State Veterinary Institute, 06126 Perugia, Italy5
Received 28 May 2002/ Returned for modification 3 August 2002/ Accepted 3 February 2003
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ABSTRACT |
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70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More recently, it has been shown that following a 5-year routine vaccination program, a novel serotype, capable of producing a generalized bacterial meningitis, has emerged (4). In this case, S. iniae probably gains access to the bloodstream and maintains a high level of bacteremia, leading to the onset of a generalized disease and meningitis, as described for other diseases (14, 27). Similarly to Streptococcus pyogenes (group A streptococcus) infection in humans, where serotype replacement in a population (24) is most likely the result of the immune status of the individuals along with the introduction of a highly virulent organism (8), the propensity of S. iniae to cause an invasive disease in fish is likely related not only to the immune status of the fish but also to the pathogenetic mechanism(s) of virulent strains. One of the features that allow S. iniae to establish an infection is related to its ability to overcome the immune response of macrophages, which play a role in initial phagocytosis and eventual killing of streptococci and other pathogens. Invasion and intracellular survival of S. iniae in host cells might thus represent an important pathogenicity mechanism in invasive infections.
To gain more insight as to the ability of S. iniae to initiate infection, we studied the various interactions between noninvasive (type I) and invasive (type II) strains of the pathogen and salmonid macrophages.
By using salmonid-specific cellular models, we demonstrate that S. iniae is capable of (i) invading and surviving in fish phagocytes and (ii) specifically inducing their apoptosis. The role of this coupled phenomenon in the infectivity of two S. iniae serotypes and in their induction of meningitis is discussed.
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MATERIALS AND METHODS |
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The established salmonid macrophage cell line RTS-11 (17), the salmonid embryonic cell line CHSE-214, and the rainbow trout gonad cell line RTG-2, used for adhesion and invasion assays, were cultured at 18°C in Dulbecco's modified Eagle medium (DMEM) (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum (GIBCO), HEPES (1%), penicillin (100 µg/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). Cell lines were subcultured every 3 weeks.
PN and blood leukocyte collection. Blood was collected in heparinized tubes and diluted (1:20) with DMEM. Pronephros phagocytes (PN) from hind-kidneys were obtained by forcing the tissues over a TG 100 separating gauze (Schleicher and Schuell, Dassel, Germany). PN and blood leukocytes were isolated through a Histopaque (d = 1.077) gradient (Sigma, St. Louis, Mo.). Cell suspensions were centrifuged for 20 min at 400 x g to remove erythrocytes and debris. The leukocyte-enriched interphase was collected, washed in phosphate-buffered saline (PBS) (15 mM Na2HPO4-145 mM NaCl [pH 7.20]), counted (by trypan blue exclusion), and resuspended in DMEM supplemented with 10% fetal calf serum.
Infection assays. For preliminary assays, RTS-11 cells were infected (multiplicity of infection [MOI], 100) for 0, 20, 40, 60, 120, or 180 min before cells were washed three times in PBS or washed and reincubated for 2 h in complete medium supplemented with ampicillin (100 µg/ml). The results showed that at time zero, the level of S. iniae adhesion to RTS cells was 0.01 to 0.03 CFU/cell. After 60 min of infection, total cell-associated bacteria were 3.8 to 6.8 CFU/cell, with only 0.4 CFU of intracellular bacteria; after 180 min, each cell harbored 2.5 to 5 CFU/cell. Therefore, in the following experiments, the RTS-11, CHSE-214, and RTG-2 cell lines were infected (MOI, 100) for 60 min for the adhesion assay and for 180 min for the invasion assay. All experiments were performed (in triplicate) at least three times.
Adhesion assay. Adhesion of S. iniae to cell lines was performed as previously described (11, 34). Mid-log-phase S. iniae bacterial suspensions were added to 2 x 105 RTS-11 macrophages and to prewashed confluent CHSE-214 and RTG-2 cells in 12-well tissue culture plates (Costar Co., Cambridge, Mass.) at an MOI of 100 bacteria per eukaryotic cell. After 15, 30, and 60 min of incubation, nonadherent bacteria were removed by washing the cells three times with PBS. For viable count determinations, infected cells were treated for 5 min with 0.05 ml of 0.25% trypsin and of 0.1% EDTA (Sigma) in Hanks balanced salt solution (GIBCO), and streptococci were harvested by adding 0.15 ml of 0.025% Triton X-100 (U.S. Biochemicals, Cleveland, Ohio) in sterile distilled water to each well. After 3 min, cell lysates were collected and serially diluted in PBS, and aliquots (in triplicates) were plated onto blood agar for assessment of bacterial CFU.
Total counts of cell-associated (invading plus surface-adherent) bacteria were determined. Adherent bacteria were quantified by subtracting the number of invasive bacteria (determined as described under "Invasion and survival assay" below) from the total cell-associated bacteria.
Invasion and survival assay. Invasion was assessed as described by Rubens et al. (33) with some minor modifications. Briefly, RTS-11, CHSE-214, and RTG-2 cells and PN were infected as described above. After 3 h, extracellular bacteria were removed by three washes with PBS, and the original volume was reconstituted with DMEM supplemented with 10% fetal calf serum and ampicillin (100 µg/ml). After the addition of ampicillin, incubation was allowed to proceed for an additional 3 h. For both S. iniae strains, the MIC (Etest; AB Biodisk, Solna, Sweden) of ampicillin is >0.016 mg/ml; incubation of S. iniae in the presence of ampicillin (100 µg/ml) for 3 h resulted in 100% killing of bacteria with no toxic effects to fish PN or cell lines.
For assays of survival of bacteria in phagocytes, PN were isolated from 100-g naïve trout as described above. Cells were collected, washed, and counted by dye exclusion. PN were infected for 3 h (as previously described), washed, and resuspended in DMEM supplemented with 10% fetal calf serum and ampicillin (100 µg/ml). Viable counts of intracellular bacteria were determined at time zero (3 h post-antibiotic addition) and after 24 and 48 h.
Opsonin-dependent invasion of trout PN. PN were isolated from 100-g naïve trout (nonvaccinated controls) and from trout specifically vaccinated against KFP 404 (with 109 CFU of formalin-killed bacteria emulsified in incomplete Freund's adjuvant [Sigma]/fish; immunization was performed as described previously [13]). Assays for assessment of PN invasion and opsonin-dependent phagocytosis were based on a previously described protocol (26), which we modified as follows. Briefly, 75 µl of KFP 404 bacterial solutions plus 25 µl of serum samples were distributed (in triplicate) in a 96-well microtiter plate and incubated for 45 min at 18°C. To obtain the maximal assay sensitivity, undiluted serum was applied to the first wells of the serial dilution (1:1, 1:5, and 1:20) and no sera were added to the last wells. Trout PN were suspended in DMEM supplemented with 0.1% gelatin and 10% fetal calf serum; 100 µl containing 2 x 105 cells (MOI, 100) was then added to each well. Cells from a given fish were combined with the serum samples obtained from the same fish. The mixture was incubated for 60 min at 18°C with shaking, and invasion was stopped by an additional 3-h incubation with ampicillin (100 µg/ml). Cells were then lysed with 0.025% Triton X-100, and intracellular bacteria were quantified by plate counting.
Survival in whole blood. Resistance to phagocytosis in whole blood was determined as described by Fuller et al. (16), with minor modifications which were found necessary due to the limited amount of blood that can be drawn from each fish. Bacterial suspensions (10 µl, containing 102 or 103 CFU) were added to 120 µl of fresh, heparinized trout or human blood in sterile glass tubes and incubated on an orbital shaker for 1.5 to 72 h at 18°C (trout blood) or 37°C (human blood). For bacterial enumeration, 20 µl of blood sample was added to 40 µl of 0.025% Triton X-100, vortexed, serially diluted, and plate counted.
Partition of bacteria in blood of diseased fish. Naïve fish were infected with S. iniae by cohabitation. Three clinically diseased fish (previously infected by intraperitoneal [i.p.] injection with 100 50% lethal doses [LD50s] of S. iniae Dan-15 or S. iniae KFP 404) were placed with 100 naïve fish (two groups of 50 fish in separated tanks); the course of the infection by cohabitation was monitored daily. Fish were considered to be diseased when they simultaneously exhibited three out of the four following clinical symptoms: lethargy, black discoloration, loss of orientation, and ocular pathologies.
To assess the number of free and ingested bacteria in the blood of diseased fish, a three-step protocol consisting of (i) fractionation between the various blood components, (ii) separation of leukocytes by Percoll purification, and (iii) enumeration of bacteria by plate counting was used. Blood was drawn from the caudal veins of diseased trout, collected in sterile heparinized tubes, and divided into three aliquots. For determination of the total number of bacteria, 20 µl of blood was lysed by addition of 40 µl of 0.025% Triton X-100 in sterile distilled water, serially diluted, and plate counted. Bacteria in serum were enumerated by plate counting of serum (obtained after full decantation of leukocytes and erythrocytes). For quantification of intracellular organisms, peripheral blood leukocytes (PBL) were purified over a 1077 Histopaque gradient and lysed with 0.025% Triton X-100, and bacteria were plate counted.
Transmission electron microscopy. Infected and control cell cultures were directly fixed in culture flasks, in cold (0 to 4°C) 1.25% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), for 45 min. Cells were then rinsed in buffer and postfixed for 45 min in 1% osmium tetroxide. After ethanol dehydration, samples were embedded in Epon resin. Ultrathin sections were stained with uranyl acetate and lead citrate according to the Reynolds method (31) and observed under an EM12 Philips transmission electron microscope at 80 kV.
Assessment of apoptosis. Apoptosis of PBL and PN was monitored by analysis of DNA fragmentation and quantified with a flow cytometer.
DNA fragmentation, visualized in gel electrophoresis as "DNA laddering," was analyzed as described previously (20), with modifications. Briefly, PBL and PN (5 x 105 per well) were infected with S. iniae as previously described. After 2 h, extracellular bacteria were removed by three washes with PBS, and the original volume was reconstituted with DMEM supplemented with 10% fetal calf serum, penicillin (100 µg/ml), and gentamicin (50 µg/ml). Incubation was allowed to proceed for an additional 22 h. Macrophages incubated with 1 µg of actinomycin D (Act D; Sigma)/ml for 24 h were used as a positive control for apoptosis (15). Cells were harvested by addition of 2 ml of 0.25% trypsin and of 0.1% EDTA (Sigma) in Hanks balanced salt solution (GIBCO) and were lysed with lysis buffer (0.2% Triton X-100, 20 mM Tris [pH 7.4], 10 mM EDTA [pH 8.0]) at room temperature for 10 min. After centrifugation, the supernatant was treated with proteinase K (100 mg/ml) for 1 h at 50°C, and then RNase (0.5 mg/ml) was added for 1 h at 50°C. Lysates were extracted twice with an equal volume of phenol-chloroform (1:1, vol/vol) and once with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) before precipitation with ethanol. Precipitates were dried and solubilized in 1x TE (10 mM Tris [pH 8.0]-1 mM EDTA). Electrophoresis was performed with a 2% agarose gel, and DNA was stained with ethidium bromide. As a negative control, noninfected cells were treated by the same procedure as the infected cells.
For flow cytometric (FCM) assessment of apoptosis, assays were performed ex vivo on PN and PBL collected from naïve trout. Leukocytes suspended in DMEM were infected (MOI, 100) as described above. FCM assays were carried out 24 h after in vitro infection on cells permeabilized and stained with propidium iodide (a fluorescent probe for DNA) in nuclear isolation medium (NIM) for 20 min in the dark at 4°C (6). Acquisitions were carried out by using a standard fluorescence-activated cell analyzer (FACScan; Becton Dickinson, Mountain View, Calif.) on cells kept in NIM. For each sample, 10,000 individual leukocytes were recorded according to the FCM procedures used for the cell cycle analysis. Debris and cell aggregates were gated out of the acquisition. To estimate the percentage of apoptotic cells, we used an FCM method based on detection of the extensive cleavage of nuclear DNA (propidium iodide red fluorescence) occurring in apoptotic cells. Apoptotic nuclei display a DNA content lower than that contained in the diploid (G0/G1) state. Data were collected and analyzed using Cellquest software.
Cells were incubated in NIM (6) for 20 min in the dark at 4°C prior to FCM analysis. We use a two-parameter dot plot of a fluorescence peak width versus fluorescence peak area signals to gate out cell aggregates from cell suspensions.
Infection of fish by free and macrophage-associated bacteria. RTS-11 macrophage monolayers were infected with S. iniae Dan-15 or S. iniae KFP 404, harvested as described above (under "Invasion and survival assay"), and suspended (103 to 107 cells/ml) in DMEM; 0.2-ml was injected i.p. into recipient fish (10 rainbow trout, 100 g each, obtained from a specific disease-free site). A second group of naïve fish was infected by i.p. injection (0.2 ml) of PBL (103, 104, 105,106, and 107 cells/ml) purified from diseased fish naturally infected by a serotype II strain. Bacterial CFU associated with RTS-11 macrophages and PBL were quantified by plating on agar. A third group was infected with (0.2-ml) mid-log-phase bacterial cultures diluted to the required inocula (102 to 106 CFU). Morbidity and mortality were monitored daily, and dead fish were subjected to bacterial examination.
Statistical analysis. Data are presented as means ± standard deviations (SD) from at least four independent experiments performed in triplicate. Results were analyzed by Student's t test. The results of partitioning of bacteria in the blood of diseased fish were analyzed by a one-way analysis of variance (SAS software, version 5). Results of infection of fish by free and macrophage-associated bacteria were analyzed by linear regression analysis (SAS software, version 5). The electron microscopy analysis and analysis of DNA fragmentation by flow cytometry and agarose gel electrophoresis were repeated six times, and data from a typical experiment are reported.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% higher CFU counts (4.8 CFU/cell) than the serotype I strain (2.5 CFU/cell), demonstrating that the efficient adhesion of the type II strain is followed by a successful intracellular invasion. PN are invaded by S. iniae (see Fig. 3) to a greater extent than RTS-11 macrophages, and as seen in RTS-11 invasion, the extent of PN invasion is also serotype dependent (11 CFU/cell for KFP 404 and 4 CFU/cell for Dan-15). The chronological events that succeeded PN invasion were monitored for 48 h. While PN invasion by the type I strain is a transitory event after which the bacterium is almost eradicated, invasion by the type II strain is accompanied by a series of events that (i) allow its survival (2.2 CFU/cell 24 h postinfection) (Fig. 2 and 3) and (ii) are followed by its multiplication (3.6 CFU/cell 48 h postinfection) (Fig. 2).
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0.4 CFU/cell after 60 min). In contrast, PN obtained from vaccinated fish and incubated for 60 min with opsonized bacteria harbored a number of bacteria per cell similar to that in RTS-11 macrophages incubated for 3 h. This finding suggests that specific antibodies increase the efficacy of phagocytosis.
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Serotype and apoptosis induction. As shown in Fig. 6, DNA fragmentation, visualized in gel electrophoresis as "DNA laddering," is a clear outcome of PN infection by S. iniae type II. The type I strain causes a less apparent apoptotic effect. Identification and quantitation of cells undergoing apoptosis was based on evaluation of the subdiploid percentage of the cell population (Fig. 7). Our data showed that S. iniae-treated leukocytes had higher percentages of nuclei with subdiploid DNA content. Percentages of cells with decreased DNA content in leukocyte cultures exposed to bacteria or left unexposed were recorded, and an apoptotic index was calculated (percentage of apoptotic cells in culture with bacteria/percentage of apoptotic cells in control). As shown in Fig. 8, the apoptosis induced in vitro with the serotype II strain (KFP 404) was stronger than that induced with the serotype I strain (Dan-15).
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DISCUSSION |
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The pathogenesis of the meningitis and septic meningitis caused by S. iniae is unclear and is possibly the result of a multistep process. It is likely that disease starts with colonization of external tissue, followed by local spread and subsequent invasion of the bloodstream. The outcome of the infection is also related to the host's reaction: failure of initial phagocytosis and killing of the pathogen will allow the establishment of the disease. The salmonid-specific models that we have constructed allow the determination of certain events during type I and type II S. iniae infections. Since the incidence of central nervous system (CNS) infection is directly correlated to the concentration of the pathogen in blood and the length of time bacteremia is maintained (5, 25, 28), the abilities of the type II strain to survive within phagocytes and to induce high levels of bacteremia are advantageous for the onset of generalized bacterial meningitis. Increased levels of bacteremia with invasion and intracellular survival of S. iniae type II strains in host circulating macrophages thus represents an important evolution of the pathogenicity mechanisms in invasive S. iniae infections.
The ability of extracellular bacteria to invade the CNS from the bloodstream has been made clear by means of in vitro and in vivo models of infection (49). The mechanisms which enable S. iniae to disseminate in the body, and more particularly in the CNS, are unclear. At one point, free bacteria would have to pass the brain-blood barrier or by translocation through or between endothelial cells and underlying tissues before entering the CNS (45). Most meningeal pathogens, such as Streptococcus pneumoniae, Escherichia coli K1, and group B streptococci, are known to interact directly with cells of the brain-blood barrier as free bacteria (39). The possibility that a similar event takes place also in S. iniae infection is strengthened by a recent report (16) showing that S. iniae causes damage to (human) BMEC monolayers, suggesting that CNS involvement may be attributed in part to the ability of S. iniae to promote cell injury and disruption of the blood-brain barrier. Another possibility, as occurs in Streptococcus suis type II (46) or Listeria monocytogenes infections of the CNS (11), is that bacteria could be carried into the CNS in association with monocytes (or phagocytes) migrating into the CNS compartment to maintain populations of resident macrophages. CNS infection can be initiated by cell-to-cell spread from infected leukocytes to the endothelium or by migration of infected leukocytes to the CNS. Our finding that the quantity of bacteria required for the establishment of experimental infection (with CNS involvement) is considerably diminished if bacteria are loaded within macrophages substantiates the idea that S. iniae can enter the CNS compartment in association with migrating monocytes, and it highlights the importance of macrophages as "Trojan horses."
Infected macrophages not only act as transporters, which assist in disseminating the bacteria throughout the organism, but since they undergo apoptotic death, they also fail in priming an immune response. Apoptosis regulates multiple physiological processes, including immune response, and reduces inflammatory tissue injury by removing damaged cells (19). Although apoptosis of Salmonella-infected macrophages does not alter antigen presentation ability (48), and apoptosis of S. pneumoniae-infected macrophages has been associated with successful clearance of bacteria (10), the ability of pathogens to promote apoptosis is considered an important mechanism for counteracting host immune defenses by avoidance of immune system-mediated killing and for initiation of infection (20, 37, 50). Apoptosis of immune cells has been associated with several streptococcal infections. Group B streptococci induce neuronal and monocyte apoptosis (15, 26), S. pneumoniae induces neuronal and neutrophil apoptosis (51), and S. pyogenes avoids host responses through apoptosis of lymphocytes and epithelial cells (38, 43).
Our results suggest that S. iniae also developed an antihost strategy based on apoptotic killing of blood leukocytes and PN. In fact, because apoptosis occurs without the release of cellular components, it reduces or suppresses inflammation (7, 18, 47). Therefore, apoptosis may be advantageous for the pathogen, as it might avoid the triggering and recruitment of nonspecific host defense mechanisms. Furthermore, macrophage death could also contribute to delaying or hindering the development of a specific immune response.
In conclusion, this study emphasizes the complexity of the strategy used by S. iniae to overcome host immune defenses. These mechanisms are shared by both serotypes, but they differ in extent: the profiles of bacteremia, intracellular survival, and induction of apoptosis by type II strains are far more efficient than those of type I strains. We suggest that the generalized meningitis induced by S. iniae type II is a consequence of its capacity to (i) survive in phagocytes and (ii) induce their apoptosis. According to this theory, the apoptotic phagocyte serves as a vector that is loaded in the blood circulation and is unloaded, after blood-brain barrier transcytosis, in the CNS.
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ACKNOWLEDGMENTS |
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This work was supported by EU funding (QLK2-CT-2000-01049) and by a joint American-Israeli grant (BARD US-3159-99).
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FOOTNOTES |
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