INFECTION AND IMMUNITY, OCt. 1992, p. 4373-4382
Vol. 60, No. 10
0019-9567/92/104373-10$02.00/0 Copyright © 1992, American Society for Microbiology
S-Layer-Mediated Association of Aeromonas salmonicida with Murine Macrophages RAFAEL A. GARDUNO, EMIL J. Y. LEE, ANiD WILLIAM W. KAY* Department of Biochemistry and Microbiology and The Canadian Bacterial Disease Network, University of Victoria, Petch Building, P.O. Box 3055, Victoria, British Columbia V8W3P6, Canada Received 27 February 1992/Accepted 22 July 1992
The interaction of Aeromonas salmonicida with the murine macrophage (M+) cell line P388D1 was used as a convenient model to study the involvement of the bacterial crystalline surface array (or A-layer) in the association with M4s. A-layer-positive (A') cells readily associated with M+s in phosphate-buffered saline, whereas A- mutants were unable to do so, even when the bacterium-M+ interaction was forced by centrifugation. M+s selectively interacted with A' cells when challenged with mixtures of A' and excess Acells. Electron microscopy indicated that in phosphate-buffered saline only A bacteria were readily internalized, although by a nonconventional mechanism, suggesting that efficient phagocytosis in the absence of opsonins was A-layer mediated. Latex beads coated with a partially assembled A-layer were more efficiently taken up than uncoated or A-protein-coated beads, indicating that an organized A-layer was essential for M4 uptake. The reduced ability of M+s plated on a substratum coated with the A-layer to bind A' bacteria also suggested that association was both A-layer and receptor mediated. In the presence of tissue culture medium, competent M+s interacted efficiently with A- bacteria and internalized them through conventional phagocytosis. A' cells were markedly cytotoxic to M+s, whereas the A-protein or A-layer was not. A- cells were cytotoxic to a lesser extent, suggesting that cytotoxicity was targeted.
Despite the plethora of prokaryotes that have been found to possess crystalline surface layers (S-layers), there is a lack of information regarding the functions of these unique two-dimensionally arrayed protein monolayers (1, 57). Particularly, S-layers of pathogenic eubacteria are naturally expected to have an important role in virulence, since surface components frequently mediate specific interactions of a pathogen with its host (46, 65).
to be appropriate for efficient phagocytosis by macrophages (Mos). In fact, the ability of A' bacteria to associate with resident peritoneal murine M4)s, as well as with head kidney tissue M4s from rainbow trout, was enhanced by the presence of the A-layer (60). These data were later confirmed and extended to the mammalian cell line BHK-21 and the salmonid cell line RTG-2 (44, 45). Peritoneal elicited-exudate cells from rainbow trout and coho or masu salmon were also used in a study showing that phagocytosis of A- cells by salmonid M4s is rather poor and that opsonization of A- bacteria with specific antibodies and homologous complement increased phagocytosis (52). Despite the above-described data, there is considerable evidence to the contrary, suggesting that the A-layer is not involved in the bacterium-host cell interaction. Early data indicated that virulent, strongly agglutinating strains of A. salmonicida (presumably A') were less easily phagocytosed than nonvirulent, nonagglutinating strains (presumably A-) by elicited or activated peritoneal M+s from brook trout (43). It was also reported that virulent, A' strains of A. salmonicida actually resist phagocytosis by M4s, in contrast to A- strains (54). Further, it has been suggested that the A-layer is not essential to virulence and reported that a supposedly A- mutant of A. salmonicida (virulent and autoagglutinating) adhered very effectively to monolayers of cells in culture (63). Others suggested that the presence of the A-layer does not correlate with enhanced adhesion to rainbow trout blood leukocytes or with virulence (24). In yet another report it was suggested that association ofA. salmonicida with two different salmonid cell lines is charge mediated, and a role for the A-layer was excluded (53). Owing to the unavailability of a M4j cell line from fish, M+s isolated from widely different sources through different isolation procedures, as well as a variety of A. salmonicida isolates, have been used in the past. Therefore, we decided to use a well-characterized MO cell line to avoid possible differences in M4 functional competence which accompany
The S-layer of the fish pathogen Aeromonas salmonicida, historically known as the A-layer (61), is composed primarily of subunits of a 50,000-Mr single protein species, the A-protein (26), whose encoding gene (vap, for virulence array protein) has been cloned and sequenced (3). Structurally, A-protein subunits are organized in a p4 symmetry array with a defined three-dimensional structure (8). This array covers the entire cell surface (48) and is thought to be anchored to the cell surface by specific lipopolysaccharide (LPS) 0-chain-dependent interactions (2). The A-layer represents the first bacterial S-layer to which specific functions have been assigned. While it protects the cells against bacteriophages (23) and confers resistance to predation by bdellovibrios (31), it is a particularly important virulence factor (23, 26, 29). Its virulence-related attributes include protection against complement killing (39) and binding of several host molecules, including porphyrins (28), immunoglobulins (47), and a surprising number of extracellular matrix proteins (7, 29). With the emergence of such functional diversity, it seemed important to determine whether the A-layer also confers invasive properties on A. salmonicida. Early reports (61) indicated that virulent, A-layer-positive (A') isolates of A. salmonicida readily associate with host cells. The A-layer was subsequently shown to confer greater cell surface hydrophobicity on A. salmonicida (60, 62), and this is thought *
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those variations. In this study, we clarified the role of the A-layer in the association of A. salmonicida with murine M+s. MATERIALS AND METHODS Bacteria. A. salmonicida A450, a well-characterized, virulent, A', LPS+ strain originally isolated from brown trout, was used as the wild type (26). The following two avirulent variants, obtained from A450 in this laboratory, were also used: A450-3, an LPS+, A- strain (23), and A450-1, an A', LPS-, A-layer-secreting strain (26). M+s. Murine M( cell line P388D1, derived from mouse strain DBA/2, was used throughout. M4s were obtained from J. L. Kluftinger and R. W. Hancock, University of British Columbia, for preliminary experiments. T. W. Pearson, University of Victoria, provided the M4s used in most of our studies. Buffers, media, and culture conditions. Filter-sterilized phosphate-buffered saline (PBS), pH 7.4, was used as the main buffer. Millonig's phosphate buffer (NaH2PO4; 55.2 g/liter, pH 7.4) was used to prepare specimens for electron microscopy (EM). Bacteria were grown in Luria broth (LB) medium at 20°C for 14 to 16 h. Bacteria used in association or cytotoxicity assays were cultured at 20°C for 48 to 72 h on tryptic soy agar (TSA) plates or on plates of TSA-hemin (10 p,g/ml) as needed. They were prepared for assays by being washed twice in PBS and having their concentration (optical density at 650 nm) adjusted as required. M4s were kept growing at 37°C in 5% CO2 in 40-ml tissue culture flasks (Falcon Laboratories) with 10 ml of complete RPMI 1640 medium. They were passaged every 48 h for use in experiments and every 96 to 120 h for maintenance of the cell line. Detachment of M+s by repeated washing was preferred over gentle scraping. Incomplete medium consisted of 450 ml of RPMI 1640 base (Sigma) (without sodium bicarbonate or glutamine), 5 ml of glutamine (30 mg/ml), 0.55 ml of 2-mercaptoethanol (3.5 p,g/ml), 5 ml of HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (1 M), and 13.35 ml of NaHCO3 (7.5%), pH 7.4. Complete medium contained fetal calf serum (Sigma Heart Line) at a final concentration of 5%. Staining and counting methods. Heat-fixed bacteria were stained by Gram stain, or with crystal violet for 1 min, as needed. M4 monolayers were methanol fixed for 5 min and stained with Wright or Giemsa stain (Sigma). Bacteria and latex beads in suspension were counted in a Petroff-Hausser counting chamber (in 5-,ul aliquots) or by using an internal standard of fungal spores (35). In the latter, 20 ,ul of a standardized fungal spore suspension was thoroughly mixed with 20 RI of the bacterial sample and spread on a clean glass slide, air dried, and stained for examination by light microscopy. Counting and calculation of the number of bacteria were done as described by Mallette (35). Mos in suspension were counted in a Neubauer hemocytometer after 1:2 dilution in 0.4% trypan blue in PBS. The percentage of phagocytosing M4s, as well as the number of particles (i.e., bacteria or latex beads) per phagocytosing M+, was determined by direct microscopy, applying a 95% confidence level (35). The two values were multiplied to obtain the number of particles associated with 100 M+s. Viable bacterial cell counts were made on Congo red plates (30 ,ug/ml) by the standard dilution plate method using two or three plates per sample. When high variability among
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plates of a single sample was observed, only the result from the plate with between 30 and 300 colonies was recorded. Congo red plates were used to distinguish A' bacteria (which give red colonies) from A- bacteria (which give white colonies) (22). The dilution medium used for viable counts was LB without glucose and containing 0.5% (vol/vol) Tween 20 to prevent autoaggregation. Survival curves. A. salmonicida survival at 37°C in PBS or LB was determined by direct viable cell counting. M+s were also tested for survival in PBS. Aliquots of M4s suspended in PBS and kept at 37°C were counted and assayed for trypan blue exclusion as the criterion for viability. EM. EM of M+s was carried out by conventional methods. Briefly, samples for transmission EM (TEM) were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in EPON 812. Thin sections were cut and stained with uranyl acetate-lead citrate for examination in a Phillips EM 300 TEM at 60 kV. Cells attached to round (10-mm-diameter) glass coverslips were also examined by scanning EM (SEM). Coverslips were fixed and dehydrated as for TEM and then critical point dried and mounted on aluminum stubs for gold coating. Specimen examination was done with a JEOL JSM 35 SEM at 20 kV. Negative staining of whole, air-dried bacteria was done on Formvar-coated grids with a saturated solution of unbuffered ammonium molybdate. Bacterium-M+ association assays. M+s were plated on 35-mm-diameter tissue culture dishes, each containing a glass coverslip (22 by 22 mm) attached to the bottom, at a ratio of 106 M+s per dish. Alternatively, 24-well tissue culture plates were inoculated with 0.5 x 106 M+s per well. Dishes or plates were incubated for up to 4 h at 37°C to allow attachment of M4s or overnight to allow attachment and growth of M4)s on the substratum. Before the experiment, M+s were washed twice with PBS and covered with 1 ml of fresh incomplete medium or PBS. Adherent M4s were scraped off of two dishes or wells to determine their number. Suspensions of washed bacteria in PBS were adjusted so that 100 RI contained a number of bacteria equal to 50 times the number of M4s per dish or well. Assays were initiated by inoculation of the dishes or wells with 100 ,Il of the corresponding bacterial suspension. In assays with 24-well plates, inoculation of wells was done sequentially (at the corresponding sampling times) so that at the end of the experiment all of the wells in a single plate were processed together. Two 35-mm dishes were usually processed at every sampling time. Supernatants from the two dishes were removed (and sampled when necessary), and the dishes were washed three times with PBS. M4s in one dish were lysed in 1 ml of deionized water for 10 min with agitation. The M+ lysate (1 ml) was collected, and the dish was washed three times with 3 ml of deionized water. Washes were collected with the lysate to produce a 1:10 dilution. The second dish was air dried, and the coverslip was stained and prepared for direct microscopy counts on monolayers. At the end of experiments with 24-well plates, supernatants from all of the wells in a plate were removed and the plate was washed twice with PBS by using a wash bottle. Plates were air dried and stained. Well bottoms were cut with a cork-boring machine and mounted on a glass slide for direct microscopy counts on monolayers. All association assays were performed at 37°C. Purification of A-layer and A-protein. Outer membrane preparations of A450, extracted with sodium lauryl sarcosinate, were prepared as previously reported (26, 48). Outer membrane preparations were subjected to serial extractions
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A450 A450-3 FIG. 1. (a) Survival curves of A' and A- A. salmonicida at 37°C in LB. Symbols: 0, A' (A450); +, A- (A450-3). Inset: electron micrograph of negatively stained A450 cells incubated at 37°C in LB. A, A-layer cap at one end of the cell. Bar, 5 pm. (b) Survival of A' and A- A. salmonicida after 24 h at 37°C. Survival was enhanced by keeping the bacterial cells in PBS.
with sodium deoxycholate (DOC) as follows. A 4.5-ml volume of an outer membrane preparation, 4.0 ml of 10 mg of lysozyme per ml, and 31.5 ml of 20 mM Tris (pH 8.0) with 5 mM EDTA were mixed and incubated for 30 min at room temperature with agitation. Forty milliliters of 4% DOC in 0.5 M NaCl with 5 mM EDTA were then added, and the preparation was incubated for 30 min at 30°C. The extracted A-layer-outer membrane pellet was separated by centrifugation at 10,000 x g for 10 min at room temperature and then washed with 20 mM phosphate buffer, pH 7.3. This preparation was labelled 1 x DOC. The DOC extraction steps were
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serially repeated to obtain the corresponding 2 x and 3 x DOC preparations. A-protein from A450 was purified to homogeneity by previously published methods (48) and adjustment of the guanidine hydrochloride concentration to 2 M. A-layer sheets from secreting mutant A450-1 were purified from culture supernatant by centrifugation at 100,000 x g for 1 h, followed by a single DOC extraction. Assays with coated latex beads. Latex beads with a 3.1-,um diameter (Seradyn, Particle Technology Division) were extensively washed with glycine-buffered saline (0.1 M glycine, 0.85% NaCl, pH 8.4). The suspension of beads was adjusted to a density of 1% solids (5.6 x 108 beads per ml), and different A-layer preparations (lx and 3x DOC and the A450-1 layer) or purified A-protein was added to achieve a final concentration of 50 ,ug/ml. Coating was allowed to occur for 3 h at room temperature under continuous gentle agitation. Beads were then washed twice with PBS and stored on ice. Confirmation of protein coating and purity was made by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of 25 ,l were boiled for 10 min with 9 ,ul of 4x sample buffer. After centrifugation in an Eppendorf microcentrifuge, a 20-pl volume of the liquid beneath the floating beads was loaded on a 12% gel for SDS-PAGE. Phagocytosis experiments with latex beads were done in PBS with 24-well tissue culture plates as described in for bacterium-M4 association assays. The beads-M4 ratio was adjusted to 5:1. Frustrated-association-phagocytosis assay. The frustratedassociation-phagocytosis assay was prepared as a regular bacterium-M4 association assay in PBS by using 35-mm dishes but with the following modifications. Half of the coverslips were coated with the purified A-layer: a 1-ml suspension of the 3 x DOC A-layer (140 ,ug/ml) in deionized sterile water was allowed to dry at room temperature on each coverslip. The coverslips were then covered with 0.5 ml of an A-layer suspension in PBS (70 p,g/ml) for 4 h. Control coverslips were similarly treated with deionized water and PBS. Coverslips were then washed with excess PBS, inoculated with 5 x 105 M+s, and incubated for 4 h prior to replacement of culture medium with 1 ml of an A450 suspension in PBS adjusted to give a bacterium-M4 ratio of 50:1. After 3 h, dishes were washed in PBS and processed for direct microscopy counts in monolayers. A. sabnonicida cytotoxicity to M4s. Cytotoxicity was evaluated in adherent M4s through bacterium-induced morphological changes. M4s and bacteria were prepared in PBS as for bacterium-M4 association assays, but M4s were inoculated on 35-mm dishes with four circular coverslips (10 mm in diameter) attached to the bottom. Two different bacterium MO cell ratios were used: 500:1 and 2,000:1. At 2 and 4 h after addition of bacteria, two coverslips were removed from each dish. One coverslip was air dried and processed for light microscopy, and the other was prepared for SEM. Also, cytotoxicity towards M4s suspended in PBS was evaluated through M4 viability, as determined by the trypan blue exclusion assay (described in the section on survival curves) by using bacterium-M4 mixtures at a cell ratio of 100:1. RESULTS Effect of experimental assay conditions on murine M+s and A. salmonicida. P388D1 M4s remained 90% viable when
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FIG. 2. Association of A4 (A450) and A- (A450-3) bacteria with M+s kept in PBS as determined by three different quantitative methods. (a) Bacterial cells associated per 100 M+s as determined by direct microscopy counts in M+ monolayers. Results of two separate experiments are shown: experiment 1; -, experiment 2; *, A'; x, A-. (b) Bacterium-M+ association as determined by direct microscopy counts using the internal-standard method. Data were obtained from samples of experiment 2 in panel a and are expressed as percentages of the bacterial cells added: counts done in M4 lysate samples; -, counts done in supernatant samples; 0, A'; x, A-. (c) Bacterium-M4 association as determined by viable cell counts. Data were obtained from M+ lysate samples taken from experiment 1 in panel a and are expressed as percentages of the viable cells added: 0, A4 bacteria; x, A- bacteria. (d to t) SEM and TEM of M4s kept in PBS with bound and internalized A4 bacteria. --
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suspended in PBS for up to 6 h (data not shown). SEM showed that, compared with control M4)s kept in complete RPMI 1640, there were no major alterations in gross morphology when M4s were kept in PBS, except for a less spread appearance. On the other hand, incubation of A. salmonicida in LB at 37°C caused a rapid decline in viability (Fig. la). Microscopic examination of these heat-stressed cultures showed that although most bacterial cells were elongated, A4 bacteria still possessed A-layers in the form of caps at the cell poles (Fig. la, inset). SDS-PAGE of membrane preparations stained for protein and LPS revealed no other significant compositional changes after 3 h at 37°C. Bacterial cells suspended in PBS were also killed at 37°C, but the final number of survivors was much higher (Fig. lb) than cells left in LB medium. EM (data not shown) revealed that these cells were elongated less (or not at all), and
A-layers were found surrounding most A4 bacteria. These data ensured a valid experimental design in which the target cells, M4s, remained viable and structurally unperturbed and adherent cells retained their surface components. Bacterium-M4 association in PBS. At all incubation times, the association of A. salmonicida with Mos (as determined through different quantitative methods) was much greater for A4 than A- strains (Fig. 2a to c). Specimens observed by SEM showed that A4 cells readily adhered to M+s and sometimes covered the entire pseudopodial surface (Fig. 2d). TEM revealed that A4 cells were also readily internalized by M4s in PBS (Fig. 2e and f), while it was not possible to identify any internalized A- cells. Forced association. It is known that A4 A. salmonicida cells in suspension autoagglutinate and consequently sediment considerably faster than A- bacteria (10, 61). To
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HOURS FIG. 3. Forced association of A' (A450) and A- (A450-3) A. sabnonicida with M+s in PBS. Association assays were done in 24-well plates in which bacteria were centrifuged (500 x g for 10 min) onto the Mi monolayer. At the different sampling times, rows of wells were washed three times in PBS and M+s were kept in fresh PBS until the end of the experiment, when the plate was air dried and processed as described in Materials and Methods. Symbols: 0, A+; x, A-.
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FIG. 4. M+ association from mixtures of Al (A450) and A(A450-3) strains ofA. salmonicida at different A--to-A cell ratios. Association with M+s was determined by viable cell counts of M+ lysate samples on Congo red agar plates (refer to Fig. 2c). Symbols: , A -to-Ak-cell ratio of 500:1; - - -, A--to-A' cell ratio of 50:1; 0, A+; x, A-.
M4s bound or internalized A- and A+ bacteria. Under these
confirm that the higher association levels observed with A+ cells were not an artifactual result of a faster sedimentation rate, both A+ and A- cell suspensions were centrifuged onto the M4 monolayer and subsequently processed to determine their levels of association with M4s (Fig. 3). The degree of M4 association with A- cells was increased, but not nearly as dramatically as for A+ cells. The global quantitative levels of M4) association for both bacterial strains were nearly an order of magnitude greater by this procedure. Thus, the difference in association of A+ and A- cells with M4)s was not due to a cell availability artifact. Selective adherence. To examine the possibility that extracellular factors from A+ cells or the association of A+ cells with M4)s may activate them towards an enhanced association with A- cells, A+-A- coincubation experiments were carried out. Two coinfection experiments were conducted by using 50:1 and 500:1 A--to-A+-cell ratios (Fig. 4). Viable cell counts of M4) lysate samples were used to monitor the level of bacterial association with M4)s. A+ and A- cells were discriminated on the basis of Congo red binding. Owing to the lethal effect of the incubation temperature upon A. salmonicida, the absolute value of these results can be regarded as an underestimate. Nevertheless, it was still evident that M4s in PBS were unable to associate with Acells, whereas A+ cells, even in the presence of a large number of A- cells, efficiently associated with these same M4)s. Thus, the association of A+ bacteria with M4s was not a cell density-determined artifact, nor did one cell type predispose M4)s to associate with the other. Effect of complex medium on bacterium-M+ Interactions. When association assays were carried out in tissue culture medium, M4s were particularly found to associate more readily with A- bacteria than previously observed (Fig. 5a and b). These more competent M+s also internalized Abacteria efficiently (Fig. 5c). Moreover, there were no apparent qualitative differences in the mechanism by which
experimental conditions,A. salnonicida was internalized by a conventional zipper mechanism (56), as depicted in Fig. 6. Assays with coated beads and substrata. A series of association assays with coated latex beads were conducted to evaluate the contribution of the A-layer alone to the processes of adherence and internalization. All of the A-layer preparations used were able to enhance the interaction of latex beads with M+s (Fig. 7a), which is all the more noteworthy because uncoated, hydrophobic latex beads are efficiently phagocytosed by M4s and for this reason are commonly used in phagocytosis assays (56). We attributed this enhancing effect to the regularly arrayed A-layer, since uncoated beads and those coated with purified monomeric A-protein behaved similarly. The A-layer prepared from A450-1 had, as determined by negative-stain EM, the greatest degree of order (data not shown) and was the most effective at promoting adherence. The possible existence of a specific adhesin-receptor interaction between the arrayed layer and the M4 surface was further examined in a frustrated-phagocytosis assay. In this type of assay, the presence of putative receptors for a particular ligand is indirectly revealed by recruiting available M4 receptors on a ligand-coated substratum (down modulation), thereby depleting available receptors and reducing subsequent bacterial cell interaction (37, 58). The global capacity for binding of A' cells was reduced 40% in M4s plated on A-layer-coated coverslips (Fig. 7b). This reduction was accompanied by a clear shift in the association profile of A' cells to M+s (Fig. 7c) (58), indicating that a specific receptor, presumably involved in the adherence process of A' A. salmonicida to M+s, was down modulated by the presence of the A-layer coating. A. salnonicida cytotoxicity to M+s. During the association assays, it was found that M4s exposed to A' bacteria appeared to be more compact (darkly stained) and to have a larger proportion of lysed cells than those exposed to Acells. These data prompted an investigation of the possible
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FIG. 5. Association of A' (A450) and A- (A450-3) A. salmonicida with M4s in RPMI 1640 tissue culture medium. (a) A' and Abacterium-M4 associations were measured after 1 h of incubation in PBS (column 1) or RPMI 1640 (column 2). The bacterium-to-M4 cell ratio was 100:1. (b and c) SEM and TEM of M+s with bound and internalized A- bacteria from panel a, column 2.
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cytotoxic effects of A' bacteria and, in particular, the role of the A-layer. Control M4s (bacterium free) and M4s challenged with either A' or A- cells (whether they were previously grown in the presence or absence of hemin) contained approximately the same proportion of cells permeable to trypan blue after an 8-h challenge (data not shown). However, there were marked morphological differences among adherent M4s treated with the different bacteria used (Fig. 8). The marked effects of A' cells upon adherent M4s were confirmed by light microscopy (Fig. 8L and M), in contrast to the less severe effects induced by Abacteria (Fig. 8H to K). Interestingly, SEM specimens indicated that A' bacteria induced a marked rounding of M4s and smoothing of their surface, presumably owing to severe cytoskeletal modifications (Fig. 8D and F). This effect was also noticeable in M4)s exposed to A- bacteria (Fig. 8E), but only at an unusually high bacterium-to-Mo cell ratio of 2,000:1 or at longer incubation times. Similar rounding and cratering effects were also seen when M4s were challenged with A- bacteria grown on TSA-hemin plates. However, the morphological changes induced by A- bacteria were never as extensive as those induced by A' bacteria, and most of the M4os exposed to A- bacteria remained intact (Fig. 8C, J, and K). Only hemin-coated A' bacteria induced massive lysis in some MO monolayers. M4s that had not yet lysed in these monolayers resembled golf balls, with craters on their surfaces, and were densely covered with bacteria (Fig. 8F and G). Especially curious was the appearance of apparent cytoskeleton ghosts, presumably consisting of a cytoskeletal structure still attached to the substratum but devoid of a cell membrane (CG in Fig. 8E and G). There was a good correlation between the light microscopy images and the detailed SEM data. DISCUSSION
Use of a well-defined system to study the role of the A-layer in M4) association appears to be justified on the basis that, perhaps, the wide discrepancies in the literature regarding this role are due to differences in the Mo sources,
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FIG. 6. TEM reconstruction of the internalization mechanism of A. salmonicida by murine M+s in RPMI 1640 tissue culture medium. Bar, 0.5 ,um for all three micrographs.
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BACTERIA/m0 FIG. 7. M4 association with A-layer-coated particles or substrata. (a) Association experiments with plain, uncoated latex beads (+) and beads coated with A-protein (x), 1x DOC A-layer preparation (0), 3x DOC A-layer preparation (0), and A-layer sheets isolated from strain A450-1 (*). The number of uncoated beads per 100 was substracted at each sampling time from the rest of the values. Each point represents the average of six independent counts from two separate experiments with triplicate samples. (b and c) Results from the frustrated-phagocytosis experiment. Each column represents the average of three independent counts from a single bacteria experiment with triplicate samples. (b) Association of with attached to glass coverslips coated with 3 x DOC A-layer preparation or to uncoated control coverslips. (c) Distribution of bacteria. according to the number of associated
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isolation methods, culture conditions, and bacterial strains used. We sought to overcome these disparities by using a well-characterized mammalian cell line, but a problem of incubation temperatures arose. The growth temperature for A. salmonicida lies between 20 and 25°C, or up to 30°C for A- bacteria (21, 23), whereas the optimal temperature for murine M4s is 37°C. We opted to use 37°C in all of our association or cytotoxicity assays and therefore keep the M4s under more favorable conditions. At the same time, the continued presence of the A-layer was confirmed for A' bacteria by EM and SDS-PAGE. Moreover, the A-layer has proved to be structurally stable at temperatures of up to 50°C (47), ensuring that no significant compromise in the adherence of A. salmonicida would take place because of major alterations on its surface. With different approaches, it was evident that the A-layer of A. salmonicida is involved in adherence to the M4 cell surface and may lead to internalization. This effect, which is analogous to the role of some characterized adhesins of bacterial pathogens (11, 20), was not merely the result of a nonspecific hydrophobic effect, as demonstrated in the latex bead and frustrated-phagocytosis experiments. It was due neither to a cell density-mediated process, as demonstrated in the mixed-strain assay; nor to a secondary artificial effect of bacterium or particle availability, as demonstrated in the mixed-strain, forced-association, and latex bead experiments; nor to the intervention of bacterial components different from the A-layer, as demonstrated by the latex bead assay. In the assays with latex beads, only the natively arrayed layer was competent in mediating enhanced adherence to M4s. Curiously, specific binding of immunoglobulins by the A-layer also requires the presence of assembled A-protein subunits (47). Differences observed in the adherence of beads coated with different A-layer preparations suggest that specific properties of the assembled layer make it more or less suitable as an adhesin. These could include high order (as in the A450-1 A-layer preparation), LPS O-polysaccharide content (higher in the 1 x DOC preparation and absent in the A450-1 A-layer), or the presence of a novel A-layer conformational pattern (16) (observed in the 3 x DOC preparation). The best evidence for the adhesin role of the A-layer was obtained from experiments carried out with PBS, an energypoor medium apparently unfavorable for the normal phagocytic process. The efficient internalization of A' bacteria inside multilayered membrane-lined phagosomes (Fig. 2e and f) indicated the presence of an unusual, pathogendirected mechanism that operates even in the absence of a normal phagocytic process, a situation similar to that observed for Legionella pneumophila (19) and salmonellae (12). IfA. salmonicida uses a pathogen-directed internalization mechanism to penetrate macrophages, it may also be able to use it to penetrate nonphagocytic cells, as has been demonstrated for invasive intracellular pathogens (13, 20, 36, 55, 64) and suggested for salmonellae (12). Preliminary results obtained with nonphagocytic fish cell lines support this contention (15). In respect to this, the unique ability of the A-layer to bind hemin (28) may be related to penetration of nonphagocytic cells as it has been demonstrated for Shigella flexneni (6). Our own data support the view that hemin-coated bacteria interact more efficiently with murine M4+s, producing, as well, a more marked cytotoxic effect. A- A. salmonicida cells were efficiently internalized by competent M4s kept in RPMI 1640 medium. Under these favorable conditions, A' and A- bacterial cells were simi-
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Vol. 60, No. 10
0019-9567/92/104373-10$02.00/0 Copyright © 1992, American Society for Microbiology
S-Layer-Mediated Association of Aeromonas salmonicida with Murine Macrophages RAFAEL A. GARDUNO, EMIL J. Y. LEE, ANiD WILLIAM W. KAY* Department of Biochemistry and Microbiology and The Canadian Bacterial Disease Network, University of Victoria, Petch Building, P.O. Box 3055, Victoria, British Columbia V8W3P6, Canada Received 27 February 1992/Accepted 22 July 1992
The interaction of Aeromonas salmonicida with the murine macrophage (M+) cell line P388D1 was used as a convenient model to study the involvement of the bacterial crystalline surface array (or A-layer) in the association with M4s. A-layer-positive (A') cells readily associated with M+s in phosphate-buffered saline, whereas A- mutants were unable to do so, even when the bacterium-M+ interaction was forced by centrifugation. M+s selectively interacted with A' cells when challenged with mixtures of A' and excess Acells. Electron microscopy indicated that in phosphate-buffered saline only A bacteria were readily internalized, although by a nonconventional mechanism, suggesting that efficient phagocytosis in the absence of opsonins was A-layer mediated. Latex beads coated with a partially assembled A-layer were more efficiently taken up than uncoated or A-protein-coated beads, indicating that an organized A-layer was essential for M4 uptake. The reduced ability of M+s plated on a substratum coated with the A-layer to bind A' bacteria also suggested that association was both A-layer and receptor mediated. In the presence of tissue culture medium, competent M+s interacted efficiently with A- bacteria and internalized them through conventional phagocytosis. A' cells were markedly cytotoxic to M+s, whereas the A-protein or A-layer was not. A- cells were cytotoxic to a lesser extent, suggesting that cytotoxicity was targeted.
Despite the plethora of prokaryotes that have been found to possess crystalline surface layers (S-layers), there is a lack of information regarding the functions of these unique two-dimensionally arrayed protein monolayers (1, 57). Particularly, S-layers of pathogenic eubacteria are naturally expected to have an important role in virulence, since surface components frequently mediate specific interactions of a pathogen with its host (46, 65).
to be appropriate for efficient phagocytosis by macrophages (Mos). In fact, the ability of A' bacteria to associate with resident peritoneal murine M4)s, as well as with head kidney tissue M4s from rainbow trout, was enhanced by the presence of the A-layer (60). These data were later confirmed and extended to the mammalian cell line BHK-21 and the salmonid cell line RTG-2 (44, 45). Peritoneal elicited-exudate cells from rainbow trout and coho or masu salmon were also used in a study showing that phagocytosis of A- cells by salmonid M4s is rather poor and that opsonization of A- bacteria with specific antibodies and homologous complement increased phagocytosis (52). Despite the above-described data, there is considerable evidence to the contrary, suggesting that the A-layer is not involved in the bacterium-host cell interaction. Early data indicated that virulent, strongly agglutinating strains of A. salmonicida (presumably A') were less easily phagocytosed than nonvirulent, nonagglutinating strains (presumably A-) by elicited or activated peritoneal M+s from brook trout (43). It was also reported that virulent, A' strains of A. salmonicida actually resist phagocytosis by M4s, in contrast to A- strains (54). Further, it has been suggested that the A-layer is not essential to virulence and reported that a supposedly A- mutant of A. salmonicida (virulent and autoagglutinating) adhered very effectively to monolayers of cells in culture (63). Others suggested that the presence of the A-layer does not correlate with enhanced adhesion to rainbow trout blood leukocytes or with virulence (24). In yet another report it was suggested that association ofA. salmonicida with two different salmonid cell lines is charge mediated, and a role for the A-layer was excluded (53). Owing to the unavailability of a M4j cell line from fish, M+s isolated from widely different sources through different isolation procedures, as well as a variety of A. salmonicida isolates, have been used in the past. Therefore, we decided to use a well-characterized MO cell line to avoid possible differences in M4 functional competence which accompany
The S-layer of the fish pathogen Aeromonas salmonicida, historically known as the A-layer (61), is composed primarily of subunits of a 50,000-Mr single protein species, the A-protein (26), whose encoding gene (vap, for virulence array protein) has been cloned and sequenced (3). Structurally, A-protein subunits are organized in a p4 symmetry array with a defined three-dimensional structure (8). This array covers the entire cell surface (48) and is thought to be anchored to the cell surface by specific lipopolysaccharide (LPS) 0-chain-dependent interactions (2). The A-layer represents the first bacterial S-layer to which specific functions have been assigned. While it protects the cells against bacteriophages (23) and confers resistance to predation by bdellovibrios (31), it is a particularly important virulence factor (23, 26, 29). Its virulence-related attributes include protection against complement killing (39) and binding of several host molecules, including porphyrins (28), immunoglobulins (47), and a surprising number of extracellular matrix proteins (7, 29). With the emergence of such functional diversity, it seemed important to determine whether the A-layer also confers invasive properties on A. salmonicida. Early reports (61) indicated that virulent, A-layer-positive (A') isolates of A. salmonicida readily associate with host cells. The A-layer was subsequently shown to confer greater cell surface hydrophobicity on A. salmonicida (60, 62), and this is thought *
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those variations. In this study, we clarified the role of the A-layer in the association of A. salmonicida with murine M+s. MATERIALS AND METHODS Bacteria. A. salmonicida A450, a well-characterized, virulent, A', LPS+ strain originally isolated from brown trout, was used as the wild type (26). The following two avirulent variants, obtained from A450 in this laboratory, were also used: A450-3, an LPS+, A- strain (23), and A450-1, an A', LPS-, A-layer-secreting strain (26). M+s. Murine M( cell line P388D1, derived from mouse strain DBA/2, was used throughout. M4s were obtained from J. L. Kluftinger and R. W. Hancock, University of British Columbia, for preliminary experiments. T. W. Pearson, University of Victoria, provided the M4s used in most of our studies. Buffers, media, and culture conditions. Filter-sterilized phosphate-buffered saline (PBS), pH 7.4, was used as the main buffer. Millonig's phosphate buffer (NaH2PO4; 55.2 g/liter, pH 7.4) was used to prepare specimens for electron microscopy (EM). Bacteria were grown in Luria broth (LB) medium at 20°C for 14 to 16 h. Bacteria used in association or cytotoxicity assays were cultured at 20°C for 48 to 72 h on tryptic soy agar (TSA) plates or on plates of TSA-hemin (10 p,g/ml) as needed. They were prepared for assays by being washed twice in PBS and having their concentration (optical density at 650 nm) adjusted as required. M4s were kept growing at 37°C in 5% CO2 in 40-ml tissue culture flasks (Falcon Laboratories) with 10 ml of complete RPMI 1640 medium. They were passaged every 48 h for use in experiments and every 96 to 120 h for maintenance of the cell line. Detachment of M+s by repeated washing was preferred over gentle scraping. Incomplete medium consisted of 450 ml of RPMI 1640 base (Sigma) (without sodium bicarbonate or glutamine), 5 ml of glutamine (30 mg/ml), 0.55 ml of 2-mercaptoethanol (3.5 p,g/ml), 5 ml of HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (1 M), and 13.35 ml of NaHCO3 (7.5%), pH 7.4. Complete medium contained fetal calf serum (Sigma Heart Line) at a final concentration of 5%. Staining and counting methods. Heat-fixed bacteria were stained by Gram stain, or with crystal violet for 1 min, as needed. M4 monolayers were methanol fixed for 5 min and stained with Wright or Giemsa stain (Sigma). Bacteria and latex beads in suspension were counted in a Petroff-Hausser counting chamber (in 5-,ul aliquots) or by using an internal standard of fungal spores (35). In the latter, 20 ,ul of a standardized fungal spore suspension was thoroughly mixed with 20 RI of the bacterial sample and spread on a clean glass slide, air dried, and stained for examination by light microscopy. Counting and calculation of the number of bacteria were done as described by Mallette (35). Mos in suspension were counted in a Neubauer hemocytometer after 1:2 dilution in 0.4% trypan blue in PBS. The percentage of phagocytosing M4s, as well as the number of particles (i.e., bacteria or latex beads) per phagocytosing M+, was determined by direct microscopy, applying a 95% confidence level (35). The two values were multiplied to obtain the number of particles associated with 100 M+s. Viable bacterial cell counts were made on Congo red plates (30 ,ug/ml) by the standard dilution plate method using two or three plates per sample. When high variability among
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plates of a single sample was observed, only the result from the plate with between 30 and 300 colonies was recorded. Congo red plates were used to distinguish A' bacteria (which give red colonies) from A- bacteria (which give white colonies) (22). The dilution medium used for viable counts was LB without glucose and containing 0.5% (vol/vol) Tween 20 to prevent autoaggregation. Survival curves. A. salmonicida survival at 37°C in PBS or LB was determined by direct viable cell counting. M+s were also tested for survival in PBS. Aliquots of M4s suspended in PBS and kept at 37°C were counted and assayed for trypan blue exclusion as the criterion for viability. EM. EM of M+s was carried out by conventional methods. Briefly, samples for transmission EM (TEM) were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in EPON 812. Thin sections were cut and stained with uranyl acetate-lead citrate for examination in a Phillips EM 300 TEM at 60 kV. Cells attached to round (10-mm-diameter) glass coverslips were also examined by scanning EM (SEM). Coverslips were fixed and dehydrated as for TEM and then critical point dried and mounted on aluminum stubs for gold coating. Specimen examination was done with a JEOL JSM 35 SEM at 20 kV. Negative staining of whole, air-dried bacteria was done on Formvar-coated grids with a saturated solution of unbuffered ammonium molybdate. Bacterium-M+ association assays. M+s were plated on 35-mm-diameter tissue culture dishes, each containing a glass coverslip (22 by 22 mm) attached to the bottom, at a ratio of 106 M+s per dish. Alternatively, 24-well tissue culture plates were inoculated with 0.5 x 106 M+s per well. Dishes or plates were incubated for up to 4 h at 37°C to allow attachment of M4s or overnight to allow attachment and growth of M4)s on the substratum. Before the experiment, M+s were washed twice with PBS and covered with 1 ml of fresh incomplete medium or PBS. Adherent M4s were scraped off of two dishes or wells to determine their number. Suspensions of washed bacteria in PBS were adjusted so that 100 RI contained a number of bacteria equal to 50 times the number of M4s per dish or well. Assays were initiated by inoculation of the dishes or wells with 100 ,Il of the corresponding bacterial suspension. In assays with 24-well plates, inoculation of wells was done sequentially (at the corresponding sampling times) so that at the end of the experiment all of the wells in a single plate were processed together. Two 35-mm dishes were usually processed at every sampling time. Supernatants from the two dishes were removed (and sampled when necessary), and the dishes were washed three times with PBS. M4s in one dish were lysed in 1 ml of deionized water for 10 min with agitation. The M+ lysate (1 ml) was collected, and the dish was washed three times with 3 ml of deionized water. Washes were collected with the lysate to produce a 1:10 dilution. The second dish was air dried, and the coverslip was stained and prepared for direct microscopy counts on monolayers. At the end of experiments with 24-well plates, supernatants from all of the wells in a plate were removed and the plate was washed twice with PBS by using a wash bottle. Plates were air dried and stained. Well bottoms were cut with a cork-boring machine and mounted on a glass slide for direct microscopy counts on monolayers. All association assays were performed at 37°C. Purification of A-layer and A-protein. Outer membrane preparations of A450, extracted with sodium lauryl sarcosinate, were prepared as previously reported (26, 48). Outer membrane preparations were subjected to serial extractions
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A450 A450-3 FIG. 1. (a) Survival curves of A' and A- A. salmonicida at 37°C in LB. Symbols: 0, A' (A450); +, A- (A450-3). Inset: electron micrograph of negatively stained A450 cells incubated at 37°C in LB. A, A-layer cap at one end of the cell. Bar, 5 pm. (b) Survival of A' and A- A. salmonicida after 24 h at 37°C. Survival was enhanced by keeping the bacterial cells in PBS.
with sodium deoxycholate (DOC) as follows. A 4.5-ml volume of an outer membrane preparation, 4.0 ml of 10 mg of lysozyme per ml, and 31.5 ml of 20 mM Tris (pH 8.0) with 5 mM EDTA were mixed and incubated for 30 min at room temperature with agitation. Forty milliliters of 4% DOC in 0.5 M NaCl with 5 mM EDTA were then added, and the preparation was incubated for 30 min at 30°C. The extracted A-layer-outer membrane pellet was separated by centrifugation at 10,000 x g for 10 min at room temperature and then washed with 20 mM phosphate buffer, pH 7.3. This preparation was labelled 1 x DOC. The DOC extraction steps were
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serially repeated to obtain the corresponding 2 x and 3 x DOC preparations. A-protein from A450 was purified to homogeneity by previously published methods (48) and adjustment of the guanidine hydrochloride concentration to 2 M. A-layer sheets from secreting mutant A450-1 were purified from culture supernatant by centrifugation at 100,000 x g for 1 h, followed by a single DOC extraction. Assays with coated latex beads. Latex beads with a 3.1-,um diameter (Seradyn, Particle Technology Division) were extensively washed with glycine-buffered saline (0.1 M glycine, 0.85% NaCl, pH 8.4). The suspension of beads was adjusted to a density of 1% solids (5.6 x 108 beads per ml), and different A-layer preparations (lx and 3x DOC and the A450-1 layer) or purified A-protein was added to achieve a final concentration of 50 ,ug/ml. Coating was allowed to occur for 3 h at room temperature under continuous gentle agitation. Beads were then washed twice with PBS and stored on ice. Confirmation of protein coating and purity was made by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of 25 ,l were boiled for 10 min with 9 ,ul of 4x sample buffer. After centrifugation in an Eppendorf microcentrifuge, a 20-pl volume of the liquid beneath the floating beads was loaded on a 12% gel for SDS-PAGE. Phagocytosis experiments with latex beads were done in PBS with 24-well tissue culture plates as described in for bacterium-M4 association assays. The beads-M4 ratio was adjusted to 5:1. Frustrated-association-phagocytosis assay. The frustratedassociation-phagocytosis assay was prepared as a regular bacterium-M4 association assay in PBS by using 35-mm dishes but with the following modifications. Half of the coverslips were coated with the purified A-layer: a 1-ml suspension of the 3 x DOC A-layer (140 ,ug/ml) in deionized sterile water was allowed to dry at room temperature on each coverslip. The coverslips were then covered with 0.5 ml of an A-layer suspension in PBS (70 p,g/ml) for 4 h. Control coverslips were similarly treated with deionized water and PBS. Coverslips were then washed with excess PBS, inoculated with 5 x 105 M+s, and incubated for 4 h prior to replacement of culture medium with 1 ml of an A450 suspension in PBS adjusted to give a bacterium-M4 ratio of 50:1. After 3 h, dishes were washed in PBS and processed for direct microscopy counts in monolayers. A. sabnonicida cytotoxicity to M4s. Cytotoxicity was evaluated in adherent M4s through bacterium-induced morphological changes. M4s and bacteria were prepared in PBS as for bacterium-M4 association assays, but M4s were inoculated on 35-mm dishes with four circular coverslips (10 mm in diameter) attached to the bottom. Two different bacterium MO cell ratios were used: 500:1 and 2,000:1. At 2 and 4 h after addition of bacteria, two coverslips were removed from each dish. One coverslip was air dried and processed for light microscopy, and the other was prepared for SEM. Also, cytotoxicity towards M4s suspended in PBS was evaluated through M4 viability, as determined by the trypan blue exclusion assay (described in the section on survival curves) by using bacterium-M4 mixtures at a cell ratio of 100:1. RESULTS Effect of experimental assay conditions on murine M+s and A. salmonicida. P388D1 M4s remained 90% viable when
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FIG. 2. Association of A4 (A450) and A- (A450-3) bacteria with M+s kept in PBS as determined by three different quantitative methods. (a) Bacterial cells associated per 100 M+s as determined by direct microscopy counts in M+ monolayers. Results of two separate experiments are shown: experiment 1; -, experiment 2; *, A'; x, A-. (b) Bacterium-M+ association as determined by direct microscopy counts using the internal-standard method. Data were obtained from samples of experiment 2 in panel a and are expressed as percentages of the bacterial cells added: counts done in M4 lysate samples; -, counts done in supernatant samples; 0, A'; x, A-. (c) Bacterium-M4 association as determined by viable cell counts. Data were obtained from M+ lysate samples taken from experiment 1 in panel a and are expressed as percentages of the viable cells added: 0, A4 bacteria; x, A- bacteria. (d to t) SEM and TEM of M4s kept in PBS with bound and internalized A4 bacteria. --
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suspended in PBS for up to 6 h (data not shown). SEM showed that, compared with control M4)s kept in complete RPMI 1640, there were no major alterations in gross morphology when M4s were kept in PBS, except for a less spread appearance. On the other hand, incubation of A. salmonicida in LB at 37°C caused a rapid decline in viability (Fig. la). Microscopic examination of these heat-stressed cultures showed that although most bacterial cells were elongated, A4 bacteria still possessed A-layers in the form of caps at the cell poles (Fig. la, inset). SDS-PAGE of membrane preparations stained for protein and LPS revealed no other significant compositional changes after 3 h at 37°C. Bacterial cells suspended in PBS were also killed at 37°C, but the final number of survivors was much higher (Fig. lb) than cells left in LB medium. EM (data not shown) revealed that these cells were elongated less (or not at all), and
A-layers were found surrounding most A4 bacteria. These data ensured a valid experimental design in which the target cells, M4s, remained viable and structurally unperturbed and adherent cells retained their surface components. Bacterium-M4 association in PBS. At all incubation times, the association of A. salmonicida with Mos (as determined through different quantitative methods) was much greater for A4 than A- strains (Fig. 2a to c). Specimens observed by SEM showed that A4 cells readily adhered to M+s and sometimes covered the entire pseudopodial surface (Fig. 2d). TEM revealed that A4 cells were also readily internalized by M4s in PBS (Fig. 2e and f), while it was not possible to identify any internalized A- cells. Forced association. It is known that A4 A. salmonicida cells in suspension autoagglutinate and consequently sediment considerably faster than A- bacteria (10, 61). To
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HOURS FIG. 3. Forced association of A' (A450) and A- (A450-3) A. sabnonicida with M+s in PBS. Association assays were done in 24-well plates in which bacteria were centrifuged (500 x g for 10 min) onto the Mi monolayer. At the different sampling times, rows of wells were washed three times in PBS and M+s were kept in fresh PBS until the end of the experiment, when the plate was air dried and processed as described in Materials and Methods. Symbols: 0, A+; x, A-.
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FIG. 4. M+ association from mixtures of Al (A450) and A(A450-3) strains ofA. salmonicida at different A--to-A cell ratios. Association with M+s was determined by viable cell counts of M+ lysate samples on Congo red agar plates (refer to Fig. 2c). Symbols: , A -to-Ak-cell ratio of 500:1; - - -, A--to-A' cell ratio of 50:1; 0, A+; x, A-.
M4s bound or internalized A- and A+ bacteria. Under these
confirm that the higher association levels observed with A+ cells were not an artifactual result of a faster sedimentation rate, both A+ and A- cell suspensions were centrifuged onto the M4 monolayer and subsequently processed to determine their levels of association with M4s (Fig. 3). The degree of M4 association with A- cells was increased, but not nearly as dramatically as for A+ cells. The global quantitative levels of M4) association for both bacterial strains were nearly an order of magnitude greater by this procedure. Thus, the difference in association of A+ and A- cells with M4)s was not due to a cell availability artifact. Selective adherence. To examine the possibility that extracellular factors from A+ cells or the association of A+ cells with M4)s may activate them towards an enhanced association with A- cells, A+-A- coincubation experiments were carried out. Two coinfection experiments were conducted by using 50:1 and 500:1 A--to-A+-cell ratios (Fig. 4). Viable cell counts of M4) lysate samples were used to monitor the level of bacterial association with M4)s. A+ and A- cells were discriminated on the basis of Congo red binding. Owing to the lethal effect of the incubation temperature upon A. salmonicida, the absolute value of these results can be regarded as an underestimate. Nevertheless, it was still evident that M4s in PBS were unable to associate with Acells, whereas A+ cells, even in the presence of a large number of A- cells, efficiently associated with these same M4)s. Thus, the association of A+ bacteria with M4s was not a cell density-determined artifact, nor did one cell type predispose M4)s to associate with the other. Effect of complex medium on bacterium-M+ Interactions. When association assays were carried out in tissue culture medium, M4s were particularly found to associate more readily with A- bacteria than previously observed (Fig. 5a and b). These more competent M+s also internalized Abacteria efficiently (Fig. 5c). Moreover, there were no apparent qualitative differences in the mechanism by which
experimental conditions,A. salnonicida was internalized by a conventional zipper mechanism (56), as depicted in Fig. 6. Assays with coated beads and substrata. A series of association assays with coated latex beads were conducted to evaluate the contribution of the A-layer alone to the processes of adherence and internalization. All of the A-layer preparations used were able to enhance the interaction of latex beads with M+s (Fig. 7a), which is all the more noteworthy because uncoated, hydrophobic latex beads are efficiently phagocytosed by M4s and for this reason are commonly used in phagocytosis assays (56). We attributed this enhancing effect to the regularly arrayed A-layer, since uncoated beads and those coated with purified monomeric A-protein behaved similarly. The A-layer prepared from A450-1 had, as determined by negative-stain EM, the greatest degree of order (data not shown) and was the most effective at promoting adherence. The possible existence of a specific adhesin-receptor interaction between the arrayed layer and the M4 surface was further examined in a frustrated-phagocytosis assay. In this type of assay, the presence of putative receptors for a particular ligand is indirectly revealed by recruiting available M4 receptors on a ligand-coated substratum (down modulation), thereby depleting available receptors and reducing subsequent bacterial cell interaction (37, 58). The global capacity for binding of A' cells was reduced 40% in M4s plated on A-layer-coated coverslips (Fig. 7b). This reduction was accompanied by a clear shift in the association profile of A' cells to M+s (Fig. 7c) (58), indicating that a specific receptor, presumably involved in the adherence process of A' A. salmonicida to M+s, was down modulated by the presence of the A-layer coating. A. salnonicida cytotoxicity to M+s. During the association assays, it was found that M4s exposed to A' bacteria appeared to be more compact (darkly stained) and to have a larger proportion of lysed cells than those exposed to Acells. These data prompted an investigation of the possible
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FIG. 5. Association of A' (A450) and A- (A450-3) A. salmonicida with M4s in RPMI 1640 tissue culture medium. (a) A' and Abacterium-M4 associations were measured after 1 h of incubation in PBS (column 1) or RPMI 1640 (column 2). The bacterium-to-M4 cell ratio was 100:1. (b and c) SEM and TEM of M+s with bound and internalized A- bacteria from panel a, column 2.
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cytotoxic effects of A' bacteria and, in particular, the role of the A-layer. Control M4s (bacterium free) and M4s challenged with either A' or A- cells (whether they were previously grown in the presence or absence of hemin) contained approximately the same proportion of cells permeable to trypan blue after an 8-h challenge (data not shown). However, there were marked morphological differences among adherent M4s treated with the different bacteria used (Fig. 8). The marked effects of A' cells upon adherent M4s were confirmed by light microscopy (Fig. 8L and M), in contrast to the less severe effects induced by Abacteria (Fig. 8H to K). Interestingly, SEM specimens indicated that A' bacteria induced a marked rounding of M4s and smoothing of their surface, presumably owing to severe cytoskeletal modifications (Fig. 8D and F). This effect was also noticeable in M4)s exposed to A- bacteria (Fig. 8E), but only at an unusually high bacterium-to-Mo cell ratio of 2,000:1 or at longer incubation times. Similar rounding and cratering effects were also seen when M4s were challenged with A- bacteria grown on TSA-hemin plates. However, the morphological changes induced by A- bacteria were never as extensive as those induced by A' bacteria, and most of the M4os exposed to A- bacteria remained intact (Fig. 8C, J, and K). Only hemin-coated A' bacteria induced massive lysis in some MO monolayers. M4s that had not yet lysed in these monolayers resembled golf balls, with craters on their surfaces, and were densely covered with bacteria (Fig. 8F and G). Especially curious was the appearance of apparent cytoskeleton ghosts, presumably consisting of a cytoskeletal structure still attached to the substratum but devoid of a cell membrane (CG in Fig. 8E and G). There was a good correlation between the light microscopy images and the detailed SEM data. DISCUSSION
Use of a well-defined system to study the role of the A-layer in M4) association appears to be justified on the basis that, perhaps, the wide discrepancies in the literature regarding this role are due to differences in the Mo sources,
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FIG. 6. TEM reconstruction of the internalization mechanism of A. salmonicida by murine M+s in RPMI 1640 tissue culture medium. Bar, 0.5 ,um for all three micrographs.
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BACTERIA/m0 FIG. 7. M4 association with A-layer-coated particles or substrata. (a) Association experiments with plain, uncoated latex beads (+) and beads coated with A-protein (x), 1x DOC A-layer preparation (0), 3x DOC A-layer preparation (0), and A-layer sheets isolated from strain A450-1 (*). The number of uncoated beads per 100 was substracted at each sampling time from the rest of the values. Each point represents the average of six independent counts from two separate experiments with triplicate samples. (b and c) Results from the frustrated-phagocytosis experiment. Each column represents the average of three independent counts from a single bacteria experiment with triplicate samples. (b) Association of with attached to glass coverslips coated with 3 x DOC A-layer preparation or to uncoated control coverslips. (c) Distribution of bacteria. according to the number of associated
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4379
isolation methods, culture conditions, and bacterial strains used. We sought to overcome these disparities by using a well-characterized mammalian cell line, but a problem of incubation temperatures arose. The growth temperature for A. salmonicida lies between 20 and 25°C, or up to 30°C for A- bacteria (21, 23), whereas the optimal temperature for murine M4s is 37°C. We opted to use 37°C in all of our association or cytotoxicity assays and therefore keep the M4s under more favorable conditions. At the same time, the continued presence of the A-layer was confirmed for A' bacteria by EM and SDS-PAGE. Moreover, the A-layer has proved to be structurally stable at temperatures of up to 50°C (47), ensuring that no significant compromise in the adherence of A. salmonicida would take place because of major alterations on its surface. With different approaches, it was evident that the A-layer of A. salmonicida is involved in adherence to the M4 cell surface and may lead to internalization. This effect, which is analogous to the role of some characterized adhesins of bacterial pathogens (11, 20), was not merely the result of a nonspecific hydrophobic effect, as demonstrated in the latex bead and frustrated-phagocytosis experiments. It was due neither to a cell density-mediated process, as demonstrated in the mixed-strain assay; nor to a secondary artificial effect of bacterium or particle availability, as demonstrated in the mixed-strain, forced-association, and latex bead experiments; nor to the intervention of bacterial components different from the A-layer, as demonstrated by the latex bead assay. In the assays with latex beads, only the natively arrayed layer was competent in mediating enhanced adherence to M4s. Curiously, specific binding of immunoglobulins by the A-layer also requires the presence of assembled A-protein subunits (47). Differences observed in the adherence of beads coated with different A-layer preparations suggest that specific properties of the assembled layer make it more or less suitable as an adhesin. These could include high order (as in the A450-1 A-layer preparation), LPS O-polysaccharide content (higher in the 1 x DOC preparation and absent in the A450-1 A-layer), or the presence of a novel A-layer conformational pattern (16) (observed in the 3 x DOC preparation). The best evidence for the adhesin role of the A-layer was obtained from experiments carried out with PBS, an energypoor medium apparently unfavorable for the normal phagocytic process. The efficient internalization of A' bacteria inside multilayered membrane-lined phagosomes (Fig. 2e and f) indicated the presence of an unusual, pathogendirected mechanism that operates even in the absence of a normal phagocytic process, a situation similar to that observed for Legionella pneumophila (19) and salmonellae (12). IfA. salmonicida uses a pathogen-directed internalization mechanism to penetrate macrophages, it may also be able to use it to penetrate nonphagocytic cells, as has been demonstrated for invasive intracellular pathogens (13, 20, 36, 55, 64) and suggested for salmonellae (12). Preliminary results obtained with nonphagocytic fish cell lines support this contention (15). In respect to this, the unique ability of the A-layer to bind hemin (28) may be related to penetration of nonphagocytic cells as it has been demonstrated for Shigella flexneni (6). Our own data support the view that hemin-coated bacteria interact more efficiently with murine M4+s, producing, as well, a more marked cytotoxic effect. A- A. salmonicida cells were efficiently internalized by competent M4s kept in RPMI 1640 medium. Under these favorable conditions, A' and A- bacterial cells were simi-
INFEcr. IMMUN.
GARDUNO ET AL.
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FIG. 8. Morphological changes induced by A. salmonicida upon adherent M)s as detected through SEM (A to G) and light microscopy (H to M). M4s were challenged with different bacterial preparations, at different cell ratios, and for different times. Panels: A and H, control M4
