- Email: [email protected]
Streptococcus suis and group B Streptococcus differ in their interactions with murine macrophages
FEMS Immunology and Medical Microbiology 21 (1998) 189^195
Streptococcus suis and group B Streptococcus di¡er in their interactions with murine macrophages Mariela A. Segura, Patrick Cleèroux, Marcelo Gottschalk * Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculteè de Meèdecine Veèteèrinaire, Universiteè de Montreèal, P.O. Box 5000, St-Hyacinthe, Queè. J2S 7C6, Canada Received 9 January 1998; revised 27 April 1998 ; accepted 7 May 1998
Abstract Streptococcus suis type 2 and group B Streptococcus type III (GBS) are important encapsulated bacterial species causing meningitis. In the present study we compared quantitatively the uptake and intracellular survival of S. suis type 2 and GBS type III with murine macrophages in non-opsonic conditions. The role of the capsule of both pathogens was also studied using previously obtained unencapsulated isogenic mutants. Encapsulated S. suis wild-type strain was practically not phagocytosed, while the unencapsulated mutant was easily ingested by macrophages. On the other hand, the well encapsulated GBS strain and its unencapsulated mutant were both phagocytosed in large numbers. Even if S. suis unencapsulated mutant showed a higher uptake rate than the parental strain, this value was always markedly lower than the numbers of ingested GBS strains. In addition, the intracellular survival of encapsulated and unencapsulated GBS strains was significantly higher than that of S. suis strains. These results suggest that interactions between GBS type III and S. suis type 2 with murine macrophages as well as the role of the capsule as an antiphagocytic factor are different for the two bacterial pathogens. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords : Streptococcus suis; Group B Streptococcus; Non-opsonic phagocytosis; Intracellular survival; Polysaccharidic capsule ; Macrophage
1. Introduction Streptococcus suis is an important pathogen which has been associated with a wide variety of infections in swine such as meningitis, septicemia, and pneumonia [1]. It has also been isolated from other animal species, as well as from humans [2,3]. To date, 35 di¡erent capsular types of S. suis have been described. S. suis capsular type 2 is considered to be * Corresponding author. Tel.: +1 (450) 773-8521 ext. 8374; Fax: +1 (450) 778-8108; E-mail: [email protected]
the most virulent as well as the most prevalent capsular type in diseased pigs [4]. Group B Streptococcus (GBS) is an important cause of pneumonia, sepsis and meningitis in neonates. GBS associated with human disease are almost invariably encapsulated, belonging to one of the nine recognized capsule serotypes: Ia, Ib, II^ VIII. Type III GBS strains account for about twothirds of the GBS isolates associated with invasive neonatal disease [5]. S. suis and GBS present several similarities. Both streptococci are well encapsulated, some capsular
0928-8244 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 4 8 - 0
FEMSIM 886 30-7-98
190
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
types are more virulent than others, they possess sialic acid in their capsule and they can cause severe meningitis. However, it is not clear whether the pathogenesis of the infection caused by the two bacterial species is similar. The capsules of a variety of bacterial species are thought to play an important role in virulence. Encapsulated organisms interact with host defense systems by several known mechanisms [6]. Among them, the sialylated polysaccharidic capsule of GBS plays a well recognized role in the resistance to phagocytosis, by modulating the alternative complement pathway, and protecting the organism from opsonophagocytic killing [7^10]. In vitro and in vivo virulence assays with unencapsulated or asialo mutants support the hypothesis that type III capsule serves as a virulence factor [11,12]. Other studies have shown that well encapsulated GBS are able to enter and persist in macrophages by evading intracellular opsonin-mediated antibacterial activities [13,14]. The role of the polysaccharidic capsule in the opsoninindependent uptake is so far unknown. S. suis is also encapsulated. In a recent work, transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain isogenic acapsular mutants from a virulent S. suis type 2 strain. These mutants were shown to be avirulent for both mice and piglets and cleared from the circulation rapidly [15]. In fact, the capsule seems to be the ¢rst critical virulence factor described so far for this bacterial species. However, avirulent ¢eld strains are also encapsulated. Other putative virulence factors have been suggested, such as cell-wall and extracellular proteins, toxins, adhesins and immunoglobulinbinding proteins [16^20], but their role in the pathogenesis of the infection is unknown. As mentioned above, S. suis also possesses sialic acid in its capsule. However, and unlike GBS, this sugar does not seem to be a critical component for the virulence [21]. It may be possible that interactions between S. suis and GBS with macrophages do di¡er. In fact, previous studies of interactions between encapsulated strains of S. suis type 2 and phagocytic cells are contradictory [22]. In the present study we compare the uptake and intracellular survival of S. suis type 2 and GBS type III with murine macrophages in non-opsonic conditions. The role of the capsule of both pathogens is
also studied using previously obtained acapsular isogenic mutants.
2. Materials and methods 2.1. Bacterial strains and growth conditions Two strains of S. suis capsular type 2 and two strains of GBS capsular type III were used: the virulent-encapsulated S. suis wild-type strain S735-SM and its avirulent-unencapsulated isogenic transposon mutant 2A [15], as well as the highly encapsulated wild-type strain of type III GBS, COH1, and its less virulent and unencapsulated isogenic transposon mutant, COH1-13 [12]. The latter strains were kindly provided by Dr. C.E. Rubens (Children's Hospital and Medical Center, Seattle, WA, USA). Bacteria were maintained as stock cultures in 50% glycerol^Todd-Hewitt broth (THB; Difco Lab., Detroit, MI, USA) at 380³C. The THB was supplemented with 10 Wg ml31 tetracycline (Tc; Sigma-Aldrich, Oakville, Canada) for growing the mutants [15]. Bacteria were grown overnight onto bovine blood agar plates at 37³C and single colonies were used as inocula for THB or THB-Tc, which were incubated for 18 h at 37³C. Working cultures were made by inoculating 0.3 ml of these cultures in 10 ml of THB at 37³C with agitation until it reached the mid-exponential phase (6 h incubation time; OD540 0.4^0.5 for S. suis strains, and 0.7^0.8 for GBS strains). Bacteria were washed twice in phosphatebu¡ered saline (PBS), pH 7.4, and diluted to approximately 107 CFU ml31 in cell culture media (without antibiotics). An accurate determination of the number of colony forming units (CFU) in the ¢nal suspension was made for each assay by plating on THB agar. 2.2. Cell lines and cell culture J774A1 murine (BALB/c) macrophage-like cell line (ATCC TIB 67, Rockville, MD) was maintained in Dulbecco's modi¢ed Eagle's medium, 1.5 g ml31 bicarbonate (DMEM). P388D1 murine (DBA/2) macrophage-like cell line (ATCC TIB 63) was maintained in Iscove's modi¢ed Dulbecco's medium (IMDM). Cell media were supplemented with 10%
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
heat-inactivated fetal bovine serum (iFBS), penicillin G (100 IU ml31 ), and streptomycin (100 Wg ml31 ) (Gibco, Burlington, Ont., Canada) and cells were grown at 37³C, 5% CO2 . For bacterial phagocytosis and survival assays, 48h culture cells were scraped up, washed once and resuspended in antibiotic-free medium at approximately 2U105 cells ml31 . 1 ml of this suspension was distributed into 24-well tissue culture plates Falcon (VWR CanLab, Quebec, Canada) and incubated for 3 h at 37³C, 5% CO2 to allow cell adhesion before assays. 2.3. Preparation of murine peritoneal macrophages Peritoneal exudate macrophages (PEM) were isolated as previously described [23]. Brie£y, ¢ve or six 6-week-old BALB/c male mice (Charles River, StConstant, Canada) were used per individual assay. Mice were inoculated intraperitoneally with 1.5 ml 3% (w/v) thioglycolate sterile broth (Sigma). PEM were recovered 4 days later by washing the peritoneal cavity with 5 ml Hanks' balanced salt solution Ca2 -Mg2 -free (HBSS, Gibco). Cells were pooled, pelleted by centrifugation for 10 min at 1000Ug, and resuspended at a ¢nal concentration of 2U105 cells ml31 in minimum essential medium (MEM, Gibco) supplemented with 10% iFBS (MEMs). 1 ml of this suspension was distributed into 24-well tissue culture plates (Falcon, VWR CanLab). Cells were allowed to adhere for 1 h at 37³C, 5% CO2 , washed with warm HBSS, and reincubated overnight in MEMs before assays. Cell purity was more than 95% as determined by non-speci¢c esterase stain and modi¢ed Wright-Giemsa stain (LeukoStat, Fisher Sci., Montreal, Canada). Cell viability was more than 99% as determined by trypan blue exclusion. 2.4. Phagocytosis assay Phagocytosis assays were performed as already described [14] with some modi¢cations. J774 cell, P388 cell and PEM plates were infected with streptococci by removing culture medium and adding 1 ml of 107 bacterial suspension per well (in respective cell culture media, supplemented with 10% iFBS), to obtain a ratio of about 100 bacteria per macrophage. Phag-
191
ocytosis was left to proceed for 30 min and 90 min at 37³C, 5% CO2 . After incubation, cell monolayers were washed twice with warm HBSS, and reincubated for 1 h with medium containing penicillin G (5 Wg ml31 ) and gentamicin (100 Wg ml31 ; Sigma) to kill extracellular bacteria. It has been demonstrated that these antibiotics do not penetrate eukaryotic cells under these conditions [14], and previous studies have shown that this concentration of antibiotics was able to kill any remaining extracellular bacteria (data not shown). In addition, supernatant controls were taken in every test to con¢rm the activity of antibiotics. After antibiotic treatment, cell monolayers were washed three times and the medium was replaced with 1 ml of sterile distilled water to lyse the macrophages. After vigorous pipetting to ensure complete cell lysis, viable intracellular streptococci were determined by quantitative plating of serial dilutions of the lysates on THB agar. Each test was done four times in independent experiments, and the number of CFU recovered per well (mean number þ S.D.) was determined. 2.5. Intracellular survival assay Survival experiments were done as described in Section 2.4, except that after 30 min of infection with streptococci, cell monolayers were washed twice with warm HBSS and antibiotic-containing medium was added and left on infected cells up to 4 h post infection. At various intervals (60, 90, 120, 180, and 240 min), infected monolayers were washed three times and the medium was replaced with 1 ml of sterile distilled water to lyse the macrophages. After vigorous pipetting to ensure complete cell lysis, viable intracellular streptococci were determined by quantitative plating of serial dilutions of the lysates on THB agar. Each test was done four times and intracellular killing was expressed as the percentage decrease in the initial number (100%) of viable intracellular bacteria at 60 min interval time [24]. 2.6. Statistics Di¡erences were analyzed for signi¢cance by using Student's unpaired t-test (two-tailed P-value).
FEMSIM 886 30-7-98
192
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
3. Results 3.1. Phagocytosis of S. suis and GBS We compared quantitatively the uptake process of S. suis and GBS wild-type strains and the two unencapsulated avirulent mutants with two di¡erent murine phagocytic cell lines (J774A1 and P388D1) and with murine PEM, at 30 min and 90 min infection times. At 30 min of phagocytosis (Fig. 1a), encapsulated S735-SM strain was practically not phagocytosed, and there was no signi¢cant di¡erence between the cell lines and PEM (P s 0.05). In contrast, encapsulated GBS strain COH1 was highly phagocytosed. Phagocytic values were more elevated with J774A1 cells, followed by PEM, while P388D1 cells showed the lowest uptake rate (P 6 0.05).
Fig. 2. Time course of intracellular survival of S. suis unencapsulated mutant 2A and GBS strains (COH1 and unencapsulated mutant COH1-13) within (a) P388D1 cells, (b) J774A1 cells, and (c) peritoneal exudate macrophages (PEM). Macrophages were allowed to ingest bacteria for 30 min, then antibiotics were added to kill extracellular bacteria and the numbers of viable intracellular bacteria were determined by quantitative plating at several post-infection times. Killing was expressed as the percentage decrease in the initial number (100%) of viable intracellular bacteria at 60 min post-infection time. These ¢gures are representative of the results of four independent experiments. Fig. 1. Phagocytosis of S. suis strains (S735-SM and the unencapsulated mutant 2A) and GBS strains (COH1 and the unencapsulated mutant COH1-13) after (a) 30 min infection time and (b) 90 min infection time. Numbers of phagocytic bacteria were determined by quantitative plating after 1 h of antibiotic treatment, and results are expressed as CFU recovered bacteria per well (means þ S.D. obtained from four independent experiments).
S. suis unencapsulated mutant 2A was highly phagocytosed compared to wild-type strain. Phagocytic values were more elevated with J774A1 cells than those obtained with PEM or P388D1 cells (P 6 0.05). However, GBS unencapsulated mutant
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
COH1-13 was slightly more phagocytosed than the wild-type COH1 strain by each of the three cell types. However, this di¡erence was markedly lower than that observed between S. suis unencapsulated mutant and its parental strain. After 90 min of phagocytosis (Fig. 1b), the uptake of encapsulated S735-SM strain was not increased with any of the three types of phagocytic cells; and values were nearly zero. In contrast, the GBS COH1 strain uptake rate was increased almost two log with PEM and nearly one log with both cell lines. P388D1 cells showed a lower uptake degree than J774A1 cells and PEM (P 6 0.05), while no signi¢cant di¡erence was noted between the latter cell types (P s 0.05). Unlike S. suis parental strain, the ingested numbers of the unencapsulated mutant 2A at 90 min were signi¢cant increased compared to phagocytic values at 30 min infection time. The increase in phagocytosis was higher with J774A1 cells than with P388D1 cells or PEM (P 6 0.05). In contrast, there were no signi¢cant di¡erences in increasing phagocytosis between the GBS unencapsulated mutant COH1-13 and the parental strain. Mutant COH1-13 strain was phagocytosed at the same rate as the parental strain by J774A1 cells and PEM (P s 0.05), but it was slightly more phagocytosed by P388D1 cells (P 6 0.05). Even if S. suis unencapsulated mutant showed a higher uptake rate than the parental strain, this value was always markedly lower than the numbers of ingested GBS strains (P 6 0.05). In general, for all four strains, the uptake rate with J774A1 cells and PEM was higher than that shown with P388D1 cells. 3.2. Intracellular survival of S. suis mutant 2A compared to GBS strains To study the intracellular survival of S. suis and GBS strains, we infected cells for 30 min and then samples were taken at di¡erent time points after the addition of antibiotic-containing medium. The few bacteria of S. suis wild-type S735-SM strain which were phagocytosed were not able to survive inside macrophages (data not shown). Mutant 2A showed a signi¢cant linear decrease (P 6 0.05) in the number of viable intracellular bacteria with all cell types. The time course of intracellular killing of mutant 2A with P388D1 cells was slower compared to J774A1 cells
193
and PEM, decreasing by 90 min to 72% of that observed at 60 min (100%), and to nearly 6% after 240 min post-infection time (Fig. 2a). In the case of J774A1 cells and PEM (Fig. 2b,c), the number of viable intracellular bacteria decreased rapidly to about 20% at 90 min; by 180 min, less than 4% survival was detected, and viable intracellular bacteria were almost not detected after 240 min post-infection time. The intracellular survival of GBS strains was signi¢cantly higher than that of S. suis mutant 2A. Results showed (Fig. 2a^c) that there were no signi¢cant di¡erences between the wild-type GBS strain COH1 and the unencapsulated mutant COH1-13 (P s 0.05) with any of the three cell types. Up to 120 min post infection, intracellular numbers of viable GBS remained almost constant. Between 180 and 240 min post infection, a quite signi¢cant decrease in the number of intracellular GBS was observed with P388D1 cells and PEM (P 6 0.05), but remained constant with J774A1 cells.
4. Discussion Surface encapsulation is one of the most important virulence factors of several pathogenic microorganisms. The bacterial capsule, by e¡ectively inhibiting phagocytosis and resisting complement-mediated bactericidal activity, may enhance bloodstream survival of the organism and facilitate intravascular replication. Indeed, the most common meningeal pathogens are all encapsulated [25]. However, the mechanisms of interaction of encapsulated bacteria with the host immune system are not at all known, and they are not generalized for all pathogens. In the present study we compared quantitatively the in vitro interactions, uptake and intracellular survival, of S. suis capsular type 2 and GBS capsular type III with murine phagocytic cells under non-opsonic conditions. The role of their capsules in these interactions was evaluated by using unencapsulated isogenic mutants of both pathogens. Viable counting techniques for monitoring phagocytic interactions have already been used for GBS [14]. The present work is the ¢rst determination of phagocytosis and time course of intracellular survival of S. suis by quantitative plating.
FEMSIM 886 30-7-98
194
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
The results of this study show that well encapsulated GBS can be phagocytosed in large numbers by murine macrophages in the absence of complement and antibodies. Our results are similar to those described by Valentin-Weigand et al. [14], who also showed that GBS COH1 strain is able to enter and persist e¤ciently in J774A1 macrophages by evading intracellular antibacterial activities. On the other hand, no di¡erences were observed between this strain and its unencapsulated mutant, which indicates that the capsule is not necessarily an antiphagocytic factor for this bacterial species. Controversially, previous reports have assumed an antiphagocytic role of GBS type III capsule since encapsulated bacteria were resistant to opsonophagocytic killing by PMN in the absence of speci¢c antibodies, while unencapsulated or asialo mutants were susceptible [11,12,26]. However, in these studies, the techniques used allowed the study of bacterial killing rather than phagocytosis, since no di¡erentiation of intracellular and extracellular bacteria was done. Di¡erent results were obtained with S. suis. The well encapsulated parental strain of S. suis type 2 was almost not phagocytosed by murine macrophages, even after 90 min of bacteria-cell contact. Interestingly, previous in vitro studies of this bacterial species with phagocytes are contradictory. Early bacterial killing studies could not demonstrate any `phagocytic activity' with S. suis type 2 [27,28], whereas more recent studies showed variable percentages of phagocytosis (7^30%), using a vital staining technique [21,23,29]. These discrepancies may be due to technical di¡erences. In fact, the results of the present study represent the ¢rst data from quantitative phagocytosis using a viable counting technique. Unlike the GBS, the high percentage of phagocytosis obtained with the unencapsulated mutant of S. suis demonstrates the antiphagocytic role of the capsule, which con¢rms previous observations [15,23,30]. Similar results were obtained when porcine monocytes were used (data not shown). The role of the capsule in intracellular bacterial survival was also studied. Both encapsulated and unencapsulated GBS strains were able not only to enter, but also to survive e¤ciently inside macrophages. To the best of our knowledge, this is the ¢rst report which demonstrates the entry and intra-
cellular survival of an unencapsulated isogenic mutant of GBS capsular type III in murine macrophages under non-opsonic conditions. It was postulated that complement has no major e¡ects on invasion and survival rates of well encapsulated GBS [14]. In addition, Rubens et al. [12] showed that unencapsulated mutants will not survive in the presence of complement, which seems to indicate that the GBS capsule plays a role in the resistance to opsonointracellular killing, but it is not an antiphagocytic factor. The few bacteria of the S. suis encapsulated parental strain recovered after 30 min of phagocytosis did not survive inside macrophages. In fact, after 60 min of reincubation time, no residual bacteria could be recovered (not shown). The survival of the unencapsulated mutant 2A was markedly reduced by more than 95% after 4 h of cell infection. Charland et al. [15] demonstrated, using murine and pig models, that mutant 2A was more susceptible to early phagocyte clearance compared with the wild-type strain, providing further evidence that the capsule is an antiphagocytic factor and plays an important role in virulence. Finally, we con¢rmed that even if continuous murine cell lines with macrophage-like properties provide a convenient model for in vitro studies of macrophage functions, some di¡erences may be observed with di¡erent lines. Results obtained in this study indicate that the J774A1 cell line is suitable for GBS- and S. suis-macrophage interaction studies, since it presents similar features to those of normal peritoneal macrophages [31]. In summary, the results of the present work suggest that interactions between GBS type III and S. suis type 2 with murine macrophages as well as the role of the capsule as an antiphagocytic factor are di¡erent for the two bacterial pathogens.
Acknowledgments We thank Julie M. Trudel for excellent technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Grant 0680154280, and by Fonds pour la Formation des Chercheurs et l'Aide aé la Recherche du Queèbec (FCAR) Grant NC-1037. M.S. is a grad-
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
uate scholarship holder from the Agence Canadienne de Deèveloppement International (ACDI). References [1] Higgins, R. and Gottschalk, M. (1998) Streptococcal diseases. In : Diseases of Swine, Chapter 38. Iowa State University Press, Ames, IA. [2] Devriese, L.A., Sustronck, B., Maenhout, T. and Haesebrouck, F. (1990) Streptococcus suis meningitis in a horse. Vet. Rec. 127, 68. [3] Trottier, S., Higgins, R., Brochu, G. and Gottschalk, M. (1991) A case of human endocarditis due to Streptococcus suis in North America [letter]. Rev. Infect. Dis. 13, 1251^1252. [4] Higgins, R. and Gottschalk, M. (1997) Distribution of Streptococcus suis capsular types in 1996. Can. Vet. J. 38, 302. [5] Baker, C.J. and Edwards, M.S. (1990) Group B streptococcal infections. In: Infectious Diseases of the Fetus and the Newborn (Klein, J.S. and Klein, J.O., Eds.), pp. 742^811. W.B. Saunders, Philadelphia, PA. [6] Kasper, D.L. (1986) Bacterial capsule ^ old dogmas and new tricks. J. Infect. Dis. 153, 407^415. [7] Edwards, M.S., Kasper, D.L., Jennings, H.J., Baker, C.J. and Nicholson-Weller, A. (1982) Capsular sialic acid prevents activation of the alternative complement pathway by type III, group B streptococci. J. Immunol. 128, 1278^1283. [8] Klegerman, M.E., Boyer, K.M., Papierniak, C.K., Levine, L. and Goto¡, S.P. (1984) Type-speci¢c capsular antigen is associated with virulence in late-onset group B streptococcal type III disease. Infect. Immun. 44, 124^129. [9] Wessels, M.R., Rubens, C.E., Benedi, V.J. and Kasper, D.L. (1989) De¢nition of a bacterial virulence factor : sialylation of the group B streptococcal capsule. Proc. Natl. Acad. Sci. USA 86, 8983^8987. [10] Shigeoka, A.O., Rote, N.S., Santos, J.I. and Hill, H.R. (1983) Assessment of the virulence factors of group B streptococci : correlation with sialic acid content. J. Infect. Dis. 147, 857^ 863. [11] Wessels, M.R., Haft, R.F., Heggen, L.M. and Rubens, C.E. (1992) Identi¢cation of a genetic locus essential for capsule sialylation in type III group B streptococci. Infect. Immun. 60, 392^400. [12] Rubens, C.E., Heggen, L.M., Haft, R.F. and Wessels, M.R. (1993) Identi¢cation of cpsD, a gene essential for type III capsule expression in group B streptococci. Mol. Microbiol. 8, 343^855. [13] Antal, J.M., Cunningham, J.V. and Goodrum, K.J. (1992) Opsonin-independent phagocytosis of group B streptococci : role of complement receptor type three. Infect. Immun. 60, 1114^1121. [14] Valentin-Weigand, P., Benkel, P., Rohde, M. and Chhatwal, G.S. (1996) Entry and intracellular survival of group B streptococci in J774 macrophages. Infect. Immun. 64, 2467^2473. [15] Charland, N., Harel, J., Kobish, M., Lacasse, S. and Gottschalk, M. (1998) Streptococcus suis serotype 2 mutants de¢cient in capsular expression. Microbiology 144, 325^332.
195
[16] Gottschalk, M., Higgins, R., Jacques, M. and Dubreuil, D. (1992) Production and characterization of two Streptococcus suis capsular type 2 mutants. Vet. Microbiol. 30, 59^71. [17] Jacobs, A.A., van den Berg, A.J., Baars, J.C., Nielsen, B. and Johannsen, L.W. (1995) Production of suilysin, the thiol-activated haemolysin of Streptococcus suis, by ¢eld isolates from diseased pigs. Vet. Rec. 137, 295^296. [18] Gottschalk, M.G., Lacouture, S. and Dubreuil, J.D. (1995) Characterization of Streptococcus suis capsular type 2 haemolysin. Microbiology 141, 189^195. [19] Tikkanen, K., Haataja, S. and Finne, J. (1996) The galactosyl(alpha 1^4)-galactose-binding adhesin of Streptococcus suis: occurrence in strains of di¡erent hemagglutination activities and induction of opsonic antibodies. Infect. Immun. 64, 3659^3665. [20] Vecht, U., Wisselink, H.J., Jellema, M.L. and Smith, H.E. (1991) Identi¢cation of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59, 3156^3162. [21] Charland, N., Kobisch, M., Martineau-Doize, B., Jacques, M. and Gottschalk, M. (1996) Role of capsular sialic acid in virulence and resistance to phagocytosis of Streptococcus suis capsular type 2. FEMS Immunol. Med. Microbiol. 14, 195^203. [22] Alexander, T. (1995) Streptococcus suis: pathogenesis and host response. In: Allen D. Leman Swine Conference (Scruton, W.C. and Cronje, R., Eds.), Vol. 22, pp. 49^53. University of Minnesota, Minneapolis, MN. [23] Brazeau, C., Gottschalk, M., Vincelette, S. and Martineaudoize, B. (1996) In vitro phagocytosis and survival of Streptococcus suis capsular type 2 inside murine macrophages. Microbiology 142, 1231^1237. [24] Shimoji, Y., Yokomizo, Y. and Mori, Y. (1996) Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages. Infect. Immun. 64, 1789^1793. [25] Tunkel, A.R. and Scheld, W.M. (1993) Pathogenesis and pathophysiology of bacterial meningitis. Clin. Microbiol. Rev. 6, 118^136. [26] Marques, M.B., Kasper, D.L., Pangburn, M.K. and Wessels, M.R. (1992) Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Infect. Immun. 60, 3986^3993. [27] Agarwal, K.K., Elliott, S.D. and Lachmann, P.J. (1969) Streptococcal infection in young pigs. III. The immunity of adult pigs investigated by the bactericidal test. J. Hyg. 67, 491^503. [28] Clifton-Hadley, F.A. (1981) Studies of Streptococcus suis Type 2 Infection in Pigs. Ph.D. Dissertation, Dept. Clin. Vet. Med., University of Cambridge, Cambridge. [29] Williams, A.E. (1990) Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates. Microb. Pathog. 8, 189^196. [30] Salasia, S.I.O., Lammler, C. and Herrmann, G. (1995) Properties of a Streptococcus suis isolate of serotype 2 and two capsular mutants. Vet. Microbiol. 45, 151^156. [31] Snyderman, R., Pike, M., Fischer, D. and Koren, H. (1977) Biologic and biochemical activities of continuous macrophage cell lines P388D1 and J774.1. J. Immunol. 119, 2060^2066.
FEMSIM 886 30-7-98
Streptococcus suis and group B Streptococcus di¡er in their interactions with murine macrophages Mariela A. Segura, Patrick Cleèroux, Marcelo Gottschalk * Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculteè de Meèdecine Veèteèrinaire, Universiteè de Montreèal, P.O. Box 5000, St-Hyacinthe, Queè. J2S 7C6, Canada Received 9 January 1998; revised 27 April 1998 ; accepted 7 May 1998
Abstract Streptococcus suis type 2 and group B Streptococcus type III (GBS) are important encapsulated bacterial species causing meningitis. In the present study we compared quantitatively the uptake and intracellular survival of S. suis type 2 and GBS type III with murine macrophages in non-opsonic conditions. The role of the capsule of both pathogens was also studied using previously obtained unencapsulated isogenic mutants. Encapsulated S. suis wild-type strain was practically not phagocytosed, while the unencapsulated mutant was easily ingested by macrophages. On the other hand, the well encapsulated GBS strain and its unencapsulated mutant were both phagocytosed in large numbers. Even if S. suis unencapsulated mutant showed a higher uptake rate than the parental strain, this value was always markedly lower than the numbers of ingested GBS strains. In addition, the intracellular survival of encapsulated and unencapsulated GBS strains was significantly higher than that of S. suis strains. These results suggest that interactions between GBS type III and S. suis type 2 with murine macrophages as well as the role of the capsule as an antiphagocytic factor are different for the two bacterial pathogens. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords : Streptococcus suis; Group B Streptococcus; Non-opsonic phagocytosis; Intracellular survival; Polysaccharidic capsule ; Macrophage
1. Introduction Streptococcus suis is an important pathogen which has been associated with a wide variety of infections in swine such as meningitis, septicemia, and pneumonia [1]. It has also been isolated from other animal species, as well as from humans [2,3]. To date, 35 di¡erent capsular types of S. suis have been described. S. suis capsular type 2 is considered to be * Corresponding author. Tel.: +1 (450) 773-8521 ext. 8374; Fax: +1 (450) 778-8108; E-mail: [email protected]
the most virulent as well as the most prevalent capsular type in diseased pigs [4]. Group B Streptococcus (GBS) is an important cause of pneumonia, sepsis and meningitis in neonates. GBS associated with human disease are almost invariably encapsulated, belonging to one of the nine recognized capsule serotypes: Ia, Ib, II^ VIII. Type III GBS strains account for about twothirds of the GBS isolates associated with invasive neonatal disease [5]. S. suis and GBS present several similarities. Both streptococci are well encapsulated, some capsular
0928-8244 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 4 8 - 0
FEMSIM 886 30-7-98
190
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
types are more virulent than others, they possess sialic acid in their capsule and they can cause severe meningitis. However, it is not clear whether the pathogenesis of the infection caused by the two bacterial species is similar. The capsules of a variety of bacterial species are thought to play an important role in virulence. Encapsulated organisms interact with host defense systems by several known mechanisms [6]. Among them, the sialylated polysaccharidic capsule of GBS plays a well recognized role in the resistance to phagocytosis, by modulating the alternative complement pathway, and protecting the organism from opsonophagocytic killing [7^10]. In vitro and in vivo virulence assays with unencapsulated or asialo mutants support the hypothesis that type III capsule serves as a virulence factor [11,12]. Other studies have shown that well encapsulated GBS are able to enter and persist in macrophages by evading intracellular opsonin-mediated antibacterial activities [13,14]. The role of the polysaccharidic capsule in the opsoninindependent uptake is so far unknown. S. suis is also encapsulated. In a recent work, transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain isogenic acapsular mutants from a virulent S. suis type 2 strain. These mutants were shown to be avirulent for both mice and piglets and cleared from the circulation rapidly [15]. In fact, the capsule seems to be the ¢rst critical virulence factor described so far for this bacterial species. However, avirulent ¢eld strains are also encapsulated. Other putative virulence factors have been suggested, such as cell-wall and extracellular proteins, toxins, adhesins and immunoglobulinbinding proteins [16^20], but their role in the pathogenesis of the infection is unknown. As mentioned above, S. suis also possesses sialic acid in its capsule. However, and unlike GBS, this sugar does not seem to be a critical component for the virulence [21]. It may be possible that interactions between S. suis and GBS with macrophages do di¡er. In fact, previous studies of interactions between encapsulated strains of S. suis type 2 and phagocytic cells are contradictory [22]. In the present study we compare the uptake and intracellular survival of S. suis type 2 and GBS type III with murine macrophages in non-opsonic conditions. The role of the capsule of both pathogens is
also studied using previously obtained acapsular isogenic mutants.
2. Materials and methods 2.1. Bacterial strains and growth conditions Two strains of S. suis capsular type 2 and two strains of GBS capsular type III were used: the virulent-encapsulated S. suis wild-type strain S735-SM and its avirulent-unencapsulated isogenic transposon mutant 2A [15], as well as the highly encapsulated wild-type strain of type III GBS, COH1, and its less virulent and unencapsulated isogenic transposon mutant, COH1-13 [12]. The latter strains were kindly provided by Dr. C.E. Rubens (Children's Hospital and Medical Center, Seattle, WA, USA). Bacteria were maintained as stock cultures in 50% glycerol^Todd-Hewitt broth (THB; Difco Lab., Detroit, MI, USA) at 380³C. The THB was supplemented with 10 Wg ml31 tetracycline (Tc; Sigma-Aldrich, Oakville, Canada) for growing the mutants [15]. Bacteria were grown overnight onto bovine blood agar plates at 37³C and single colonies were used as inocula for THB or THB-Tc, which were incubated for 18 h at 37³C. Working cultures were made by inoculating 0.3 ml of these cultures in 10 ml of THB at 37³C with agitation until it reached the mid-exponential phase (6 h incubation time; OD540 0.4^0.5 for S. suis strains, and 0.7^0.8 for GBS strains). Bacteria were washed twice in phosphatebu¡ered saline (PBS), pH 7.4, and diluted to approximately 107 CFU ml31 in cell culture media (without antibiotics). An accurate determination of the number of colony forming units (CFU) in the ¢nal suspension was made for each assay by plating on THB agar. 2.2. Cell lines and cell culture J774A1 murine (BALB/c) macrophage-like cell line (ATCC TIB 67, Rockville, MD) was maintained in Dulbecco's modi¢ed Eagle's medium, 1.5 g ml31 bicarbonate (DMEM). P388D1 murine (DBA/2) macrophage-like cell line (ATCC TIB 63) was maintained in Iscove's modi¢ed Dulbecco's medium (IMDM). Cell media were supplemented with 10%
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
heat-inactivated fetal bovine serum (iFBS), penicillin G (100 IU ml31 ), and streptomycin (100 Wg ml31 ) (Gibco, Burlington, Ont., Canada) and cells were grown at 37³C, 5% CO2 . For bacterial phagocytosis and survival assays, 48h culture cells were scraped up, washed once and resuspended in antibiotic-free medium at approximately 2U105 cells ml31 . 1 ml of this suspension was distributed into 24-well tissue culture plates Falcon (VWR CanLab, Quebec, Canada) and incubated for 3 h at 37³C, 5% CO2 to allow cell adhesion before assays. 2.3. Preparation of murine peritoneal macrophages Peritoneal exudate macrophages (PEM) were isolated as previously described [23]. Brie£y, ¢ve or six 6-week-old BALB/c male mice (Charles River, StConstant, Canada) were used per individual assay. Mice were inoculated intraperitoneally with 1.5 ml 3% (w/v) thioglycolate sterile broth (Sigma). PEM were recovered 4 days later by washing the peritoneal cavity with 5 ml Hanks' balanced salt solution Ca2 -Mg2 -free (HBSS, Gibco). Cells were pooled, pelleted by centrifugation for 10 min at 1000Ug, and resuspended at a ¢nal concentration of 2U105 cells ml31 in minimum essential medium (MEM, Gibco) supplemented with 10% iFBS (MEMs). 1 ml of this suspension was distributed into 24-well tissue culture plates (Falcon, VWR CanLab). Cells were allowed to adhere for 1 h at 37³C, 5% CO2 , washed with warm HBSS, and reincubated overnight in MEMs before assays. Cell purity was more than 95% as determined by non-speci¢c esterase stain and modi¢ed Wright-Giemsa stain (LeukoStat, Fisher Sci., Montreal, Canada). Cell viability was more than 99% as determined by trypan blue exclusion. 2.4. Phagocytosis assay Phagocytosis assays were performed as already described [14] with some modi¢cations. J774 cell, P388 cell and PEM plates were infected with streptococci by removing culture medium and adding 1 ml of 107 bacterial suspension per well (in respective cell culture media, supplemented with 10% iFBS), to obtain a ratio of about 100 bacteria per macrophage. Phag-
191
ocytosis was left to proceed for 30 min and 90 min at 37³C, 5% CO2 . After incubation, cell monolayers were washed twice with warm HBSS, and reincubated for 1 h with medium containing penicillin G (5 Wg ml31 ) and gentamicin (100 Wg ml31 ; Sigma) to kill extracellular bacteria. It has been demonstrated that these antibiotics do not penetrate eukaryotic cells under these conditions [14], and previous studies have shown that this concentration of antibiotics was able to kill any remaining extracellular bacteria (data not shown). In addition, supernatant controls were taken in every test to con¢rm the activity of antibiotics. After antibiotic treatment, cell monolayers were washed three times and the medium was replaced with 1 ml of sterile distilled water to lyse the macrophages. After vigorous pipetting to ensure complete cell lysis, viable intracellular streptococci were determined by quantitative plating of serial dilutions of the lysates on THB agar. Each test was done four times in independent experiments, and the number of CFU recovered per well (mean number þ S.D.) was determined. 2.5. Intracellular survival assay Survival experiments were done as described in Section 2.4, except that after 30 min of infection with streptococci, cell monolayers were washed twice with warm HBSS and antibiotic-containing medium was added and left on infected cells up to 4 h post infection. At various intervals (60, 90, 120, 180, and 240 min), infected monolayers were washed three times and the medium was replaced with 1 ml of sterile distilled water to lyse the macrophages. After vigorous pipetting to ensure complete cell lysis, viable intracellular streptococci were determined by quantitative plating of serial dilutions of the lysates on THB agar. Each test was done four times and intracellular killing was expressed as the percentage decrease in the initial number (100%) of viable intracellular bacteria at 60 min interval time [24]. 2.6. Statistics Di¡erences were analyzed for signi¢cance by using Student's unpaired t-test (two-tailed P-value).
FEMSIM 886 30-7-98
192
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
3. Results 3.1. Phagocytosis of S. suis and GBS We compared quantitatively the uptake process of S. suis and GBS wild-type strains and the two unencapsulated avirulent mutants with two di¡erent murine phagocytic cell lines (J774A1 and P388D1) and with murine PEM, at 30 min and 90 min infection times. At 30 min of phagocytosis (Fig. 1a), encapsulated S735-SM strain was practically not phagocytosed, and there was no signi¢cant di¡erence between the cell lines and PEM (P s 0.05). In contrast, encapsulated GBS strain COH1 was highly phagocytosed. Phagocytic values were more elevated with J774A1 cells, followed by PEM, while P388D1 cells showed the lowest uptake rate (P 6 0.05).
Fig. 2. Time course of intracellular survival of S. suis unencapsulated mutant 2A and GBS strains (COH1 and unencapsulated mutant COH1-13) within (a) P388D1 cells, (b) J774A1 cells, and (c) peritoneal exudate macrophages (PEM). Macrophages were allowed to ingest bacteria for 30 min, then antibiotics were added to kill extracellular bacteria and the numbers of viable intracellular bacteria were determined by quantitative plating at several post-infection times. Killing was expressed as the percentage decrease in the initial number (100%) of viable intracellular bacteria at 60 min post-infection time. These ¢gures are representative of the results of four independent experiments. Fig. 1. Phagocytosis of S. suis strains (S735-SM and the unencapsulated mutant 2A) and GBS strains (COH1 and the unencapsulated mutant COH1-13) after (a) 30 min infection time and (b) 90 min infection time. Numbers of phagocytic bacteria were determined by quantitative plating after 1 h of antibiotic treatment, and results are expressed as CFU recovered bacteria per well (means þ S.D. obtained from four independent experiments).
S. suis unencapsulated mutant 2A was highly phagocytosed compared to wild-type strain. Phagocytic values were more elevated with J774A1 cells than those obtained with PEM or P388D1 cells (P 6 0.05). However, GBS unencapsulated mutant
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
COH1-13 was slightly more phagocytosed than the wild-type COH1 strain by each of the three cell types. However, this di¡erence was markedly lower than that observed between S. suis unencapsulated mutant and its parental strain. After 90 min of phagocytosis (Fig. 1b), the uptake of encapsulated S735-SM strain was not increased with any of the three types of phagocytic cells; and values were nearly zero. In contrast, the GBS COH1 strain uptake rate was increased almost two log with PEM and nearly one log with both cell lines. P388D1 cells showed a lower uptake degree than J774A1 cells and PEM (P 6 0.05), while no signi¢cant di¡erence was noted between the latter cell types (P s 0.05). Unlike S. suis parental strain, the ingested numbers of the unencapsulated mutant 2A at 90 min were signi¢cant increased compared to phagocytic values at 30 min infection time. The increase in phagocytosis was higher with J774A1 cells than with P388D1 cells or PEM (P 6 0.05). In contrast, there were no signi¢cant di¡erences in increasing phagocytosis between the GBS unencapsulated mutant COH1-13 and the parental strain. Mutant COH1-13 strain was phagocytosed at the same rate as the parental strain by J774A1 cells and PEM (P s 0.05), but it was slightly more phagocytosed by P388D1 cells (P 6 0.05). Even if S. suis unencapsulated mutant showed a higher uptake rate than the parental strain, this value was always markedly lower than the numbers of ingested GBS strains (P 6 0.05). In general, for all four strains, the uptake rate with J774A1 cells and PEM was higher than that shown with P388D1 cells. 3.2. Intracellular survival of S. suis mutant 2A compared to GBS strains To study the intracellular survival of S. suis and GBS strains, we infected cells for 30 min and then samples were taken at di¡erent time points after the addition of antibiotic-containing medium. The few bacteria of S. suis wild-type S735-SM strain which were phagocytosed were not able to survive inside macrophages (data not shown). Mutant 2A showed a signi¢cant linear decrease (P 6 0.05) in the number of viable intracellular bacteria with all cell types. The time course of intracellular killing of mutant 2A with P388D1 cells was slower compared to J774A1 cells
193
and PEM, decreasing by 90 min to 72% of that observed at 60 min (100%), and to nearly 6% after 240 min post-infection time (Fig. 2a). In the case of J774A1 cells and PEM (Fig. 2b,c), the number of viable intracellular bacteria decreased rapidly to about 20% at 90 min; by 180 min, less than 4% survival was detected, and viable intracellular bacteria were almost not detected after 240 min post-infection time. The intracellular survival of GBS strains was signi¢cantly higher than that of S. suis mutant 2A. Results showed (Fig. 2a^c) that there were no signi¢cant di¡erences between the wild-type GBS strain COH1 and the unencapsulated mutant COH1-13 (P s 0.05) with any of the three cell types. Up to 120 min post infection, intracellular numbers of viable GBS remained almost constant. Between 180 and 240 min post infection, a quite signi¢cant decrease in the number of intracellular GBS was observed with P388D1 cells and PEM (P 6 0.05), but remained constant with J774A1 cells.
4. Discussion Surface encapsulation is one of the most important virulence factors of several pathogenic microorganisms. The bacterial capsule, by e¡ectively inhibiting phagocytosis and resisting complement-mediated bactericidal activity, may enhance bloodstream survival of the organism and facilitate intravascular replication. Indeed, the most common meningeal pathogens are all encapsulated [25]. However, the mechanisms of interaction of encapsulated bacteria with the host immune system are not at all known, and they are not generalized for all pathogens. In the present study we compared quantitatively the in vitro interactions, uptake and intracellular survival, of S. suis capsular type 2 and GBS capsular type III with murine phagocytic cells under non-opsonic conditions. The role of their capsules in these interactions was evaluated by using unencapsulated isogenic mutants of both pathogens. Viable counting techniques for monitoring phagocytic interactions have already been used for GBS [14]. The present work is the ¢rst determination of phagocytosis and time course of intracellular survival of S. suis by quantitative plating.
FEMSIM 886 30-7-98
194
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
The results of this study show that well encapsulated GBS can be phagocytosed in large numbers by murine macrophages in the absence of complement and antibodies. Our results are similar to those described by Valentin-Weigand et al. [14], who also showed that GBS COH1 strain is able to enter and persist e¤ciently in J774A1 macrophages by evading intracellular antibacterial activities. On the other hand, no di¡erences were observed between this strain and its unencapsulated mutant, which indicates that the capsule is not necessarily an antiphagocytic factor for this bacterial species. Controversially, previous reports have assumed an antiphagocytic role of GBS type III capsule since encapsulated bacteria were resistant to opsonophagocytic killing by PMN in the absence of speci¢c antibodies, while unencapsulated or asialo mutants were susceptible [11,12,26]. However, in these studies, the techniques used allowed the study of bacterial killing rather than phagocytosis, since no di¡erentiation of intracellular and extracellular bacteria was done. Di¡erent results were obtained with S. suis. The well encapsulated parental strain of S. suis type 2 was almost not phagocytosed by murine macrophages, even after 90 min of bacteria-cell contact. Interestingly, previous in vitro studies of this bacterial species with phagocytes are contradictory. Early bacterial killing studies could not demonstrate any `phagocytic activity' with S. suis type 2 [27,28], whereas more recent studies showed variable percentages of phagocytosis (7^30%), using a vital staining technique [21,23,29]. These discrepancies may be due to technical di¡erences. In fact, the results of the present study represent the ¢rst data from quantitative phagocytosis using a viable counting technique. Unlike the GBS, the high percentage of phagocytosis obtained with the unencapsulated mutant of S. suis demonstrates the antiphagocytic role of the capsule, which con¢rms previous observations [15,23,30]. Similar results were obtained when porcine monocytes were used (data not shown). The role of the capsule in intracellular bacterial survival was also studied. Both encapsulated and unencapsulated GBS strains were able not only to enter, but also to survive e¤ciently inside macrophages. To the best of our knowledge, this is the ¢rst report which demonstrates the entry and intra-
cellular survival of an unencapsulated isogenic mutant of GBS capsular type III in murine macrophages under non-opsonic conditions. It was postulated that complement has no major e¡ects on invasion and survival rates of well encapsulated GBS [14]. In addition, Rubens et al. [12] showed that unencapsulated mutants will not survive in the presence of complement, which seems to indicate that the GBS capsule plays a role in the resistance to opsonointracellular killing, but it is not an antiphagocytic factor. The few bacteria of the S. suis encapsulated parental strain recovered after 30 min of phagocytosis did not survive inside macrophages. In fact, after 60 min of reincubation time, no residual bacteria could be recovered (not shown). The survival of the unencapsulated mutant 2A was markedly reduced by more than 95% after 4 h of cell infection. Charland et al. [15] demonstrated, using murine and pig models, that mutant 2A was more susceptible to early phagocyte clearance compared with the wild-type strain, providing further evidence that the capsule is an antiphagocytic factor and plays an important role in virulence. Finally, we con¢rmed that even if continuous murine cell lines with macrophage-like properties provide a convenient model for in vitro studies of macrophage functions, some di¡erences may be observed with di¡erent lines. Results obtained in this study indicate that the J774A1 cell line is suitable for GBS- and S. suis-macrophage interaction studies, since it presents similar features to those of normal peritoneal macrophages [31]. In summary, the results of the present work suggest that interactions between GBS type III and S. suis type 2 with murine macrophages as well as the role of the capsule as an antiphagocytic factor are di¡erent for the two bacterial pathogens.
Acknowledgments We thank Julie M. Trudel for excellent technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Grant 0680154280, and by Fonds pour la Formation des Chercheurs et l'Aide aé la Recherche du Queèbec (FCAR) Grant NC-1037. M.S. is a grad-
FEMSIM 886 30-7-98
M.A. Segura et al. / FEMS Immunology and Medical Microbiology 21 (1998) 189^195
uate scholarship holder from the Agence Canadienne de Deèveloppement International (ACDI). References [1] Higgins, R. and Gottschalk, M. (1998) Streptococcal diseases. In : Diseases of Swine, Chapter 38. Iowa State University Press, Ames, IA. [2] Devriese, L.A., Sustronck, B., Maenhout, T. and Haesebrouck, F. (1990) Streptococcus suis meningitis in a horse. Vet. Rec. 127, 68. [3] Trottier, S., Higgins, R., Brochu, G. and Gottschalk, M. (1991) A case of human endocarditis due to Streptococcus suis in North America [letter]. Rev. Infect. Dis. 13, 1251^1252. [4] Higgins, R. and Gottschalk, M. (1997) Distribution of Streptococcus suis capsular types in 1996. Can. Vet. J. 38, 302. [5] Baker, C.J. and Edwards, M.S. (1990) Group B streptococcal infections. In: Infectious Diseases of the Fetus and the Newborn (Klein, J.S. and Klein, J.O., Eds.), pp. 742^811. W.B. Saunders, Philadelphia, PA. [6] Kasper, D.L. (1986) Bacterial capsule ^ old dogmas and new tricks. J. Infect. Dis. 153, 407^415. [7] Edwards, M.S., Kasper, D.L., Jennings, H.J., Baker, C.J. and Nicholson-Weller, A. (1982) Capsular sialic acid prevents activation of the alternative complement pathway by type III, group B streptococci. J. Immunol. 128, 1278^1283. [8] Klegerman, M.E., Boyer, K.M., Papierniak, C.K., Levine, L. and Goto¡, S.P. (1984) Type-speci¢c capsular antigen is associated with virulence in late-onset group B streptococcal type III disease. Infect. Immun. 44, 124^129. [9] Wessels, M.R., Rubens, C.E., Benedi, V.J. and Kasper, D.L. (1989) De¢nition of a bacterial virulence factor : sialylation of the group B streptococcal capsule. Proc. Natl. Acad. Sci. USA 86, 8983^8987. [10] Shigeoka, A.O., Rote, N.S., Santos, J.I. and Hill, H.R. (1983) Assessment of the virulence factors of group B streptococci : correlation with sialic acid content. J. Infect. Dis. 147, 857^ 863. [11] Wessels, M.R., Haft, R.F., Heggen, L.M. and Rubens, C.E. (1992) Identi¢cation of a genetic locus essential for capsule sialylation in type III group B streptococci. Infect. Immun. 60, 392^400. [12] Rubens, C.E., Heggen, L.M., Haft, R.F. and Wessels, M.R. (1993) Identi¢cation of cpsD, a gene essential for type III capsule expression in group B streptococci. Mol. Microbiol. 8, 343^855. [13] Antal, J.M., Cunningham, J.V. and Goodrum, K.J. (1992) Opsonin-independent phagocytosis of group B streptococci : role of complement receptor type three. Infect. Immun. 60, 1114^1121. [14] Valentin-Weigand, P., Benkel, P., Rohde, M. and Chhatwal, G.S. (1996) Entry and intracellular survival of group B streptococci in J774 macrophages. Infect. Immun. 64, 2467^2473. [15] Charland, N., Harel, J., Kobish, M., Lacasse, S. and Gottschalk, M. (1998) Streptococcus suis serotype 2 mutants de¢cient in capsular expression. Microbiology 144, 325^332.
195
[16] Gottschalk, M., Higgins, R., Jacques, M. and Dubreuil, D. (1992) Production and characterization of two Streptococcus suis capsular type 2 mutants. Vet. Microbiol. 30, 59^71. [17] Jacobs, A.A., van den Berg, A.J., Baars, J.C., Nielsen, B. and Johannsen, L.W. (1995) Production of suilysin, the thiol-activated haemolysin of Streptococcus suis, by ¢eld isolates from diseased pigs. Vet. Rec. 137, 295^296. [18] Gottschalk, M.G., Lacouture, S. and Dubreuil, J.D. (1995) Characterization of Streptococcus suis capsular type 2 haemolysin. Microbiology 141, 189^195. [19] Tikkanen, K., Haataja, S. and Finne, J. (1996) The galactosyl(alpha 1^4)-galactose-binding adhesin of Streptococcus suis: occurrence in strains of di¡erent hemagglutination activities and induction of opsonic antibodies. Infect. Immun. 64, 3659^3665. [20] Vecht, U., Wisselink, H.J., Jellema, M.L. and Smith, H.E. (1991) Identi¢cation of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59, 3156^3162. [21] Charland, N., Kobisch, M., Martineau-Doize, B., Jacques, M. and Gottschalk, M. (1996) Role of capsular sialic acid in virulence and resistance to phagocytosis of Streptococcus suis capsular type 2. FEMS Immunol. Med. Microbiol. 14, 195^203. [22] Alexander, T. (1995) Streptococcus suis: pathogenesis and host response. In: Allen D. Leman Swine Conference (Scruton, W.C. and Cronje, R., Eds.), Vol. 22, pp. 49^53. University of Minnesota, Minneapolis, MN. [23] Brazeau, C., Gottschalk, M., Vincelette, S. and Martineaudoize, B. (1996) In vitro phagocytosis and survival of Streptococcus suis capsular type 2 inside murine macrophages. Microbiology 142, 1231^1237. [24] Shimoji, Y., Yokomizo, Y. and Mori, Y. (1996) Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages. Infect. Immun. 64, 1789^1793. [25] Tunkel, A.R. and Scheld, W.M. (1993) Pathogenesis and pathophysiology of bacterial meningitis. Clin. Microbiol. Rev. 6, 118^136. [26] Marques, M.B., Kasper, D.L., Pangburn, M.K. and Wessels, M.R. (1992) Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Infect. Immun. 60, 3986^3993. [27] Agarwal, K.K., Elliott, S.D. and Lachmann, P.J. (1969) Streptococcal infection in young pigs. III. The immunity of adult pigs investigated by the bactericidal test. J. Hyg. 67, 491^503. [28] Clifton-Hadley, F.A. (1981) Studies of Streptococcus suis Type 2 Infection in Pigs. Ph.D. Dissertation, Dept. Clin. Vet. Med., University of Cambridge, Cambridge. [29] Williams, A.E. (1990) Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates. Microb. Pathog. 8, 189^196. [30] Salasia, S.I.O., Lammler, C. and Herrmann, G. (1995) Properties of a Streptococcus suis isolate of serotype 2 and two capsular mutants. Vet. Microbiol. 45, 151^156. [31] Snyderman, R., Pike, M., Fischer, D. and Koren, H. (1977) Biologic and biochemical activities of continuous macrophage cell lines P388D1 and J774.1. J. Immunol. 119, 2060^2066.
FEMSIM 886 30-7-98