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Department of Infectious Diseases, Research Institute, International Medical Center of Japan
Department of Microbiology and Immunology, Tokyo Women's Medical University, Tokyo
Molecular Bacteriology Section, National Institute of Animal Health, Ibaraki, Japan
We analyzed the in vivo dynamics of peritoneal exudate cells (PECs) in mice injected with group A streptococcus (GAS). A live low-virulence strain, as well as heat-killed low- and high-virulence strains, significantly increased the number of PECs (primarily neutrophils), whereas a live high-virulence strain did not. When coinjected with thioglycollate, the live high-virulence strain, as well as most other GAS strains, suppressed the ability of thioglycollate to induce neutrophil exudation. This suppression was due to a cytocidal effect of GAS on exuded neutrophils rather than an inhibition of neutrophil migration. In addition, GAS enhanced the apoptosis of neutrophils. These cytocidal effects were significantly reduced by the deletion of functional streptolysin S from GAS. Our findings suggest that, in addition to the production of antiphagocytic factors and survival inside phagocytes, GAS uses a more aggressive methodthe elimination of neutrophilsto evade the host's innate immune system.
Neutrophils are a critical participant in the innate immune system. To escape destruction by neutrophils, bacterial pathogens have developed several mechanisms. Among these pathogens is group A streptococcus (GAS), which is responsible for a wide spectrum of human diseases, including streptococcal toxic shock syndrome (STSS). Many molecules of GAS are thought to be involved in helping the bacterium evade phagocytic ingestion by neutrophils [13]. In addition, GAS has been found to survive intact in professional phagocytic cells, which enables the bacterium to evade detection by the host defense system [4, 5]. GAS produces several cytotoxins, including streptolysin S (SLS), which is encoded by the sagA gene and has lethal effects on experimental animals [68]. In addition to an exotoxin type of SLS, a cell-bound type of SLS has shown strong cytotoxic effects on various host cells, including neutrophils [9]. Despite these results, which show that GAS has several strategies by which it can evade the action of the immune system, it is unclear which of these strategies is most critical for evasion.
Although GAS is not pathogenic in mice, several studies have used mice as a model to analyze the pathogenic mechanism of GAS infection. We used a mouse model to compare the virulence of different M types of GAS and found that GAS virulence in mice reflected, at least in part, the pathologic progression of STSS in humans [10]. We therefore used this mouse model of the host innate immune system to analyze GAS pathogenesis in humans, by injecting mice with GAS and investigating the dynamics of the induction of peritoneal exudate cells (PECs).
MATERIALS AND METHODS
Bacterial strains.
The GAS strains used are listed in table 1 and were handled as described elsewhere [11]. Kanamycin was used at a concentration of 500 g/mL for GAS and of 50 g/mL for Escherichia coli, spectinomycin was used at a concentration of 50 g/mL for both GAS and E. coli, and ampicillin was used at a concentration of 100 g/mL for E. coli. Heat-killed GAS strains were obtained by heating the bacteria at 70°C for 30 min.
Injection of mice with GAS and isolation of PECs.
All mouse experiments were performed according to the guidelines of the Ethics Review Committee of Animal Experiments, Tokyo Women's Medical University. GAS suspensions in PBS (0.5 mL), at the indicated cell densities, were injected intraperitoneally into each of 3 female 67-week-old ddY mice. The number of colony-forming units of injected bacteria was determined by incubating each GAS sample on SRBC agar (Nissui) plates. To recover PECs, the peritoneal cavity of each mouse was rinsed with 3 or 5 mL of PBS, and the PEC density was determined by counting trypan bluestained cells on a hemocytometer; data are presented as the total number of PECs per mouse peritoneal cavity. The morphological characteristics, viability, and apoptosis of each PEC suspension were determined by Giemsa staining and by acridine orange and ethidium bromide staining [12], respectively. At least 200 cells were counted for each sample.
Flow cytometry.
The neutrophil content and apoptosis of PEC populations were analyzed by flow cytometry. Cells were incubated with phycoerythrin-conjugated antimouse neutrophil antibody (Caltag Laboratories) or with fluorescein isothiocyanate (FITC)conjugated Annexin V by use of the Annexin VFITC Apoptosis Detection Kit (BioVision). At least 5000 cells/sample were analyzed by use of EPICS XL (Beckman Coulter).
Plasmid construction.
A thermosensitive suicide vector, pSET4s [13], and pLZ12-Km2 [14] were used to destroy and restore, respectively, functional SLS. The oligonucleotide primers 5-ccacggatccagggtttacatattaatcatt-3 (primer 1), 5-gtgggtcgactataacttccgctaccacctt-3 (primer 2), and 5-gtgggtcgactaccacatagagatgctaaat-3 (primer 3) were designed on the basis of the sagA region sequence of an M3 GAS strain (GenBank accession number AE014146). Primers 1 and 2 were used to amplify a fragment encoding the N-terminal region of sagA, including the 5 noncoding region for SLS destruction, and primers 1 and 3 were used to amplify a fragment containing the entire sagA open-reading frame for the restoration of SLS. Each polymerase chain reaction product was ligated into a TOPO-TA vector and expanded by use of a TOPO-TA cloning kit (Invitrogen), and each was sequenced. The former and latter fragments were subcloned into pSET4s and pLZ12-Km2, respectively, which resulted in pSET4s-SLSdest and pLZ-SLS.
Derivation of SLS expression mutants.
One-milliliter aliquots of GAS precultures were diluted with 10 mL of prewarmed brain-heart infusion (BHI) broth (Becton Dickinson) and incubated for 2 h. Bacterial cells were harvested by centrifugation, washed 4 times with 10 mL of ice-chilled 10% glycerol, and suspended in 500 L of 10% glycerol. Aliquots (100 L) of the bacterial suspensions were mixed with plasmid DNA in ice-chilled 2-mm gap cuvettes, and the mixtures were electroporated at 2500 V by use of a TransPorator Plus (BTX).
Bacteria electroporated with pSET4s-SLSdest were cultured for 2 h at 28°C in 5 mL of BHI broth, harvested, spread onto Todd-Hewitt (Becton Dickinson) agar that contained 0.2% yeast extract (THY agar) and spectinomycin, and cultured overnight at 28°C. The resulting transformants were recovered from the plates with 1 mL of BHI and cultured in 5 mL of BHI that contained spectinomycin, for 2 h at 28°C. Twenty microliters of each culture was transferred to 2 mL of fresh BHI medium that contained spectinomycin and cultured for another 4 h at 37°C, which is a nonpermissive temperature for the duplication of pSET4s derivatives. The bacteria were harvested, spread on THY agar that contained spectinomycin, and cultured overnight at 37°C to select the transformants.
SLS-deficient bacteria electroporated with pLZ-SLS were cultured for 1 h at 37°C in BHI medium, harvested, and spread onto THY agar that contained kanamycin and spectinomycin to select the transformants. The disruption and reintroduction of sagA was confirmed by Southern-hybridization analysis of PstI-digested chromosomal DNA from each mutant. Although the sagA probe excised from pLZ-SLS hybridized to 1 band in parent strains, it hybridized to 2 bands in SLS-deficient mutants (because of the introduction of a PstI site in pSET4s) and to 3 bands in the SLS-complemented strain (data not shown).
Characterization of the GAS mutants.
A sufficient amount of preculture of each mutant was transferred to prewarmed BHI broth to yield an OD600 of 0.002, and the generation time was determined by monitoring the optical density at 600 nm. The activity of streptolysin O (SLO) and SLS was determined by an SRBC hemolysis assay, except that, in the latter, 2-mercaptoethanol was omitted from the reaction and the GAS mutants were cultured in BHI medium that contained 1% Tween 80 [10]. The hyaluronic acid contents of each culture were determined by use of Stains-all dye [10]. M protein and streptococcal pyrogenic exotoxin (SPE)B were analyzed by Western blotting that used antibodies against the M proteinconstant region [15] and SPE-B (Toxin Technology), respectively. LD50 values were determined as described elsewhere [10].
In vitro assessment of viability, apoptosis, and active caspase level of neutrophils.
Neutrophils were obtained by injecting 1012-week-old female C57BL/6 mice with thioglycollate 812 h before the collection of samples. The PEC response of C57BL/6 mice to GAS was similar to that of ddY mice (data not shown). The neutrophil populations were >95% pure, as determined by flow cytometry. Human neutrophils were prepared from human blood by use of Mono-Poly Resolving Medium (Dainippon Pharmaceutical). The human neutrophil preparations contained >95% neutrophils, according to the results of morphological analysis. Aliquots of 5 × 106 neutrophils in 2 mL of RPMI 1640 medium supplemented with 10% fetal calf serum were placed in each well of a 24-well plate and cultured with 1 × 107 cfu of one of the GAS strains. PEC viability and apoptosis were determined by the fluorescence dye method and by flow cytometry by use of Annexin V, respectively, as described above. Cell lysates prepared from 1 × 106 cells collected at the time points indicated were analyzed by Western blotting that used anticaspase 7 antibody (Stressgen Biotechnologies).
RESULTS
Effects of GAS injection on PEC dynamics in mice.
To determine the effects of injected GAS on PEC dynamics in mice, we used 2 M3 GAS strains: M3-1, which has low virulence, and M3-f, which has high virulence [10]. After the injection of live or heat-killed GAS, we determined the total number of PECs over time. Mice injected with live or heat-killed M3-1 or with heat-killed M3-f showed increases in the number of PECs, with the peak amount, at 12 h, being 4-fold higher than that observed in uninoculated mice (figure 1A). In contrast, mice injected with live M3-f showed no increase in the number of PECs and began to die 12 h after injection (figure 1B). Increased numbers of PECs were not observed in mice injected with PBS or zymosan A (data not shown). Morphological analyses showed that PECs from uninoculated mice were composed mainly of lymphocytes, whereas >90% of the PECs obtained from mice injected with live or with heat-killed M3-1 or heat-killed M3-f were neutrophils (data not shown). Flow-cytometric analysis showed that only 11% of PECs from uninoculated mice reacted with monoclonal antibody 7/4, an antibody that reacts with mouse neutrophils [1618], whereas >90% of PECs from mice injected with GAS preparations reacted with this antibody (figure 1C). When we compared the ability of heat-killed M3-l to induce neutrophils with that of thioglycollate medium (an inducer of peritoneal neutrophils), we found that both induced similar levels of PECs 12 h after injection (figure 1C and 1D). These results indicate that GAS bacterial cells have the ability to induce neutrophils in mice.
Effect of GAS on PECs induced by neutrophil-inducing agents.
Because neutrophils migrate to the site of infection, we were not surprised to find that a low-virulence GAS strain induced neutrophil exudation. Our finding, however, that M3-f had the opposite effect on PEC induction (figure 1) led us to test the effects on the number of PECs of coinjecting live M3-f and heat-killed M3-1 or thioglycollate. We found that the coinjection of M3-f and heat-killed M3-1 or thioglycollate induced a significantly lower number of PECs after 12 h than did injection of M3-1 or thioglycollate alone (figure 1D), which suggests that live M3-f suppresses the effects of these PEC inducers. We therefore focused on the mechanism by which M3-f counteracts the effects of thioglycollate.
First, we analyzed the number, viability, and population of PECs obtained from mice injected with thioglycollate and M3-f. In contrast to what was observed with thioglycollate alone, we found that the coinjection of thioglycollate and M3-f induced an increased number of PECs for the first 6 h, followed by a significant decrease (figure 2A). Approximately 90% of the PECs from mice injected with thioglycollate alone were viable 12 h after injection. In mice injected with M3-f plus thioglycollate, however, cell viability was reduced to 30% 12 h after injection (figure 2B). PEC preparations from mice injected with thioglycollate and thioglycollate plus M3-f consisted of 90% neutrophils 12 h after injection (figure 2C). We observed no significant differences in the time course of neutrophil induction in these 2 groups of mice, which indicates that the kinetics of thioglycollate-induced neutrophil migration to the peritoneal cavity is not altered by GAS. Because PECs from mice coinjected with thioglycollate and M3-f contained a high proportion of nonviable cells, our results strongly suggest that this reduction in the number of PECs resulted from the cytocidal effects of GAS on neutrophils rather than from its inhibition of neutrophil migration.
Second, we analyzed whether the effect of M3-f on the thioglycollate-induced increase in PECs was specific to this GAS strain. We therefore injected mice with 3 different doses of each of 8 M3 GAS clinical isolates, which varied in virulence from 3 × 108 to 8 × 103 cfu/mouse [10], along with thioglycollate, and the PEC induction level was measured (data not shown). At 2 × 104 cfu/mouse, no GAS strain, except M3-f, significantly altered the number of PECs induced by thioglycollate. At 4 × 105 cfu/mouse, the 4 more-virulent GAS strains reduced the number of PECs induced by thioglycollate, whereas the 4 less-virulent strains had no effect. At 3 × 106 cfu/mouse, all strains except M3-1 reduced the number of PECs induced by thioglycollate. Similar reducing effects were observed when mice were injected with 1 M4, 1 M12, or 1 M1 GAS strain. These results indicate that almost all GAS strains can reduce the number of PECs induced by thioglycollate and that this property is closely associated with virulence, at least in the M3 GAS strains.
SLS involvement in the cytocidal effects of GAS on neutrophils.
We found that the injection into mice of GAS culture supernatants that contain SLO, SLS, superantigenic toxins, M proteins, and nicotinamide adenine dinucleotide glycohydrolase had no effect on the number of PECs induced by thioglycollate (data not shown). We therefore hypothesized that a factor associated with growing bacterial cells might be critical for the PEC-reducing effect of GAS. We began by testing SLS. We designed functional SLSdeficient mutants of 2 M3 GAS strainsM3-f and M3-2, which were designated M3-f SLS and M3-2 SLS, respectively. Sequencing analyses of the sagA regions of these bacteria showed that integration of pSET4s-SLSdest led to replacement of the C-terminal 4-aa residues of SLS with 35-aa residues encoding the C terminus of a catalase derived from the vector and that this replacement did not carry any conserved domain, according to an analysis by use of Prosite (http://kr.expasy.org/prosite/). To exclude any possible influence of the polar effect, we reintroduced intact SLS into the SLS-deficient mutants and obtained an SLS-complemented strain for the M3-2 SLS mutant that was designated M3-2 SLS::SLS, but this was not done for the M3-f SLS mutant. Generation times, SLO production, and hyaluronic acid contents of the mutant strains were comparable to those of the parent strains (table 1). In contrast, we could not detect any SLS activity in the SLS-deficient strains, which indicates that genetic manipulation of the strains caused the deletion of functional SLS. In the sagA-complemented strain, SLS activity was recovered. When we analyzed the amounts of M protein and SPE-B in the culture supernatants of the GAS strains by Western blotting, we found no significant differences between the parent and mutant strains (data not shown). These findings strongly suggest that the pleiotropic effects of the sagA manipulation, which have been reported in an M49 strain [19] and an M6 strain [20] but not in other GAS strains [6, 21], are minimal in our SLS mutants. SLS deficiency in GAS has been reported to cause a diminution in virulence, as measured by LD50, in mice [6]. We found that the SLS-deficient mutant of M3-f had a 10-fold lower virulence level than did the parent strain. In contrast, a lower level of reduction in virulence was observed in the M3-2 SLS-deficient mutant, compared with its parent strain. This reduction, however, was abolished in the sagA-complemented strain, M3-2 SLS::SLS.
We then injected the GAS strains, with or without thioglycollate, into mice and determined the number of induced PECs after 12 h (figure 3). The injection of M3-f alone did not increase the number of PECs, whereas injection of its SLS-deficient strain increased the number of PECs by 2-fold. The injection of M3-2, which is one of the low-virulence strains, induced an 3-fold increase in the number of PECs that was enhanced by SLS deficiency but returned to the level observed in the parent strain by SLS complementation, although the differences among the M3-2 strains did not differ significantly. The parent strains, M3-f and M3-2, significantly reduced the number of PECs induced by thioglycollate. When either of the SLS-deficient mutants M3-f SLS or M3-2 SLS was injected together with thioglycollate, the mutant attenuated the effect of thioglycollate on the number of PECs by 30%50%, compared with its parent strain. This attenuation was abolished, however, by the SLS-complemented strain M3-2 SLS::SLS. Taken together, these results indicate that SLS is a critical factor in the cytocidal effects of GAS on neutrophils in vivo.
When we incubated neutrophils from C57BL/6 mice with the GAS strains in vitroto test their effects on the viability, apoptosis, and phagocytic ability of these neutrophils (figure 4)we found that, although neutrophil viability remained >90% during an 8-h incubation in the absence of GAS, it was significantly reduced, to almost 0%, after incubation with M3-f or M3-2 (figure 4A). Incubation with the SLS-deficient mutants significantly attenuated this reduced viability, but this reduction was restored by incubation with the sagA-complemented strain M3-2 SLS::SLS.
GAS has been reported to accelerate neutrophil apoptosis [22]. Incubation of neutrophils with M3-f or M3-2 increased the percentage of Annexin Vpositive cells to 6% and 4.5%, respectively (figure 4B). The percentage of Annexin Vpositive cells was significantly lower when the neutrophils were incubated with SLS-deficient GAS strains but was not altered by incubation with M3-2 SLS::SLS. In neutrophils incubated with M3-f, M3-2, and M3-2 SLS::SLS, the percentages of apoptotic cells or nonapoptotic dead cells (figure 4C) and the amounts of activated caspase 7 (figure 4D) increased more rapidly than did those in neutrophils incubated with their SLS-deficient mutants. These results indicate that, in addition to increasing nonapoptotic cell death, GAS accelerates neutrophil apoptosis, at least in part by a mechanism associated with SLS activation of the caspase system.
We then assayed the apoptosis of human neutrophils after incubation with the GAS strains in the presence or absence of human plasma (figure 5). In the presence of human plasma, the percentage of Annexin Vpositive cells was increased, up to 5%, during incubation with GAS, and the mutation status of SLS did not affect this increase significantly (figure 5A). In the absence of human plasma, however, the percentage of Annexin Vpositive cells induced by incubation with GAS strains was significantly lower than that when the neutrophils were incubated with SLS-deficient GAS strains (figure 5B). This decrease in Annexin Vpositive cells was not observed in neutrophils incubated with M3-2 SLS::SLS. These results indicate that human plasma masks the effects of the SLS deficiency of GAS in the acceleration of neutrophil apoptosis.
DISCUSSION
Only a limited number of studies have examined the interaction of GAS with the host innate immune system in vivo. Our in vivo results clearly indicate that the GAS bacterial cell itself induces PECs, which are composed mainly of neutrophils. This is in good agreement with results in guinea pigs, in which it has been shown that OK432, which is prepared from a GAS strain, has strong neutrophil-inducing activity [23, 24]. These findings were expected, primarily because neutrophils are exuded to the site of infection.
We also showed that live M3 GAS strains dose-dependently reduced the number of neutrophils induced by thioglycollate. Our results strongly suggest that GAS reduces neutrophil induction by eradicating neutrophils that migrate to the peritoneal cavity rather than by inhibiting neutrophil migration. This eradication of neutrophils was mediated by the induction of nonapoptotic cell death, as well as by the acceleration of neutrophil apoptosis, which we observed in vitro. Neutrophils have been found to readily undergo apoptosis [25], and those that have migrated to the site of inflammation are rapidly eliminated by macrophages that recognize their apoptotic state [26, 27]. These observations suggest that neutrophils encountered by GAS undergo apoptosis more rapidly than usual [22] and are eliminated from the peritoneal cavity, thus reducing the number of PECs in this space.
Because more-virulent M3 GAS strains require a smaller number of bacteria for suppression of the thioglycollate induction of neutrophils, the differing ability of M3 GAS strains to kill neutrophils is due, at least in part, to the difference in virulence [10]. Thus, our findings suggest strongly that the cytocidal effect of GAS on neutrophils is a critical mechanism by which bacteria evade being killed by neutrophils in vivo. This effect has also been observed for M4, M12, and M1 GAS strains, which indicates that virtually all GAS strains are able to attenuate the activity of neutrophil-inducing agents.
We also have shown that the cytocidal effect of GAS on neutrophils was mediated at least partly by SLS. Exotoxin-type, but not cell boundtype, SLS can be inactivated by the components of plasma [9], which may explain the need for living GAS cells in their cytocidal effects on neutrophils in vivo. It has been reported, however, that SLS is not involved in bacterial resistance to phagocytosis or in the lysis of human neutrophils [21, 28]. The discrepancy between these sets of results is due to the presence or absence of human serum (figure 5). Human serum usually contains antibodies to several GAS components [29, 30], which thus enhances the biological functions of neutrophils. Therefore, our findings in a mouse model can be applied to the interaction between GAS and human neutrophils.
Several products of GAS other than SLS have been shown to induce cytocidal effects, including apoptosis, on host cells [3134], and many molecules of GAS are up- or down-regulated during neutrophil apoptosis [35]. Our results do not exclude the possibility that molecules other than SLS contribute to the cytocidal effects of GAS on neutrophils that we observed in the present study. For example, SLS-deficient mutants can accelerate neutrophil apoptosis, which must be due to GAS molecules other than SLS. SLS may cooperate with these other molecules to induce neutrophil apoptosis [21, 28].
In conclusion, our in vivo results show that GAS has a strong capacity to injure neutrophils. Several reports have indicated that GAS produces molecules with antiphagocytic activity and can survive inside phagocytic cells. Although our results do not exclude these mechanisms from contributing to GAS pathogenicity, our findings strongly suggest that GAS uses an even more aggressive methodthe elimination of neutrophils encountering the bacteriumto evade host innate immunity.
Acknowledgments
We thank Dr. J. Suzuki (Azabu University), for helpful discussions, and Dr. N. Okada (Kitasato University), for providing pLZ-Km12.
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