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Section of Infectious Diseases, Department of Medicine,1 Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, Houston, Texas 770302
Received 16 December 2004/ Returned for modification 14 January 2005/ Accepted 31 January 2005
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ABSTRACT |
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107 CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A2. Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface. |
INTRODUCTION |
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The oropharynx is the major site of entry for GAS into the human body and its main portal of transmission (19, 43, 46). Saliva is ubiquitous in the human oropharynx and is an essential part of both the acquired and innate immune defense systems (25, 39). Landmark experiments conducted more than 60 years ago demonstrated the important role played by saliva in the establishment of GAS infection and the subsequent transmission of infectious organisms (18-20). These studies revealed that large numbers of live GAS were present in the saliva of individuals with GAS pharyngitis (19). Moreover, it was established that a major route for dissemination of GAS from infected individuals into the environment was via dispersal of aerosolized saliva (20). Other investigators have reported that pharyngitis patients with detectable levels of GAS in their saliva were more likely to transmit the organism than were individuals whose saliva did not contain GAS (26, 58). These observations, together with the known importance of saliva in host defense, identify the crucial significance of the GAS-saliva interaction. Despite this knowledge, the GAS-saliva interaction has not been extensively investigated in recent years.
To increase our understanding of this interaction, we studied the growth of GAS in saliva collected from healthy human volunteers. We used genetically diverse GAS strains to determine whether the ability of GAS to grow in saliva is widespread within the species. Western immunoblot analysis was used to test the hypothesis that proven and putative extracellular virulence proteins are produced during cultivation of GAS in saliva and to examine the relationship between virulence factor production and growth phenotypes. Finally, isogenic mutant strains were analyzed to determine whether inactivation of genes encoding extracellular virulence factors or transcriptional regulators altered the growth pattern of GAS in saliva.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Collection of saliva from healthy human volunteers. Saliva was collected on ice from healthy volunteers under a Baylor College of Medicine Institutional Review Board human subjects protocol using a modification of the method described by DeJong et al. (8). Saliva production was stimulated by chewing on paraffin wax, and 2.5 mmol of dithiothreitol (DTT) (Fisher Scientific, Pittsburgh, Pa.) was added to aid subsequent filtration. The saliva was clarified by centrifugation at 40,000 x g for 15 min and sterilized by filtration through a 0.20-µm filter (Nalgene Labware, Rochester, NY). The saliva was frozen at –20°C and thawed immediately prior to use. Pooled saliva collected from at least four donors was used to minimize effects of donor variation on study results.
Growth of GAS in human saliva.
Bacteria were incubated at 37°C with 5% CO2. A schematic of the procedures used to grow GAS is shown in Fig. 1. GAS was grown overnight on BSA to confirm colony morphology and purity. Bacteria were inoculated into THY, grown overnight, diluted 1:50 with fresh THY broth, and grown to mid-logarithmic phase (optical density at 600 nm [OD600] of 0.5). A 1:50 dilution of the mid-logarithmic-phase culture was made into 10 ml of saliva to create saliva medium A. After 4.5 h of growth in saliva medium A, a second 1:50 dilution was made in 10 ml of fresh saliva (saliva medium B). Aliquots were removed from both saliva media every 1.5 h for the first 9 h and then daily thereafter and assessed for both OD600 and number of CFU. For determination of CFU, samples were serially diluted 10-fold in phosphate-buffered saline (PBS) (Sigma) and plated in duplicate on BSA. The plates were incubated overnight, and colonies were counted. Each experiment was performed at least in quadruplicate.
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Addition of purified proteins to saliva. Streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB) were cloned, overexpressed, and purified as described previously (23, 27). The purified proteins were added as appropriate to saliva at physiologic concentrations, 5 µg/ml for Sic and 20 µg/ml for SpeB (16, 52).
Statistical analysis. Differences between GAS strains grown in saliva were analyzed by determining the concentration (log10 CFU/ml) at baseline and at maximum growth in saliva medium B for each strain and then using a paired Student's t test to compare values. The ability of a particular GAS strain to persist in saliva was analyzed using a paired Student's t test to compare concentrations (log10 CFU/ml) in saliva medium B at 168 h. Statistical significance was assigned at a two-sided P value of 0.05 using Bonferroni's adjustment for multiple comparisons.
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RESULTS |
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Growth of serotype M1 strain MGAS5005 in saliva.
We used serotype M1 strain MGAS5005 for our initial growth studies because it is genetically representative of M1 strains that cause a high proportion of invasive and noninvasive GAS infections and its genome has recently been sequenced (24, 40, 51). This strain's growth pattern in saliva was highly reproducible (Fig. 2A). There was a rapid increase of 1 to 1.5 log10 CFU/ml when mid-exponential-growth-phase organisms were transferred to saliva. A peak value of 2.5 x 107 CFU/ml was reached within a few hours. Surprisingly, a prolonged stationary phase was observed, characterized by a cell density of 1 x 107 CFU/ml that persisted for up to 28 days (Fig. 2B).
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1.5 to 2.0 log10 CFU/ml over the next 24 h, despite starting from an inoculum that was nearly 1.0 log10 less than the starting inoculum in saliva medium A. The resulting density (1 x 107 CFU/ml) was essentially identical to that of GAS grown in saliva medium A. In addition, a similar prolonged stationary phase was observed in saliva medium B. We next determined the ability of serotype M1 strain MGAS5005 to grow in response to a fresh nutrient supply added after prolonged culture. Organisms growing in saliva for 7 days were subcultured at a 1:50 dilution into fresh saliva. The organisms grew 2.0 log10 CFU/ml to reach 107 CFU/ml within 18 h. Together, these data indicate that serotype M1 strain MGAS5005 not only grew in human saliva to a cell density of 107 CFU/ml but also maintained the ability to proliferate in response to a fresh supply of saliva, even after a prolonged stationary phase. Addition of glucose or control of pH did not alter the saliva growth phenotype. Because the concentration of glucose in human saliva is only about 1% of that in blood or tissue, it has been suggested that the level of free glucose in saliva is insufficient to support bacterial cell division to values exceeding 1 x 107 CFU/ml (42, 48). We added exogenous glucose to saliva to achieve glucose levels equal to that found in human blood (100 mg/dl) but observed no difference in the maximum density of GAS (Fig. 2A). The normal pH of human saliva is between 6 and 7, and the presence of bicarbonate provides significant buffering capacity (25). The pH of the saliva during growth of GAS ranged between 6.5 and 7.5. We used exogenous bicarbonate to maintain the culture media between pH 7 and 7.1 but observed no difference in maximum growth achieved by GAS in the pH-stabilized saliva compared with saliva where no effort was made to control pH (Fig. 2A). Finally, we were able to filter a limited amount of saliva without adding DTT. Serotype M1 strain MGAS5005 grown in saliva filtered without DTT had no change in growth phenotype compared to results when the usual DTT-filtered saliva was used. Taken together, we conclude that glucose levels, pH, or the addition of DTT did not affect the growth phenotype of serotype M1 strain MGAS5005 in human saliva.
Prolonged stationary phase not observed in other laboratory media. The steady, high-density persistence of serotype M1 strain MGAS5005 in saliva for at least 28 days was very unexpected. We studied the growth of serotype M1 strain MGAS5005 in nutrient-rich laboratory media (THY) and in nutrient-limited CDM. In both THY and CDM, there was a rapid decline of viable organisms after the first 24 h, and we were unable to detect viable organisms after more than 5 days (Fig. 2B). These results indicate that the interaction of serotype M1 strain MGAS5005 with saliva led to the unusually prolonged stationary phase.
Growth of GAS in saliva leads to decreased killing by ampicillin. To achieve a prolonged steady cell density during cultivation in saliva, serotype M1 strain MGAS5005 either had to closely balance cell death with cell renewal or had to enter a state of decreased cellular turnover. Given the limited nutrients available in saliva, we hypothesized that decreased cellular turnover was responsible for the prolonged stationary phase observed during growth in saliva. To test this hypothesis, we grew serotype M1 strain MGAS5005 in saliva for 72 h and then added various concentrations of ampicillin, a drug which kills actively dividing cells. After 72 h of growth in saliva, the minimum bactericidal concentration of ampicillin was nearly 100-fold higher than that in either THY or CDM (Fig. 3). These data support our hypothesis that cultivation of GAS in saliva leads to diminished cellular turnover.
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All of the GAS strains tested grew in saliva medium B to a density of 107 CFU/ml within 24 h (Fig. 4A and B). However, the ability of various strains to persist in saliva varied markedly. The serotype M1 strains persisted at the highest levels, whereas the serotype M3, M9, M11, M12, M28, M33, M73, and M77 strains persisted at slightly lower densities, 5.5 x 105 CFU/ml (Fig. 4A and B). In addition, within about 10 days we detected no viable organisms for the serotype M6 strain JRS4 and the serotype M43 and M94 strains (Fig. 4B). The lack of persistence of the serotype M6 strain JRS4 was surprising in that M6 serotypes commonly cause pharyngitis whereas M43 and M94 serotype strains rarely cause pharyngitis. Strain JRS4 was isolated in 1971, and hence, we hypothesized that its prolonged time in laboratory conditions had rendered it less able to grow in saliva. We tested this hypothesis by growing two other serotype M6 strains that were recently isolated (Table 1) and found that these two strains had a prolonged stationary phase compared to JRS4 (Fig. 4B). Taken together, these data show GAS strains of diverse serotypes generally achieve similar maximum densities during cultivation in saliva. There were some differences among the abilities of various GAS strains to persist in saliva, but we could draw no definitive correlation between the growth phenotype of a particular strain in saliva and the propensity of that strain's particular serotype to cause pharyngitis.
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GAS produces a variety of extracellular virulence factors during growth in saliva. Having found that Sic and SpeB were produced during growth in saliva, we hypothesized that other proven and putative extracellular virulence proteins were produced by GAS during cultivation in saliva. Using Western immunoblot analysis, we found growth-phase-dependent production of several known and putative GAS virulence factors, such as streptococcal Mac protein (Mac), streptococcal pyrogenic exotoxin A (SpeA), streptococcal phospholipase A2 (SlaA), and SPy2191. In light of our earlier findings with the sic and speB isogenic mutant strains, we also examined the growth of mac, SlaA, and SPy2191 isogenic mutant strains. We found that the mac, SlaA, and SPy2191 isogenic mutant strains and their parental wild-type strains had similar growth phenotypes (Table 2). Taken together with the Sic and SpeB protein production data, we conclude that during growth in saliva, GAS elaborates a wide range of extracellular proteins, at least two of which directly impact the ability of the organism to grow and persist in saliva.
Evaluation of effect of transcriptional regulators on growth of GAS in saliva. The entry of GAS into a prolonged stationary phase following growth in saliva combined with our finding of growth-phase-dependent production of multiple virulence factors suggests that GAS sensed and responded to the environmental conditions it encountered in saliva. To better understand how GAS altered its gene regulation during interaction with saliva, we studied the growth pattern of 11 isogenic mutant strains with deletions in various known and putative transcriptional regulators (17, 44, 57; unpublished data). In previous work, no apparent differences were identified in the growth of these isogenic mutant strains in THY compared to that of the wild type (17, 44, 57). All of the 11 isogenic mutant strains studied here had growth phenotypes similar to that of their parental wild-type strain in terms of maximum growth achieved and the ability to persist during the stationary phase (Table 2). We conclude that the 11 transcriptional regulators we studied are not crucial for maximal growth or persistence of serotype M1 strain MGAS5005 in human saliva.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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107 CFU/ml of saliva, a finding that is very similar to that observed for Streptococcus gordonii (42). The ability of GAS to grow in human saliva to this density is consistent with reported densities of 5 x 106 organisms per milliliter of saliva in patients with symptomatic GAS pharyngitis (19, 26, 46). The factors limiting the growth of GAS in human saliva in vitro and in vivo are not understood. It has been suggested that the level of free glucose in saliva is not sufficient to support cell division of oral streptococci to values exceeding 1 x 107 CFU/ml (42). However, similar to other investigators, we found that the addition of glucose did not increase the density of organisms, suggesting that the concentration of glucose alone is not likely to be a limiting factor for growth (9). We also found that pH or the addition of DTT did not affect the maximum growth achieved in vitro.
The ability of human saliva to restrict the density of microorganisms such as GAS to 107 CFU/ml is in striking contrast to the growth-promoting ability of other body fluids. Bacteria growing in blood, urine, or cerebrospinal fluid can reach densities of 109 CFU/ml and higher (7, 29, 45). The presence of multiple antimicrobial peptides and the limited nutrients available in saliva for the growth of microorganisms likely contribute to this phenomenon (1, 25). The observation that saliva limits microbial proliferation confirms the fundamental contribution of saliva to innate immune defense of the human oropharynx.
The persistence in saliva for at least 28 days at near-maximal growth levels of all serotype M1 strains studied was very unexpected (Fig. 2). This persistence contrasted with our findings, also observed by other investigators (53), that in nutrient-rich media (THY) and in nutrient-limited media (CDM), GAS had a rapid decrease in viable organisms after reaching stationary phase. We speculate that when a cell density of 1 x 107 CFU/ml is reached in saliva, GAS enters a state of restricted metabolic activity, thereby allowing persistence at a relatively high density, a phenomenon which would be expected to enhance the likelihood of transmission to a new host. The idea of restricted metabolic activity (and presumably decreased cell wall turnover) is supported by the observation that during the stationary phase in saliva, GAS had decreased susceptibility to killing by ampicillin.
Growth and persistence of GAS strains in saliva. Although there is significant geographic variation in the relative frequency of pharyngitis caused by distinct M-protein serotypes, several M types tend to predominate in many areas (21, 49). For example, serotype M1 strains are the leading cause of GAS pharyngitis in many studies (11, 49). Thus, it is noteworthy that we found that serotype M1 strains persisted in saliva at higher densities and for longer periods than did the strains of all other serotypes studied. We speculate that the persistence of serotype M1 strains in human saliva contributes to the prevalent nature of serotype M1 strains as a cause of pharyngitis. It is possible that the production of Sic by these strains is an important factor in this regard (see below).
Overall, we did not observe a simple relationship between the magnitude of persistence in saliva and reported abundance of a GAS serotype in human infections. For example, strains of serotypes that rarely cause pharyngitis, such as M9, M11, and M33, persisted at levels similar to those of strains of serotypes that often cause pharyngitis. The difficulty of using individual strains to generally characterize a serotype is illustrated by our results with the M6 serotype strains, where there were marked differences in the abilities of individual strains to persist in saliva. Taken together, our results suggest that the growth and persistence of a GAS strain of a particular serotype in human saliva are not alone sufficient to account for that serotype's relative abundance in pharyngitis cases, although they may contribute. It will be important to investigate this issue with a larger sample of GAS strains.
The role of Sic and SpeB in the growth of GAS in human saliva. GAS elaborates at least two extracellular proteins, SpeB and Sic, that have been shown to inhibit or inactivate the purified forms of antimicrobial peptides present in saliva (13, 14, 16, 47). Sic is produced mainly by serotype M1 strains, whereas virtually all strains have the speB gene, although protein expression levels are variable (16, 27, 52). Sic appears to function through binding inhibition, whereas the cysteine protease SpeB can actively degrade peptides involved in innate immunity (14, 47). We tested the hypothesis that the activities of SpeB and Sic contributed to the ability of GAS to proliferate in saliva, using isogenic mutant strains in which SpeB and Sic were not produced. Both the sic and speB isogenic mutant strains had decreased growth in human saliva, and this deficiency was reversed by the addition of the respective purified protein to the culture medium. Moreover, GAS strains that failed to persist in saliva did not produce detectable amounts of SpeB during culture in saliva. Inactivation of Sic has previously been shown to lead to a decreased ability of GAS to colonize the mouse oropharynx (35). Our results suggest that the failure of the sic mutant strain to successfully colonize the oropharynx may have been due in part to its inability to persist at high levels in saliva. Taken together, we conclude that Sic and SpeB are crucial to a successful GAS interaction with human saliva.
GAS extracellular protein production during growth in saliva. Having established a role for Sic and SpeB for GAS-saliva interaction, we next sought to determine if GAS secreted other extracellular proteins during growth in saliva. We found that several proven and putative extracellular virulence proteins (e.g., SpeA, Mac, SlaA, and SPy2191) were produced during growth in human saliva (Table 2). Moreover, these proteins were produced in a growth-phase-dependent fashion, suggesting that transcriptional regulation of GAS virulence factor production occurred during growth in saliva.
Taken altogether, our results shed new light on a key early event in the life cycle of GAS, namely, the interaction between GAS and human saliva. Recent reports have documented upregulation of several GAS genes in response to contact with human epithelial cells in vitro, another important event in the life cycle of this pathogen (2, 5, 6, 41). However, by showing that GAS responded in a complex fashion to growth in human saliva, our results suggest that molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface. Thus, further study of GAS-saliva interaction is likely to provide new insights into the molecular strategies used by this common human pathogen to successfully colonize the human oropharynx, produce disease, and disseminate to a new host.
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ACKNOWLEDGMENTS |
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This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, grant U01-AI60595 to J.M.M.
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FOOTNOTES |
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| J. Bacteriol. | J. Virol. |
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, dashed line; saliva medium B, 
, solid line; THY medium, 
, dashed line; CDM, •, solid line; saliva plus glucose, 
, dashed line; saliva with pH controlled, X, solid line. (B) Growth over 28 days (d) for five serotype M1 strains in saliva medium B. Symbols: MGAS5005, 
, solid line; MGAS9415, 
, solid line; saliva with 1.024 µg/ml ampicillin, •, dashed line; THY with 0.016 ampicillin, 
, solid line); serotype M12 strain MGAS9429 (
, solid line), serotype M9 strain MGAS12487 (X, dashed line), serotype M43 strain MGAS9464 (
