Maltodextrin Utilization Plays a Key Role in the Ability of Group A St
http://www.100md.com
《感染与免疫杂志》
Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, Texas 77030
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
Analysis of multiple group A Streptococcus (GAS) genomes shows that genes encoding proteins involved in carbohydrate utilization comprise some 15% of the core GAS genome. Yet there is a limited understanding of how carbohydrate utilization contributes to GAS pathogenesis. Previous genome-wide GAS studies led us to a focused investigation of MalE, a putative maltodextrin-binding protein. Analysis of 28 strains of 22 distinct M protein serotypes showed that MalE is highly conserved among diverse GAS strains. malE transcript levels were significantly increased during growth in human saliva compared to growth in a chemically defined glucose-containing medium or a nutrient-rich medium. MalE was accessible to antibody binding, indicating that it is expressed on the GAS cell surface. Moreover, growth in human saliva appeared to increase MalE surface expression compared to growth in a nutrient-rich medium. Analysis of a malE isogenic mutant strain revealed decreased growth in human saliva compared to wild-type GAS. Radiolabeled carbohydrate binding assays showed that MalE was required for the binding of maltose but not glucose. The malE isogenic mutant strain colonized a lower percentage of GAS-challenged mice compared to wild-type and genetically complemented strains. Furthermore, decreased numbers of CFU were recovered from mice infected with the malE strain compared to those infected with wild-type GAS. These data demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Further investigation into carbohydrate transport and metabolism pathways may yield novel insights into GAS pathogenesis.
INTRODUCTION
Many human bacterial pathogens devote large portions of their genome to genes encoding known or putative proteins involved in carbohydrate transport and metabolism (12, 37, 58). Therefore, it is reasonable to speculate that the utilization of carbohydrates is essential to the ability of pathogenic bacteria to cause disease in humans. Indeed, recent investigations in diverse human pathogens have demonstrated the vital contribution of carbohydrate metabolism to microbial pathogenesis (20, 23, 33, 54). Carbohydrate transport and metabolism genes are often conserved among pathogenic bacteria (12, 56, 58). Consequently, an increased understanding of specific carbohydrate metabolism and transport pathways may lead to insights into shared mechanisms by which a broad range of microbes infect humans.
Group A Streptococcus (GAS) causes an assortment of human diseases ranging from uncomplicated pharyngeal and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome (6). Similar to other human-adapted pathogens, genes encoding proteins putatively involved in carbohydrate transport and metabolism make up about 15% of the core GAS genome (2, 4, 52, 57, 58). Although they account for a substantial portion of the GAS genome, however, there has been a relative dearth of research into how the utilization and transport of carbohydrates contribute to GAS pathogenesis (22, 42).
The mucosal surface of the human oropharynx is a major site of GAS colonization and infection (38). To successfully colonize and infect the human oropharynx, GAS must overcome multiple aspects of the acquired and innate immune systems including secretory immunoglobulin, antimicrobial peptides, and a limited nutrient supply (1, 8, 48, 55). Recently, we have begun to examine the interaction of GAS and human saliva, a key mediator of acquired and innate immunity in the human oropharynx (48). We employed expression microarray analysis to generate a genome-wide view of GAS gene transcript levels during interaction with human saliva (49). The transcript levels of genes encoding known and putative proteins involved in carbohydrate transport and metabolism were markedly elevated early in the GAS-saliva interaction. Analysis of the GAS transcriptome during experimental pharyngitis in non-human primates similarly showed that carbohydrate metabolism gene transcript levels were abundant during the initial colonization phase (61). Taken together, these data suggest that genes involved in carbohydrate metabolism and transport may be central to early steps in GAS pharyngeal colonization and infection.
Previously, two putative GAS carbohydrate transport proteins were shown to be immunogenic in humans recovering from GAS infection and in mice experimentally infected with GAS (28). One of these proteins, M5005_Spy_1058, is a putative maltodextrin-binding protein that has been annotated as MalE based on homology with the MalE protein encoded by Escherichia coli (5, 52). Genome-wide transcriptome analyses of GAS in human blood and in human saliva showed marked upregulation of malE (16, 49). The high transcript levels of malE during GAS-human fluid interaction suggest that MalE may be critical to GAS during colonization or infection of humans. In this work we sought to characterize the cellular location, transcriptional regulation, and contribution to GAS growth of MalE. We also tested the hypothesis that MalE participates in the ability of GAS to colonize the oropharynx of mice. Our findings suggest that maltodextrin transport via MalE is a key component of the initial colonization of the oropharynx by GAS.
MATERIALS AND METHODS
Bacterial strains and culture media. The genome of serotype M1 strain MGAS5005 has been fully sequenced (52). This strain has been extensively used for in vitro and animal models of GAS infection including transcriptome analysis during growth in human saliva (49, 51, 52, 61). The other bacterial strains used in this study are listed in Table 1. GAS was grown on Trypticase soy agar containing 5% bovine serum albumin (BSA; Becton Dickinson) or in Todd-Hewitt broth containing 0.2% (wt/vol) yeast extract (THY; Difco). Carbohydrates were added at a 1.0% (wt/vol) concentration to a carbohydrate-free preparation of a commercially available chemically defined medium (CDM; JR Biosciences) (62). For this article, we will use glucose medium to refer to the carbohydrate-free CDM with 1% glucose added and maltose medium to refer to the carbohydrate-free CDM with 1% maltose added. Spectinomycin (Sigma) was added to THY agar or broth at 150 μg/ml, and chloramphenicol (Sigma) was added at 4 μg/ml to spectinomycin THY agar or broth when appropriate. Blue-white screening was performed by adding 50 μg/ml of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) to the chloramphenicol-spectinomycin THY agar.
Growth of GAS in human saliva. GAS was grown in human saliva as previously described (48). Saliva was collected on ice from healthy volunteers as previously described under a Baylor College of Medicine Institutional Review Board human subjects protocol (48). Pooled saliva collected from at least four donors was used to minimize the effects of donor variation on study results.
DNA sequence analysis. Chromosomal DNA was isolated using a phenol-chloroform extraction method as previously described (41). DNA sequencing primers were designed on the basis of the serotype M1 strain MGAS5005 genome (Table 2) (52). The malE open reading frame M5005_Spy_1058 corresponds to open reading frame SPy1294 in serotype M1 strain SF370 (12). Sequence data obtained from both DNA strands with an Applied Biosystems 3700 automated sequencer were assembled with Sequencher 4.5. Comparative sequence alignment of the inferred amino acid sequences was performed with CLUSTALW.
RNA isolation and transcript level analysis. Serotype M1 strain MGAS5005 was grown in various media to early- and late-logarithmic growth phases. RNA was isolated with a Fast Prep Blue Kit (Q/BioGene) and purified using an RNeasy kit (QIAGEN) as previously described (49). The concentration and quality of RNA were assessed with an Agilent 2100 Bioanalyzer and analysis of the A260/A280 ratio. cDNAs were created from the RNA using SuperscriptIII (Invitrogen) according to the manufacturer's instructions. TaqMan real-time quantitative reverse transcription-PCR (QRT-PCR) was performed in quadruplicate with an ABI Thermocycler 7700 (Applied Biosystems) using the change in cycle threshold for analysis, as previously described (49). TaqMan primers and probes for malE and the internal control gene proS are listed in Table 2.
Detection of MalE on the GAS cell surface. Surface localization of MalE was assessed with a FACSCaliber flow cytometer (BD Biosciences) using affinity-purified MalE-specific antibodies (Bethyl Laboratories) (28, 42). Antibody against streptococcal phospholipase A2 (SlaA), a GAS protein not encoded by strain MGAS5005, was used as a control for nonspecific antibody binding. Strain MGAS5005 and the malE isogenic mutant strain (see below) were grown to exponential phase in either THY medium or human saliva, harvested by centrifugation, washed once in Dulbecco's phosphate-buffered saline (PBS), pH 7.2, and suspended in Dulbecco's PBS at 108 CFU/ml. Anti-MalE or anti-SlaA antibody was added at a final concentration of 0.05 μg/100 μl to 100 μl of bacterial suspension and incubated for 30 min on ice. Samples were washed with Pharmingen stain buffer (BD Biosciences) and stained with phycoerythrin-conjugated donkey anti-rabbit immunoglobulin G (Jackson ImmunoResearch) (1:100 dilution) for 30 min on ice. The cells were washed again with stain buffer and analyzed via flow cytometry.
Creation of malE isogenic mutant strain. The malE isogenic mutant strain was created from parental serotype M1 strain MGAS5005 using the PCR-mediated method of Kuwayama et al. (25). Primers were designed to amplify the 5' and 3' ends of the malE gene region along with nucleotide sequences that were complementary to the 5' or 3' portion of the spectinomycin (spc) resistance cassette (Table 2). A third set of primers with nucleotide sequences complementary to the 5' and 3' ends of the malE gene region was used to amplify the spc cassette from plasmid pSL60 (30). Fusion PCR was then used to link the 5' and 3' malE gene region PCR products to the spc cassette via the overlapping nucleotide sequence regions (9, 25). This resulted in a PCR product where the spc cassette, flanked by the 5' and 3' malE gene regions, was now inserted in the place of nearly the entire malE open reading frame. The gene disruption PCR construct was used to transform competent GAS cells with spectinomycin resistance used as the selection mechanism. The malE isogenic mutant strain was analyzed by Southern hybridization and DNA sequencing to confirm that the proper genetic construct was obtained (Fig. 1).
Complementation of malE isogenic mutant strain. For complementation purposes, we utilized plasmid pDC123 which includes the chloramphenicol acetyltransferase (cat) gene and the phoZ gene that allows for blue-white screening via alkaline phosphatase activity (27). The complete malE gene along with 250 bp upstream and 15 bp downstream was amplified from strain MGAS5005 chromosomal DNA using primers that introduced EcoR1-cut sites at the 5' and 3' ends (Table 2). The resulting PCR product was digested with EcoR1 (New England Biolabs) and cloned into the EcoR1 site of plasmid pDC123. The resulting plasmid, named pDCmalE, was used to transform competent malE cells. Chloramphenicol resistance and blue-white screening were used to choose transformed strains for further analysis. Presence of the desired pDCmalE construct was confirmed using PCR and DNA sequencing. pDC123 lacking the malE gene was used to transform strain MGAS5005 and the malE isogenic mutant strain as controls.
Carbohydrate binding assays. Strain MGAS5005, the malE isogenic mutant strain, and the compmalE (complemented mutant) strain were grown in either glucose or maltose medium to mid-exponential phase, collected by centrifugation, washed, and suspended to an optical density at 600 nm (OD600) of 0.5 in 150 μl of CDM lacking carbohydrates. A total of 25 μl of 280 μM 1.85 MBq [14C]maltose or [14C]glucose (GE Healthcare) was added to obtain a final concentration of 40 μM radiolabeled carbohydrate. At various times 40-μl samples were removed and filtered on a 0.45-μm-pore-size nitrocellulose membrane (Millipore) which was then rinsed twice with CDM lacking carbohydrates. The radioactivity trapped on each filter was then determined by liquid scintillation counting (Beckman Model LS7500). Samples were taken every minute for the first 4 min during which carbohydrate binding rates were found to be linear. The amount of protein in the cultures was determined by Bradford assay (Bio-Rad). All experiments were performed in quadruplicate.
Mouse colonization experiments. All animal experiments were performed under a protocol approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Mouse throat colonization studies were conducted with adult (18 to 20 g) female outbred CD-1 Swiss mice (Harlan Sprague-Dawley, Inc.) as described previously (30). Strain MGAS5005, the malE isogenic mutant strain, and the compmalE strain were grown in THY medium and harvested at an OD600 of 0.5. The cells were washed once and suspended in sterile, pyrogen-free PBS to a density of 1.5 x 108 CFU in 100 μl. Both nares of each mouse were inoculated with 50 μl of the GAS suspension. This dose of MGAS5005 has been previously shown to result in the colonization of 75% of inoculated animals (30). The mouse throats were swabbed prior to inoculation and then daily thereafter. Throat swabs were plated onto BSA and grown overnight, and beta-hemolytic colonies were tested for the presence of GAS carbohydrate antigen via latex agglutination (BD Biosciences). Blood collected from dead animals by cardiac puncture was cultured on BSA. In addition, compmalE colonies recovered from colonized and dead mice were tested for loss of the complementary plasmid by culturing in THY broth with chloramphenicol.
Statistical analysis. Flow cytometry and RNA transcript level comparisons were performed using a Student's two-sided t test. Growth comparisons were done using analysis of variance. Radiolabeled carbohydrate binding rates were compared using linear regression. The chi-square test was used to assess statistical differences in throat colonization rates among the animal groups infected with strain MGAS5005, its malE isogenic mutant derivative, and the compmalE strain. Analysis of variance was used to test for differences in number of CFU recovered from animals infected with the three strains. Statistical significance was assigned at a two-sided P value of 0.05 using Bonferroni's adjustment for multiple comparisons when appropriate. Statistical calculations were performed with NCSS software, version 2004 (Kaysville, Utah).
RESULTS
Description of the malE gene region and homology of MalE among various GAS strains. MalE has been identified as a putative lipoprotein based on the presence and composition of its amino-terminal secretion signal sequence (28, 53). Analysis of the serotype M1 strain MGAS5005 genome (52) shows that downstream of malE are two genes, malF and malG, whose products are predicted to form a permease for solute transport (Fig. 2). Together with MalE, MalF and MalG are predicted to comprise two parts of an ATP binding cassette transport system, a common mechanism by which bacteria import substrates (18). The putative ATP-binding portion of the ATP binding cassette transport system is located outside of the malE genome region, an arrangement that is not unusual in gram-positive bacteria (39, 45). Upstream of malE and divergently transcribed are malR, which encodes a putative transcriptional regulator, and malP and malQ, which encode enzymes putatively involved in generating various forms of glucose from maltodextrins (Fig. 2). The identical gene arrangement is present in all 12 GAS strains sequenced to date (2-4, 12, 17, 34, 50).
MalE is the only protein encoded by this gene region that is putatively surface exposed and thus possibly under selective pressure from the host immune system. Some GAS surface-exposed proteins are highly variable among various strains, whereas others are well conserved (31, 42). We sequenced the malE gene from 28 strains comprising 22 different M serotypes (Table 1). The malE gene was present in all strains tested. There was an average of 3.2 amino acid replacements per 415 amino acid sites (Table 1). These data show that MalE is highly conserved among diverse GAS strains, indicating that information generated regarding MalE function in a particular GAS strain may be broadly applicable to diverse GAS strains.
malE transcript level is increased in maltose medium compared to glucose medium and nutrient-rich medium. Many bacteria employ carbon catabolite repression, a phenomenon where the use of sugars other than glucose is minimized when glucose is available (19). By adding specific sugars to carbohydrate-free CDM, we tested the hypothesis that growth in maltose medium would lead to increased transcript levels of malE compared to growth in THY or glucose medium. As assessed by real-time QRT-PCR, during the mid-exponential phase of growth the transcript level of malE in the maltose medium showed a 35-fold increase compared with the glucose medium and 95-fold increase compared with the THY medium (P < 0.001 for both) (Fig. 3). At entry into stationary phase, the malE transcript level was fourfold and threefold higher in the maltose medium compared to the glucose and THY media, respectively (P = 0.024 and P = 0.008). These data show that a significant increase in malE transcript level occurred during growth in a medium containing maltose, suggesting that malE transcription is repressed in a nutrient-rich environment but induced in the presence of maltose.
malE transcript level is increased during growth in human saliva compared to growth in a nutrient-rich medium. Previous expression microarray analysis found that malE transcript levels were high during growth of GAS in human saliva, especially in the exponential growth phase (49). Thus, we tested the hypothesis that the interaction of GAS with saliva leads to increased malE transcript levels compared to growth in a nutrient-rich medium. Similar to the situation during growth in maltose, the malE transcript level in saliva at mid-exponential phase was 45-fold higher than growth in glucose medium and 120-fold higher than growth in THY medium (P < 0.001 for each) (Fig. 3). Upon entry into stationary phase in saliva, the transcript levels of malE decreased but remained 13-fold and 10-fold higher than growth in glucose or THY medium, respectively (P < 0.001 for each). Taken together, we conclude that growth of GAS human saliva led to a significant increase in the malE transcript level, suggesting that MalE is important for the successful interaction of GAS with human saliva.
Growth in human saliva increased the amount of MalE on the GAS cell surface. We used flow cytometry to test two hypotheses. First, we predicted that MalE would be expressed on the GAS cell surface. Second, we expected that the increased transcript levels of malE during growth in human saliva would result in increased MalE on the GAS cell surface compared to growth in THY medium. We used anti-streptococcal phospholipase A2 (SlaA) antibody as the control antibody because SlaA is not produced by strain MGAS5005 (4, 52). The flow cytometry data showed no significant increase above the control level in the amount of bound anti-MalE when the malE isogenic mutant strain was examined, suggesting that the anti-MalE antibody is specific for MalE (Fig. 4A). When wild-type strain MGAS5005 was studied, there was an increase in binding above the control level for anti-MalE during the mid-exponential growth phase in THY medium (Fig. 4B). Moreover, there was an increase in bound anti-MalE at mid-exponential phase during growth in saliva compared to growth in THY medium (Fig. 4C) (P < 0.001). These data are consistent with the in silico analysis predicting that MalE is present on the GAS cell surface and therefore accessible to antibody. Also, the increased transcript level of malE observed during growth in saliva appeared to result in increased MalE levels on the GAS cell surface.
The malE isogenic mutant strain had decreased growth in maltose medium compared to wild-type. To directly test the role played by MalE in various GAS activities, we created a nonpolar malE isogenic mutant strain from parental M1 strain MGAS5005 and genetically complemented the mutant strain to create strain compmalE (see Material and Methods). We tested the hypothesis that MalE is important for the utilization of maltose but not glucose by comparing the growth patterns of wild-type GAS, the malE isogenic mutant strain, and the compmalE strain in various media. There was no growth for any strain in the carbohydrate-free CDM when no exogenous carbohydrate was added (data not shown). We observed no differences when the three strains were grown in THY medium or during growth in glucose medium (Fig. 5A, 5B) (P = 0.791 for THY; P = 0.972 for glucose medium). However, the malE isogenic mutant strain was significantly less able to grow in maltose medium compared to the wild-type and complemented strains (Fig. 5C) (P = 0.001). There was no significant difference in growth between the wild-type and the compmalE strain during growth in maltose medium (P = 0.611). These data show that MalE is needed for GAS to optimally grow in maltose medium.
The malE isogenic mutant strain grew less in human saliva compared to wild type. A key finding of our earlier GAS-saliva investigations was that diverse GAS strains were readily able to grow in human saliva and, contrary to growth in laboratory media, persisted at maximal CFU counts for up to 30 days (48). The observation that the malE transcript level increased 100-fold during growth in saliva compared to growth in THY medium suggested that, similar to its role in a maltose medium, MalE might be critical to the ability of GAS to grow in human saliva. We tested this hypothesis by comparing the growth of strain MGAS5005, the malE isogenic mutant strain, and the compmalE strain in saliva pooled from healthy donors. As previously described, GAS saliva growth experiments are performed using CFU analysis rather than density readings because saliva can aggregate GAS and thereby interfere with OD readings (8, 48). Whereas both the wild-type and complemented mutant strains grew 2.5 log10 CFU/ml from baseline and persisted at a density of 1 x 107 CFU/ml, the malE isogenic mutant strain only grew 1.0 log10 CFU/ml and persisted at 5.5 x 105 CFU/ml (Fig. 5D) (P value for growth = 0.002; P value for persistence = 0.007). The addition of glucose at 100 mg/dl to the saliva growth medium restored the growth of the malE isogenic mutant strain to wild-type levels (data not shown). Therefore, we conclude that MalE is key to the optimal growth and persistence of GAS in human saliva.
The malE isogenic mutant strain was deficient in maltose, but not glucose, binding. In earlier portions of this work, we found that MalE was expressed on the GAS cell surface and that a malE isogenic mutant strain grew poorly in maltose medium. To test the hypothesis that MalE mediates maltose transport, we studied GAS binding of 14C-radiolabeled carbohydrates. We found no difference among the ability of the wild-type, malE, or compmalE strains to bind glucose (P = 0.541) (Fig. 6A). In contrast, in comparison to the wild-type and the complemented malE strain, the malE mutant strain showed a 95% decrease in maltose binding (P < 0.001) (Fig. 6B). These results show that the presence of MalE is required for maximal binding of maltose by GAS but not for the binding of glucose. These findings are in concordance with the earlier data demonstrating that MalE is needed for the optimum growth of GAS in maltose medium but not in glucose medium.
The malE isogenic mutant strain was unable to efficiently colonize the mouse oropharynx. Although mice are not normally susceptible to pharyngeal infection by GAS, temporary colonization of the mouse oropharynx occurs following nasopharyngeal inoculation (30, 35, 43). We used adult outbred CD-1 mice to test the hypothesis that MalE is needed for optimum GAS colonization of the oropharynx. Each group of 30 mice was challenged intranasally with 1.5 x 108 CFU GAS in a total of 100 μl (50 μl per nostril). The oropharynx of each mouse was then swabbed daily using a fine-tip, sterile cotton applicator (Fisher), which was then used to inoculate a BSA plate. The plates were incubated for 24 h and examined for beta-hemolytic colonies, which were tested for the presence of GAS carbohydrate antigen via latex agglutination (BD Biosciences). At each time point tested, significantly more mice were colonized with the MGAS5005 and the compmalE strains compared to the malE isogenic mutant strain (Fig. 7A). Moreover, at each time point tested, the number of GAS CFU was significantly higher in the animals infected with the wild-type and complemented strains compared to the isogenic mutant strain (Fig. 7B). These data support our hypothesis that MalE is critical in the ability of GAS to successfully colonize the oropharynx.
As we were unable to maintain chloramphenicol selection on the compmalE strain during infection, we assayed for loss of the complementary plasmid. A total of 43 of 50 colonies recovered from animals infected with the complemented malE strain remained chloramphenicol resistant. Therefore, even in the absence of chloramphenicol pressure, the majority of compmalE strains maintained the pDCmalE plasmid, providing further evidence of the critical nature of MalE during oropharyngeal colonization.
DISCUSSION
We recently initiated a genome-wide investigation of GAS-human saliva interaction to gain insight into how unstudied areas of the GAS genome contribute to GAS pathogenesis (48, 49). Analysis of the GAS transcriptome in saliva combined with other recently published data led us to focus on the gene/gene product M5005_Spy_1058 or MalE (16, 28, 49, 61). Previous genome-wide investigations showed that malE was highly transcribed during GAS interaction with human blood and had dynamic transcript levels during the colonization of the non-human primate oropharynx (16, 61). Moreover, MalE is known to be immunogenic in mice and humans infected with GAS (28). Finally, genome-wide investigations of pathogens related to GAS, i.e., Streptococcus pneumoniae and group B Streptococcus, also suggested that MalE homologues might be involved in bacterial pathogenesis (24, 36).
We first determined whether MalE is highly conserved among diverse GAS strains. The publication of multiple full-length GAS genomes has led to the observation that about 85 to 90% of the GAS genome is shared by various strains while the remaining 10 to 15% is highly variable (4, 17, 50). To be broadly applicable to GAS pathogenesis, a gene/gene product needs to be present in the majority of infecting GAS strains. Our analysis of 28 strains of 22 distinct M serotypes shows that the malE gene was present among all tested strains. Also, the MalE amino acid sequence was highly conserved among diverse strains (Table 1). Therefore, our findings regarding the role played by MalE in a particular GAS strain are likely to be relevant to the broader GAS population.
Like many other bacteria, GAS encodes a homologue of catabolite control protein A (CcpA) (19, 23, 52). The preferential utilization of glucose in low G+C gram-positive bacteria is thought to be at least partially mediated by CcpA acting at catabolite responsive element (cre) sites (44). Our findings of low malE transcript levels in the presence of glucose or in a nutrient-rich medium (THY) suggest that malE is subject to such repression. Glucose levels in saliva are very low; therefore, glucose-mediated catabolite repression is unlikely to occur during GAS-saliva interaction (47). However, the importance of glucose-mediated catabolite repression in malE transcription is debatable as malE transcript levels were previously shown to be quite high during growth in human blood, a glucose-rich medium (16).
As an addition to CcpA-mediated repression, further regulation of many genes involved in the metabolism of nonglucose sugars occurs via sugar-specific transcriptional regulators that respond to induction with the sugar of interest (14, 44, 60). MalR, the putative transcriptional regulator encoded immediately adjacent to MalE (Fig. 2), is a putative member of the LacI family of transcriptional repressors and may be responsible for malE repression in the absence of maltose or another inducing agent (40). The fact that malE transcript levels during growth in saliva are lower in the stationary phase compared to the exponential phase suggests that consumption and/or degradation of the inducing substance(s) is occurring (Fig. 3). Whether maltose or some other substance acts as to induce malE transcription during growth in saliva is currently being examined.
The high transcript levels of malE during growth in saliva suggested that MalE was important for optimal GAS-saliva interaction. Indeed, we determined that the growth of the malE isogenic mutant strain in saliva was significantly lower than growth of the wild type (Fig. 3). Previous investigations have shown that the well-described GAS virulence factors streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB) are also needed for the optimum growth of strain MGAS5005 in human saliva (48). Presumably Sic and SpeB are important for GAS-saliva interaction due to their ability to inactivate innate immune peptides (13, 46). The data generated during this investigation suggest that MalE is critical to GAS-saliva interaction through its contribution to nutrient uptake. Given that saliva has relatively low glucose levels, it seems reasonable to speculate that MalE is needed for GAS to utilize other carbon sources present in human saliva (21, 48). This hypothesis is supported by our data demonstrating that adding exogenous glucose to saliva abrogated the growth defect of the malE isogenic mutant strain.
Having found that MalE is important for the growth of GAS in human saliva ex vivo, we tested the in vivo contribution of MalE to GAS pathogenesis using a mouse oropharynx colonization model. In our study the MalE-deficient strain colonized the mouse oropharynx at a lower level than did the wild type within 24 h of inoculation. This finding suggests that MalE is important at the earliest stages of GAS colonization. That MalE might participate during the initial processes of GAS host-pathogen interaction is supported by the high malE gene transcript level early during GAS-saliva interaction and the finding of dynamic malE gene transcript levels during the colonization stage in the non-human primate oropharynx (49, 61).
There are multiple pathways by which MalE may contribute to the ability of GAS to colonize the oropharynx. Presently, we know that MalE is critical for the utilization of maltose by GAS. As many bacterial maltose-binding proteins also bind longer maltodextrins (e.g., maltotriose and maltotetraose), it is likely that MalE mediates the uptake of carbohydrates in addition to maltose (11, 39). Therefore, one possibility is that maltodextrins are critical carbon sources for GAS in the oropharynx. Such a supposition is supported by the fact that -amylase, an enzyme that breaks down starch into maltodextrins, has the highest levels of all human enzymes in saliva (21). Moreover, GAS produces at least two extracellular starch-degrading enzymes, a cell-surface-attached pullulanase and an actively secreted cyclomaltodextrin glucanotransferase (12, 22, 29). Thus, GAS and human enzymes present in the oropharynx are capable of creating nutrients whose utilization are at least partially mediated by MalE.
In addition to directly affecting growth, nutrient acquisition may also influence the production of virulence factors required for optimal colonization/infection of the oropharynx (49). Investigations in S. pneumoniae and Vibrio cholerae have shown that deletions in carbon metabolism genes, including the maltose operon, led to decreased production of such well-known virulence factors as capsular polysaccharide and cholera toxin (15, 26). Similarly, inactivation of a two-component gene regulatory system (sptR/S) affecting carbohydrate metabolism in GAS resulted in lower gene transcript levels of several key virulence factors including sic and speB during interaction with human saliva (49). Analysis of the key GAS transcriptional regulator mga promoter region shows that a cre site is present, suggesting a direct mechanism by which carbohydrate levels might influence a broad array of virulence factors (10). Therefore, nutrient uptake mediated by MalE may be critical not only for growth but also for adequate production of classical virulence factors needed for pharyngeal colonization.
Recently there has been increased appreciation of the role of basic metabolic processes in bacterial pathogenesis. The ability of Mycobacterium tuberculosis to create latent infection was shown to be affected by inactivation of isocitrate lyase, an enzyme involved in the glyoxylate shunt pathway (32). In an organism more closely related to GAS, two independent investigations have shown that CcpA was key to the virulence of S. pneumoniae in a bacteremia and a nasopharyngeal colonization model (15, 23). The importance of CcpA is thought to be due to its contribution to the preferential utilization of carbon sources (59). Importantly, the S. pneumoniae MalE homologue was one of the eight proteins identified as differentially regulated by CcpA, suggesting that differences in virulence seen in a CcpA-deficient S. pneumoniae strain might be at least partially secondary to effects on MalE (23).
In conclusion, we used results of transcriptome analysis to focus on the putative maltodextrin-binding protein MalE. Our data demonstrate that MalE is central to the ability of GAS to utilize maltose and participates in GAS colonization of the oropharynx. These findings lay the groundwork for a new line of investigation into GAS pathogenesis, namely, how carbohydrate utilization contributes to the ability of GAS to infect humans. As many of the nutrient acquisition mechanisms utilized by GAS appear to be shared by other human mucosal pathogens, such studies may yield broadly applicable insights into microbial host-pathogen interaction.
ACKNOWLEDGMENTS
This work was supported by American Heart Association grant 0565133Y (S.A.S.) and National Institutes of Health grant K12 RR 17655-04 (S.A.S.).
FOOTNOTES
Corresponding author. Mailing address: Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, TX 77025. Phone: (713) 441-5890. Fax: (713) 790-6460. E-mail: jmmusser@tmh.tmc.edu.
REFERENCES
1. Amerongen, A. V., and E. C. Veerman. 2002. Saliva—the defender of the oral cavity. Oral Dis. 8:12-22.
2. Banks, D. J., S. F. Porcella, K. D. Barbian, S. B. Beres, L. E. Philips, J. M. Voyich, F. R. DeLeo, J. M. Martin, G. A. Somerville, and J. M. Musser. 2004. Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain. J. Infect. Dis. 190:727-738.
3. Beres, S. B., E. W. Richter, M. J. Nagiec, P. Sumby, S. F. Porcella, F. R. DeLeo, and J. M. Musser. 2006. Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus. Proc. Natl. Acad. Sci. USA 103:7059-7064.
4. Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M. Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, D. S. Campbell, T. M. Smith, J. K. McCormick, D. Y. Leung, P. M. Schlievert, and J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99:10078-10083.
5. Boos, W., and H. Shuman. 1998. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol. Mol. Biol. Rev. 62:204-229.
6. Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685-694.
7. Chaussee, M. S., R. O. Watson, J. C. Smoot, and J. M. Musser. 2001. Identification of Rgg-regulated exoproteins of Streptococcus pyogenes. Infect. Immun. 69:822-831.
8. Courtney, H. S., and D. L. Hasty. 1991. Aggregation of group A streptococci by human saliva and effect of saliva on streptococcal adherence to host cells. Infect. Immun. 59:1661-1666.
9. Dalton, T. L., and J. R. Scott. 2004. CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J. Bacteriol. 186:3928-3937.
10. Deutscher, J., R. Herro, A. Bourand, I. Mijakovic, and S. Poncet. 2005. P-Ser-HPr—a link between carbon metabolism and the virulence of some pathogenic bacteria. Biochim. Biophys. Acta 1754:118-125.
11. Dippel, R., and W. Boos. 2005. The maltodextrin system of Escherichia coli: metabolism and transport. J. Bacteriol. 187:8322-8331.
12. Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658-4663.
13. Frick, I. M., P. Akesson, M. Rasmussen, A. Schmidtchen, and L. Bjorck. 2003. SIC, a secreted protein of Streptococcus pyogenes that inactivates antibacterial peptides. J. Biol. Chem. 278:16561-16566.
14. Gering, M., and R. Bruckner. 1996. Transcriptional regulation of the sucrase gene of Staphylococcus xylosus by the repressor ScrR. J. Bacteriol. 178:462-469.
15. Giammarinaro, P., and J. C. Paton. 2002. Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae. Infect. Immun. 70:5454-5461.
16. Graham, M. R., K. Virtaneva, S. F. Porcella, W. T. Barry, B. B. Gowen, C. R. Johnson, F. A. Wright, and J. M. Musser. 2005. Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies. Am. J. Pathol. 166:455-465.
17. Green, N. M., S. Zhang, S. F. Porcella, M. J. Nagiec, K. D. Barbian, S. B. Beres, R. B. Lefebvre, and J. M. Musser. 2005. Genome sequence of a serotype M28 strain of group A Streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity. J. Infect. Dis. 192:760-770.
18. Holland, I. B., and M. A. Blight. 1999. ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. J. Mol. Biol. 293:381-399.
19. Hueck, C. J., and W. Hillen. 1995. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria Mol. Microbiol. 15:395-401.
20. Hufnagel, M., S. Koch, R. Creti, L. Baldassarri, and J. Huebner. 2004. A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice. J. Infect. Dis. 189:420-430.
21. Humphrey, S. P., and R. T. Williamson. 2001. A review of saliva: normal composition, flow, and function. J. Prosthet. Dent. 85:162-169.
22. Hytonen, J., S. Haataja, and J. Finne. 2003. Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase. Infect. Immun. 71:784-793.
23. Iyer, R., N. S. Baliga, and A. Camilli. 2005. Catabolite control protein A (CcpA) contributes to virulence and regulation of sugar metabolism in Streptococcus pneumoniae. J. Bacteriol. 187:8340-8349.
24. Jones, A. L., K. M. Knoll, and C. E. Rubens. 2000. Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis. Mol. Microbiol. 37:1444-1455.
25. Kuwayama, H., S. Obara, T. Morio, M. Katoh, H. Urushihara, and Y. Tanaka. 2002. PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res. 30:e2.
26. Lang, H., G. Jonson, J. Holmgren, and E. T. Palva. 1994. The maltose regulon of Vibrio cholerae affects production and secretion of virulence factors. Infect. Immun. 62:4781-4788.
27. Lee, M. H., A. Nittayajarn, R. P. Ross, C. B. Rothschild, D. Parsonage, A. Claiborne, and C. E. Rubens. 1999. Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins. J. Bacteriol. 181:5790-5799.
28. Lei, B., M. Liu, G. L. Chesney, and J. M. Musser. 2004. Identification of new candidate vaccine antigens made by Streptococcus pyogenes: purification and characterization of 16 putative extracellular lipoproteins. J. Infect. Dis. 189:79-89.
29. Lei, B., S. Mackie, S. Lukomski, and J. M. Musser. 2000. Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68:6807-6818.
30. Lukomski, S., N. P. Hoe, I. Abdi, J. Rurangirwa, P. Kordari, M. Liu, S. J. Dou, G. G. Adams, and J. M. Musser. 2000. Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Infect. Immun. 68:535-542.
31. Matsumoto, M., N. P. Hoe, M. Liu, S. B. Beres, G. L. Sylva, C. M. Brandt, G. Haase, and J. M. Musser. 2003. Intrahost sequence variation in the streptococcal inhibitor of complement gene in patients with human pharyngitis. J. Infect. Dis. 187:604-612.
32. McKinney, J. D., K. Honer zu Bentrup, E. J. Munoz-Elias, A. Miczak, B. Chen, W. T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs, Jr., and D. G. Russell. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735-738.
33. Munoz-Elias, E. J., and J. D. McKinney. 2005. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11:638-644.
34. Nakagawa, I., K. Kurokawa, A. Yamashita, M. Nakata, Y. Tomiyasu, N. Okahashi, S. Kawabata, K. Yamazaki, T. Shiba, T. Yasunaga, H. Hayashi, M. Hattori, and S. Hamada. 2003. Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution. Genome Res. 13:1042-1055.
35. Nelson, D., L. Loomis, and V. A. Fischetti. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc. Natl. Acad. Sci. USA 98:4107-4112.
36. Orihuela, C. J., J. N. Radin, J. E. Sublett, G. Gao, D. Kaushal, and E. I. Tuomanen. 2004. Microarray analysis of pneumococcal gene expression during invasive disease. Infect. Immun. 72:5582-5596.
37. Parkhill, J., G. Dougan, K. D. James, N. R. Thomson, D. Pickard, J. Wain, C. Churcher, K. L. Mungall, S. D. Bentley, M. T. Holden, M. Sebaihia, S. Baker, D. Basham, K. Brooks, T. Chillingworth, P. Connerton, A. Cronin, P. Davis, R. M. Davies, L. Dowd, N. White, J. Farrar, T. Feltwell, N. Hamlin, A. Haque, T. T. Hien, S. Holroyd, K. Jagels, A. Krogh, T. S. Larsen, S. Leather, S. Moule, P. O'Gaora, C. Parry, M. Quail, K. Rutherford, M. Simmonds, J. Skelton, K. Stevens, S. Whitehead, and B. G. Barrell. 2001. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413:848-852.
38. Peter, G., and A. L. Smith. 1977. Group A streptococcal infections of the skin and pharynx (second of two parts). N. Engl. J. Med. 297:365-370.
39. Puyet, A., and M. Espinosa. 1993. Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae. Correlation to the Escherichia coli maltose regulon. J. Mol. Biol. 230:800-811.
40. Puyet, A., A. M. Ibanez, and M. Espinosa. 1993. Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features. J. Biol. Chem. 268:25402-25408.
41. Reda, K. B., V. Kapur, J. A. Mollick, J. G. Lamphear, J. M. Musser, and R. R. Rich. 1994. Molecular characterization and phylogenetic distribution of the streptococcal superantigen gene (ssa) from Streptococcus pyogenes. Infect. Immun. 62:1867-1874.
42. Reid, S. D., N. M. Green, G. L. Sylva, J. M. Voyich, E. T. Stenseth, F. R. DeLeo, T. Palzkill, D. E. Low, H. R. Hill, and J. M. Musser. 2002. Postgenomic analysis of four novel antigens of group A Streptococcus: growth phase-dependent gene transcription and human serologic response. J. Bacteriol. 184:6316-6324.
43. Sabharwal, H., F. Michon, D. Nelson, W. Dong, K. Fuchs, R. C. Manjarrez, A. Sarkar, C. Uitz, A. Viteri-Jackson, R. S. R. Suarez, M. Blake, and J. B. Zabriskie. 2006. Group A Streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection. J. Infect. Dis. 193:129-135.
44. Saier, M. H., Jr., S. Chauvaux, G. M. Cook, J. Deutscher, I. T. Paulsen, J. Reizer, and J. J. Ye. 1996. Catabolite repression and inducer control in gram-positive bacteria. Microbiology 142:217-230.
45. Scheffel, F., R. Fleischer, and E. Schneider. 2004. Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius. Biochim. Biophys. Acta 1656:57-65.
46. Schmidtchen, A., I. M. Frick, E. Andersson, H. Tapper, and L. Bjorck. 2002. Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Mol. Microbiol. 46:157-168.
47. Schumann, G. B. 1994. Clinical utility of body fluid analyses: an overview. Clin. Lab. Sci. 7:28-31.
48. Shelburne, S. A., III, C. Granville, M. Tokuyama, I. Sitkiewicz, P. Patel, and J. M. Musser. 2005. Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva. Infect. Immun. 73:4723-4731.
49. Shelburne, S. A., P. Sumby, I. Sitkiewicz, C. N. Granville, F. R. DeLeo, and J. M. Musser. 2005. Central role of a two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva. Proc. Natl. Acad. Sci. USA 102:16037-16042.
50. Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee, G. L. Sylva, D. E. Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins, S. B. Beres, D. S. Campbell, T. M. Smith, Q. Zhang, V. Kapur, J. A. Daly, L. G. Veasy, and J. M. Musser. 2002. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc. Natl. Acad. Sci. USA 99:4668-4673.
51. Sumby, P., K. D. Barbian, D. J. Gardner, A. R. Whitney, D. M. Welty, R. D. Long, J. R. Bailey, M. J. Parnell, N. P. Hoe, G. G. Adams, F. R. Deleo, and J. M. Musser. 2005. Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc. Natl. Acad. Sci. USA 102:1679-1684.
52. Sumby, P., S. F. Porcella, A. G. Madrigal, K. D. Barbian, K. Virtaneva, S. M. Ricklefs, D. E. Sturdevant, M. R. Graham, J. Vuopio-Varkila, N. P. Hoe, and J. M. Musser. 2005. Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events. J. Infect. Dis. 192:771-782.
53. Sutcliffe, I. C., and D. J. Harrington. 2002. Pattern searches for the identification of putative lipoprotein genes in gram-positive bacterial genomes. Microbiology 148:2065-2077.
54. Tchawa Yimga, M., M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen. 2006. Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. Infect. Immun. 74:1130-1140.
55. Tenovuo, J. 2002. Antimicrobial agents in saliva—protection for the whole body. J. Dent. Res. 81:807-809.
56. Tettelin, H., V. Masignani, M. J. Cieslewicz, C. Donati, D. Medini, N. L. Ward, S. V. Angiuoli, J. Crabtree, A. L. Jones, A. S. Durkin, R. T. Deboy, T. M. Davidsen, M. Mora, M. Scarselli, I. Margarit y Ros, J. D. Peterson, C. R. Hauser, J. P. Sundaram, W. C. Nelson, R. Madupu, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, S. A. Sullivan, S. C. Daugherty, D. H. Haft, J. Selengut, M. L. Gwinn, L. Zhou, N. Zafar, H. Khouri, D. Radune, G. Dimitrov, K. Watkins, K. J. O'Connor, S. Smith, T. R. Utterback, O. White, C. E. Rubens, G. Grandi, L. C. Madoff, D. L. Kasper, J. L. Telford, M. R. Wessels, R. Rappuoli, and C. M. Fraser. 2005. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome". Proc. Natl. Acad. Sci. USA 102:13950-13955.
57. Tettelin, H., V. Masignani, M. J. Cieslewicz, J. A. Eisen, S. Peterson, M. R. Wessels, I. T. Paulsen, K. E. Nelson, I. Margarit, T. D. Read, L. C. Madoff, A. M. Wolf, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. T. DeBoy, A. S. Durkin, J. F. Kolonay, R. Madupu, M. R. Lewis, D. Radune, N. B. Fedorova, D. Scanlan, H. Khouri, S. Mulligan, H. A. Carty, R. T. Cline, S. E. Van Aken, J. Gill, M. Scarselli, M. Mora, E. T. Iacobini, C. Brettoni, G. Galli, M. Mariani, F. Vegni, D. Maione, D. Rinaudo, R. Rappuoli, J. L. Telford, D. L. Kasper, G. Grandi, and C. M. Fraser. 2002. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl. Acad. Sci. USA 99:12391-12396.
58. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson, J. Heidelberg, R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M. Gwinn, J. F. Kolonay, W. C. Nelson, J. D. Peterson, L. A. Umayam, O. White, S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H. Khouri, A. M. Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V. Feldblyum, S. Angiuoli, T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus, F. Yang, H. O. Smith, J. C. Venter, B. A. Dougherty, D. A. Morrison, S. K. Hollingshead, and C. M. Fraser. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498-506.
59. Titgemeyer, F., and W. Hillen. 2002. Global control of sugar metabolism: a gram-positive solution. Antonie Leeuwenhoek 82:59-71.
60. van Wezel, G. P., J. White, P. Young, P. W. Postma, and M. J. Bibb. 1997. Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes. Mol. Microbiol. 23:537-549.
61. Virtaneva, K., S. F. Porcella, M. R. Graham, R. M. Ireland, C. A. Johnson, S. M. Ricklefs, I. Babar, L. D. Parkins, R. A. Romero, G. J. Corn, D. J. Gardner, J. R. Bailey, M. J. Parnell, and J. M. Musser. 2005. Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques. Proc. Natl. Acad. Sci. USA 102:9014-9019.
62. Vise, P. D., K. Kodali, N. Hoe, A. Paszczynski, J. M. Musser, and G. W. Daughdrill. 2003. Stable isotope labeling of a group A Streptococcus virulence factor using a chemically defined growth medium. Protein Expr. Purif. 32:232-238.(Samuel A. Shelburne III, Paul Sumby, Iza)
Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, Texas 77030
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
Analysis of multiple group A Streptococcus (GAS) genomes shows that genes encoding proteins involved in carbohydrate utilization comprise some 15% of the core GAS genome. Yet there is a limited understanding of how carbohydrate utilization contributes to GAS pathogenesis. Previous genome-wide GAS studies led us to a focused investigation of MalE, a putative maltodextrin-binding protein. Analysis of 28 strains of 22 distinct M protein serotypes showed that MalE is highly conserved among diverse GAS strains. malE transcript levels were significantly increased during growth in human saliva compared to growth in a chemically defined glucose-containing medium or a nutrient-rich medium. MalE was accessible to antibody binding, indicating that it is expressed on the GAS cell surface. Moreover, growth in human saliva appeared to increase MalE surface expression compared to growth in a nutrient-rich medium. Analysis of a malE isogenic mutant strain revealed decreased growth in human saliva compared to wild-type GAS. Radiolabeled carbohydrate binding assays showed that MalE was required for the binding of maltose but not glucose. The malE isogenic mutant strain colonized a lower percentage of GAS-challenged mice compared to wild-type and genetically complemented strains. Furthermore, decreased numbers of CFU were recovered from mice infected with the malE strain compared to those infected with wild-type GAS. These data demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Further investigation into carbohydrate transport and metabolism pathways may yield novel insights into GAS pathogenesis.
INTRODUCTION
Many human bacterial pathogens devote large portions of their genome to genes encoding known or putative proteins involved in carbohydrate transport and metabolism (12, 37, 58). Therefore, it is reasonable to speculate that the utilization of carbohydrates is essential to the ability of pathogenic bacteria to cause disease in humans. Indeed, recent investigations in diverse human pathogens have demonstrated the vital contribution of carbohydrate metabolism to microbial pathogenesis (20, 23, 33, 54). Carbohydrate transport and metabolism genes are often conserved among pathogenic bacteria (12, 56, 58). Consequently, an increased understanding of specific carbohydrate metabolism and transport pathways may lead to insights into shared mechanisms by which a broad range of microbes infect humans.
Group A Streptococcus (GAS) causes an assortment of human diseases ranging from uncomplicated pharyngeal and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome (6). Similar to other human-adapted pathogens, genes encoding proteins putatively involved in carbohydrate transport and metabolism make up about 15% of the core GAS genome (2, 4, 52, 57, 58). Although they account for a substantial portion of the GAS genome, however, there has been a relative dearth of research into how the utilization and transport of carbohydrates contribute to GAS pathogenesis (22, 42).
The mucosal surface of the human oropharynx is a major site of GAS colonization and infection (38). To successfully colonize and infect the human oropharynx, GAS must overcome multiple aspects of the acquired and innate immune systems including secretory immunoglobulin, antimicrobial peptides, and a limited nutrient supply (1, 8, 48, 55). Recently, we have begun to examine the interaction of GAS and human saliva, a key mediator of acquired and innate immunity in the human oropharynx (48). We employed expression microarray analysis to generate a genome-wide view of GAS gene transcript levels during interaction with human saliva (49). The transcript levels of genes encoding known and putative proteins involved in carbohydrate transport and metabolism were markedly elevated early in the GAS-saliva interaction. Analysis of the GAS transcriptome during experimental pharyngitis in non-human primates similarly showed that carbohydrate metabolism gene transcript levels were abundant during the initial colonization phase (61). Taken together, these data suggest that genes involved in carbohydrate metabolism and transport may be central to early steps in GAS pharyngeal colonization and infection.
Previously, two putative GAS carbohydrate transport proteins were shown to be immunogenic in humans recovering from GAS infection and in mice experimentally infected with GAS (28). One of these proteins, M5005_Spy_1058, is a putative maltodextrin-binding protein that has been annotated as MalE based on homology with the MalE protein encoded by Escherichia coli (5, 52). Genome-wide transcriptome analyses of GAS in human blood and in human saliva showed marked upregulation of malE (16, 49). The high transcript levels of malE during GAS-human fluid interaction suggest that MalE may be critical to GAS during colonization or infection of humans. In this work we sought to characterize the cellular location, transcriptional regulation, and contribution to GAS growth of MalE. We also tested the hypothesis that MalE participates in the ability of GAS to colonize the oropharynx of mice. Our findings suggest that maltodextrin transport via MalE is a key component of the initial colonization of the oropharynx by GAS.
MATERIALS AND METHODS
Bacterial strains and culture media. The genome of serotype M1 strain MGAS5005 has been fully sequenced (52). This strain has been extensively used for in vitro and animal models of GAS infection including transcriptome analysis during growth in human saliva (49, 51, 52, 61). The other bacterial strains used in this study are listed in Table 1. GAS was grown on Trypticase soy agar containing 5% bovine serum albumin (BSA; Becton Dickinson) or in Todd-Hewitt broth containing 0.2% (wt/vol) yeast extract (THY; Difco). Carbohydrates were added at a 1.0% (wt/vol) concentration to a carbohydrate-free preparation of a commercially available chemically defined medium (CDM; JR Biosciences) (62). For this article, we will use glucose medium to refer to the carbohydrate-free CDM with 1% glucose added and maltose medium to refer to the carbohydrate-free CDM with 1% maltose added. Spectinomycin (Sigma) was added to THY agar or broth at 150 μg/ml, and chloramphenicol (Sigma) was added at 4 μg/ml to spectinomycin THY agar or broth when appropriate. Blue-white screening was performed by adding 50 μg/ml of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) to the chloramphenicol-spectinomycin THY agar.
Growth of GAS in human saliva. GAS was grown in human saliva as previously described (48). Saliva was collected on ice from healthy volunteers as previously described under a Baylor College of Medicine Institutional Review Board human subjects protocol (48). Pooled saliva collected from at least four donors was used to minimize the effects of donor variation on study results.
DNA sequence analysis. Chromosomal DNA was isolated using a phenol-chloroform extraction method as previously described (41). DNA sequencing primers were designed on the basis of the serotype M1 strain MGAS5005 genome (Table 2) (52). The malE open reading frame M5005_Spy_1058 corresponds to open reading frame SPy1294 in serotype M1 strain SF370 (12). Sequence data obtained from both DNA strands with an Applied Biosystems 3700 automated sequencer were assembled with Sequencher 4.5. Comparative sequence alignment of the inferred amino acid sequences was performed with CLUSTALW.
RNA isolation and transcript level analysis. Serotype M1 strain MGAS5005 was grown in various media to early- and late-logarithmic growth phases. RNA was isolated with a Fast Prep Blue Kit (Q/BioGene) and purified using an RNeasy kit (QIAGEN) as previously described (49). The concentration and quality of RNA were assessed with an Agilent 2100 Bioanalyzer and analysis of the A260/A280 ratio. cDNAs were created from the RNA using SuperscriptIII (Invitrogen) according to the manufacturer's instructions. TaqMan real-time quantitative reverse transcription-PCR (QRT-PCR) was performed in quadruplicate with an ABI Thermocycler 7700 (Applied Biosystems) using the change in cycle threshold for analysis, as previously described (49). TaqMan primers and probes for malE and the internal control gene proS are listed in Table 2.
Detection of MalE on the GAS cell surface. Surface localization of MalE was assessed with a FACSCaliber flow cytometer (BD Biosciences) using affinity-purified MalE-specific antibodies (Bethyl Laboratories) (28, 42). Antibody against streptococcal phospholipase A2 (SlaA), a GAS protein not encoded by strain MGAS5005, was used as a control for nonspecific antibody binding. Strain MGAS5005 and the malE isogenic mutant strain (see below) were grown to exponential phase in either THY medium or human saliva, harvested by centrifugation, washed once in Dulbecco's phosphate-buffered saline (PBS), pH 7.2, and suspended in Dulbecco's PBS at 108 CFU/ml. Anti-MalE or anti-SlaA antibody was added at a final concentration of 0.05 μg/100 μl to 100 μl of bacterial suspension and incubated for 30 min on ice. Samples were washed with Pharmingen stain buffer (BD Biosciences) and stained with phycoerythrin-conjugated donkey anti-rabbit immunoglobulin G (Jackson ImmunoResearch) (1:100 dilution) for 30 min on ice. The cells were washed again with stain buffer and analyzed via flow cytometry.
Creation of malE isogenic mutant strain. The malE isogenic mutant strain was created from parental serotype M1 strain MGAS5005 using the PCR-mediated method of Kuwayama et al. (25). Primers were designed to amplify the 5' and 3' ends of the malE gene region along with nucleotide sequences that were complementary to the 5' or 3' portion of the spectinomycin (spc) resistance cassette (Table 2). A third set of primers with nucleotide sequences complementary to the 5' and 3' ends of the malE gene region was used to amplify the spc cassette from plasmid pSL60 (30). Fusion PCR was then used to link the 5' and 3' malE gene region PCR products to the spc cassette via the overlapping nucleotide sequence regions (9, 25). This resulted in a PCR product where the spc cassette, flanked by the 5' and 3' malE gene regions, was now inserted in the place of nearly the entire malE open reading frame. The gene disruption PCR construct was used to transform competent GAS cells with spectinomycin resistance used as the selection mechanism. The malE isogenic mutant strain was analyzed by Southern hybridization and DNA sequencing to confirm that the proper genetic construct was obtained (Fig. 1).
Complementation of malE isogenic mutant strain. For complementation purposes, we utilized plasmid pDC123 which includes the chloramphenicol acetyltransferase (cat) gene and the phoZ gene that allows for blue-white screening via alkaline phosphatase activity (27). The complete malE gene along with 250 bp upstream and 15 bp downstream was amplified from strain MGAS5005 chromosomal DNA using primers that introduced EcoR1-cut sites at the 5' and 3' ends (Table 2). The resulting PCR product was digested with EcoR1 (New England Biolabs) and cloned into the EcoR1 site of plasmid pDC123. The resulting plasmid, named pDCmalE, was used to transform competent malE cells. Chloramphenicol resistance and blue-white screening were used to choose transformed strains for further analysis. Presence of the desired pDCmalE construct was confirmed using PCR and DNA sequencing. pDC123 lacking the malE gene was used to transform strain MGAS5005 and the malE isogenic mutant strain as controls.
Carbohydrate binding assays. Strain MGAS5005, the malE isogenic mutant strain, and the compmalE (complemented mutant) strain were grown in either glucose or maltose medium to mid-exponential phase, collected by centrifugation, washed, and suspended to an optical density at 600 nm (OD600) of 0.5 in 150 μl of CDM lacking carbohydrates. A total of 25 μl of 280 μM 1.85 MBq [14C]maltose or [14C]glucose (GE Healthcare) was added to obtain a final concentration of 40 μM radiolabeled carbohydrate. At various times 40-μl samples were removed and filtered on a 0.45-μm-pore-size nitrocellulose membrane (Millipore) which was then rinsed twice with CDM lacking carbohydrates. The radioactivity trapped on each filter was then determined by liquid scintillation counting (Beckman Model LS7500). Samples were taken every minute for the first 4 min during which carbohydrate binding rates were found to be linear. The amount of protein in the cultures was determined by Bradford assay (Bio-Rad). All experiments were performed in quadruplicate.
Mouse colonization experiments. All animal experiments were performed under a protocol approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Mouse throat colonization studies were conducted with adult (18 to 20 g) female outbred CD-1 Swiss mice (Harlan Sprague-Dawley, Inc.) as described previously (30). Strain MGAS5005, the malE isogenic mutant strain, and the compmalE strain were grown in THY medium and harvested at an OD600 of 0.5. The cells were washed once and suspended in sterile, pyrogen-free PBS to a density of 1.5 x 108 CFU in 100 μl. Both nares of each mouse were inoculated with 50 μl of the GAS suspension. This dose of MGAS5005 has been previously shown to result in the colonization of 75% of inoculated animals (30). The mouse throats were swabbed prior to inoculation and then daily thereafter. Throat swabs were plated onto BSA and grown overnight, and beta-hemolytic colonies were tested for the presence of GAS carbohydrate antigen via latex agglutination (BD Biosciences). Blood collected from dead animals by cardiac puncture was cultured on BSA. In addition, compmalE colonies recovered from colonized and dead mice were tested for loss of the complementary plasmid by culturing in THY broth with chloramphenicol.
Statistical analysis. Flow cytometry and RNA transcript level comparisons were performed using a Student's two-sided t test. Growth comparisons were done using analysis of variance. Radiolabeled carbohydrate binding rates were compared using linear regression. The chi-square test was used to assess statistical differences in throat colonization rates among the animal groups infected with strain MGAS5005, its malE isogenic mutant derivative, and the compmalE strain. Analysis of variance was used to test for differences in number of CFU recovered from animals infected with the three strains. Statistical significance was assigned at a two-sided P value of 0.05 using Bonferroni's adjustment for multiple comparisons when appropriate. Statistical calculations were performed with NCSS software, version 2004 (Kaysville, Utah).
RESULTS
Description of the malE gene region and homology of MalE among various GAS strains. MalE has been identified as a putative lipoprotein based on the presence and composition of its amino-terminal secretion signal sequence (28, 53). Analysis of the serotype M1 strain MGAS5005 genome (52) shows that downstream of malE are two genes, malF and malG, whose products are predicted to form a permease for solute transport (Fig. 2). Together with MalE, MalF and MalG are predicted to comprise two parts of an ATP binding cassette transport system, a common mechanism by which bacteria import substrates (18). The putative ATP-binding portion of the ATP binding cassette transport system is located outside of the malE genome region, an arrangement that is not unusual in gram-positive bacteria (39, 45). Upstream of malE and divergently transcribed are malR, which encodes a putative transcriptional regulator, and malP and malQ, which encode enzymes putatively involved in generating various forms of glucose from maltodextrins (Fig. 2). The identical gene arrangement is present in all 12 GAS strains sequenced to date (2-4, 12, 17, 34, 50).
MalE is the only protein encoded by this gene region that is putatively surface exposed and thus possibly under selective pressure from the host immune system. Some GAS surface-exposed proteins are highly variable among various strains, whereas others are well conserved (31, 42). We sequenced the malE gene from 28 strains comprising 22 different M serotypes (Table 1). The malE gene was present in all strains tested. There was an average of 3.2 amino acid replacements per 415 amino acid sites (Table 1). These data show that MalE is highly conserved among diverse GAS strains, indicating that information generated regarding MalE function in a particular GAS strain may be broadly applicable to diverse GAS strains.
malE transcript level is increased in maltose medium compared to glucose medium and nutrient-rich medium. Many bacteria employ carbon catabolite repression, a phenomenon where the use of sugars other than glucose is minimized when glucose is available (19). By adding specific sugars to carbohydrate-free CDM, we tested the hypothesis that growth in maltose medium would lead to increased transcript levels of malE compared to growth in THY or glucose medium. As assessed by real-time QRT-PCR, during the mid-exponential phase of growth the transcript level of malE in the maltose medium showed a 35-fold increase compared with the glucose medium and 95-fold increase compared with the THY medium (P < 0.001 for both) (Fig. 3). At entry into stationary phase, the malE transcript level was fourfold and threefold higher in the maltose medium compared to the glucose and THY media, respectively (P = 0.024 and P = 0.008). These data show that a significant increase in malE transcript level occurred during growth in a medium containing maltose, suggesting that malE transcription is repressed in a nutrient-rich environment but induced in the presence of maltose.
malE transcript level is increased during growth in human saliva compared to growth in a nutrient-rich medium. Previous expression microarray analysis found that malE transcript levels were high during growth of GAS in human saliva, especially in the exponential growth phase (49). Thus, we tested the hypothesis that the interaction of GAS with saliva leads to increased malE transcript levels compared to growth in a nutrient-rich medium. Similar to the situation during growth in maltose, the malE transcript level in saliva at mid-exponential phase was 45-fold higher than growth in glucose medium and 120-fold higher than growth in THY medium (P < 0.001 for each) (Fig. 3). Upon entry into stationary phase in saliva, the transcript levels of malE decreased but remained 13-fold and 10-fold higher than growth in glucose or THY medium, respectively (P < 0.001 for each). Taken together, we conclude that growth of GAS human saliva led to a significant increase in the malE transcript level, suggesting that MalE is important for the successful interaction of GAS with human saliva.
Growth in human saliva increased the amount of MalE on the GAS cell surface. We used flow cytometry to test two hypotheses. First, we predicted that MalE would be expressed on the GAS cell surface. Second, we expected that the increased transcript levels of malE during growth in human saliva would result in increased MalE on the GAS cell surface compared to growth in THY medium. We used anti-streptococcal phospholipase A2 (SlaA) antibody as the control antibody because SlaA is not produced by strain MGAS5005 (4, 52). The flow cytometry data showed no significant increase above the control level in the amount of bound anti-MalE when the malE isogenic mutant strain was examined, suggesting that the anti-MalE antibody is specific for MalE (Fig. 4A). When wild-type strain MGAS5005 was studied, there was an increase in binding above the control level for anti-MalE during the mid-exponential growth phase in THY medium (Fig. 4B). Moreover, there was an increase in bound anti-MalE at mid-exponential phase during growth in saliva compared to growth in THY medium (Fig. 4C) (P < 0.001). These data are consistent with the in silico analysis predicting that MalE is present on the GAS cell surface and therefore accessible to antibody. Also, the increased transcript level of malE observed during growth in saliva appeared to result in increased MalE levels on the GAS cell surface.
The malE isogenic mutant strain had decreased growth in maltose medium compared to wild-type. To directly test the role played by MalE in various GAS activities, we created a nonpolar malE isogenic mutant strain from parental M1 strain MGAS5005 and genetically complemented the mutant strain to create strain compmalE (see Material and Methods). We tested the hypothesis that MalE is important for the utilization of maltose but not glucose by comparing the growth patterns of wild-type GAS, the malE isogenic mutant strain, and the compmalE strain in various media. There was no growth for any strain in the carbohydrate-free CDM when no exogenous carbohydrate was added (data not shown). We observed no differences when the three strains were grown in THY medium or during growth in glucose medium (Fig. 5A, 5B) (P = 0.791 for THY; P = 0.972 for glucose medium). However, the malE isogenic mutant strain was significantly less able to grow in maltose medium compared to the wild-type and complemented strains (Fig. 5C) (P = 0.001). There was no significant difference in growth between the wild-type and the compmalE strain during growth in maltose medium (P = 0.611). These data show that MalE is needed for GAS to optimally grow in maltose medium.
The malE isogenic mutant strain grew less in human saliva compared to wild type. A key finding of our earlier GAS-saliva investigations was that diverse GAS strains were readily able to grow in human saliva and, contrary to growth in laboratory media, persisted at maximal CFU counts for up to 30 days (48). The observation that the malE transcript level increased 100-fold during growth in saliva compared to growth in THY medium suggested that, similar to its role in a maltose medium, MalE might be critical to the ability of GAS to grow in human saliva. We tested this hypothesis by comparing the growth of strain MGAS5005, the malE isogenic mutant strain, and the compmalE strain in saliva pooled from healthy donors. As previously described, GAS saliva growth experiments are performed using CFU analysis rather than density readings because saliva can aggregate GAS and thereby interfere with OD readings (8, 48). Whereas both the wild-type and complemented mutant strains grew 2.5 log10 CFU/ml from baseline and persisted at a density of 1 x 107 CFU/ml, the malE isogenic mutant strain only grew 1.0 log10 CFU/ml and persisted at 5.5 x 105 CFU/ml (Fig. 5D) (P value for growth = 0.002; P value for persistence = 0.007). The addition of glucose at 100 mg/dl to the saliva growth medium restored the growth of the malE isogenic mutant strain to wild-type levels (data not shown). Therefore, we conclude that MalE is key to the optimal growth and persistence of GAS in human saliva.
The malE isogenic mutant strain was deficient in maltose, but not glucose, binding. In earlier portions of this work, we found that MalE was expressed on the GAS cell surface and that a malE isogenic mutant strain grew poorly in maltose medium. To test the hypothesis that MalE mediates maltose transport, we studied GAS binding of 14C-radiolabeled carbohydrates. We found no difference among the ability of the wild-type, malE, or compmalE strains to bind glucose (P = 0.541) (Fig. 6A). In contrast, in comparison to the wild-type and the complemented malE strain, the malE mutant strain showed a 95% decrease in maltose binding (P < 0.001) (Fig. 6B). These results show that the presence of MalE is required for maximal binding of maltose by GAS but not for the binding of glucose. These findings are in concordance with the earlier data demonstrating that MalE is needed for the optimum growth of GAS in maltose medium but not in glucose medium.
The malE isogenic mutant strain was unable to efficiently colonize the mouse oropharynx. Although mice are not normally susceptible to pharyngeal infection by GAS, temporary colonization of the mouse oropharynx occurs following nasopharyngeal inoculation (30, 35, 43). We used adult outbred CD-1 mice to test the hypothesis that MalE is needed for optimum GAS colonization of the oropharynx. Each group of 30 mice was challenged intranasally with 1.5 x 108 CFU GAS in a total of 100 μl (50 μl per nostril). The oropharynx of each mouse was then swabbed daily using a fine-tip, sterile cotton applicator (Fisher), which was then used to inoculate a BSA plate. The plates were incubated for 24 h and examined for beta-hemolytic colonies, which were tested for the presence of GAS carbohydrate antigen via latex agglutination (BD Biosciences). At each time point tested, significantly more mice were colonized with the MGAS5005 and the compmalE strains compared to the malE isogenic mutant strain (Fig. 7A). Moreover, at each time point tested, the number of GAS CFU was significantly higher in the animals infected with the wild-type and complemented strains compared to the isogenic mutant strain (Fig. 7B). These data support our hypothesis that MalE is critical in the ability of GAS to successfully colonize the oropharynx.
As we were unable to maintain chloramphenicol selection on the compmalE strain during infection, we assayed for loss of the complementary plasmid. A total of 43 of 50 colonies recovered from animals infected with the complemented malE strain remained chloramphenicol resistant. Therefore, even in the absence of chloramphenicol pressure, the majority of compmalE strains maintained the pDCmalE plasmid, providing further evidence of the critical nature of MalE during oropharyngeal colonization.
DISCUSSION
We recently initiated a genome-wide investigation of GAS-human saliva interaction to gain insight into how unstudied areas of the GAS genome contribute to GAS pathogenesis (48, 49). Analysis of the GAS transcriptome in saliva combined with other recently published data led us to focus on the gene/gene product M5005_Spy_1058 or MalE (16, 28, 49, 61). Previous genome-wide investigations showed that malE was highly transcribed during GAS interaction with human blood and had dynamic transcript levels during the colonization of the non-human primate oropharynx (16, 61). Moreover, MalE is known to be immunogenic in mice and humans infected with GAS (28). Finally, genome-wide investigations of pathogens related to GAS, i.e., Streptococcus pneumoniae and group B Streptococcus, also suggested that MalE homologues might be involved in bacterial pathogenesis (24, 36).
We first determined whether MalE is highly conserved among diverse GAS strains. The publication of multiple full-length GAS genomes has led to the observation that about 85 to 90% of the GAS genome is shared by various strains while the remaining 10 to 15% is highly variable (4, 17, 50). To be broadly applicable to GAS pathogenesis, a gene/gene product needs to be present in the majority of infecting GAS strains. Our analysis of 28 strains of 22 distinct M serotypes shows that the malE gene was present among all tested strains. Also, the MalE amino acid sequence was highly conserved among diverse strains (Table 1). Therefore, our findings regarding the role played by MalE in a particular GAS strain are likely to be relevant to the broader GAS population.
Like many other bacteria, GAS encodes a homologue of catabolite control protein A (CcpA) (19, 23, 52). The preferential utilization of glucose in low G+C gram-positive bacteria is thought to be at least partially mediated by CcpA acting at catabolite responsive element (cre) sites (44). Our findings of low malE transcript levels in the presence of glucose or in a nutrient-rich medium (THY) suggest that malE is subject to such repression. Glucose levels in saliva are very low; therefore, glucose-mediated catabolite repression is unlikely to occur during GAS-saliva interaction (47). However, the importance of glucose-mediated catabolite repression in malE transcription is debatable as malE transcript levels were previously shown to be quite high during growth in human blood, a glucose-rich medium (16).
As an addition to CcpA-mediated repression, further regulation of many genes involved in the metabolism of nonglucose sugars occurs via sugar-specific transcriptional regulators that respond to induction with the sugar of interest (14, 44, 60). MalR, the putative transcriptional regulator encoded immediately adjacent to MalE (Fig. 2), is a putative member of the LacI family of transcriptional repressors and may be responsible for malE repression in the absence of maltose or another inducing agent (40). The fact that malE transcript levels during growth in saliva are lower in the stationary phase compared to the exponential phase suggests that consumption and/or degradation of the inducing substance(s) is occurring (Fig. 3). Whether maltose or some other substance acts as to induce malE transcription during growth in saliva is currently being examined.
The high transcript levels of malE during growth in saliva suggested that MalE was important for optimal GAS-saliva interaction. Indeed, we determined that the growth of the malE isogenic mutant strain in saliva was significantly lower than growth of the wild type (Fig. 3). Previous investigations have shown that the well-described GAS virulence factors streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB) are also needed for the optimum growth of strain MGAS5005 in human saliva (48). Presumably Sic and SpeB are important for GAS-saliva interaction due to their ability to inactivate innate immune peptides (13, 46). The data generated during this investigation suggest that MalE is critical to GAS-saliva interaction through its contribution to nutrient uptake. Given that saliva has relatively low glucose levels, it seems reasonable to speculate that MalE is needed for GAS to utilize other carbon sources present in human saliva (21, 48). This hypothesis is supported by our data demonstrating that adding exogenous glucose to saliva abrogated the growth defect of the malE isogenic mutant strain.
Having found that MalE is important for the growth of GAS in human saliva ex vivo, we tested the in vivo contribution of MalE to GAS pathogenesis using a mouse oropharynx colonization model. In our study the MalE-deficient strain colonized the mouse oropharynx at a lower level than did the wild type within 24 h of inoculation. This finding suggests that MalE is important at the earliest stages of GAS colonization. That MalE might participate during the initial processes of GAS host-pathogen interaction is supported by the high malE gene transcript level early during GAS-saliva interaction and the finding of dynamic malE gene transcript levels during the colonization stage in the non-human primate oropharynx (49, 61).
There are multiple pathways by which MalE may contribute to the ability of GAS to colonize the oropharynx. Presently, we know that MalE is critical for the utilization of maltose by GAS. As many bacterial maltose-binding proteins also bind longer maltodextrins (e.g., maltotriose and maltotetraose), it is likely that MalE mediates the uptake of carbohydrates in addition to maltose (11, 39). Therefore, one possibility is that maltodextrins are critical carbon sources for GAS in the oropharynx. Such a supposition is supported by the fact that -amylase, an enzyme that breaks down starch into maltodextrins, has the highest levels of all human enzymes in saliva (21). Moreover, GAS produces at least two extracellular starch-degrading enzymes, a cell-surface-attached pullulanase and an actively secreted cyclomaltodextrin glucanotransferase (12, 22, 29). Thus, GAS and human enzymes present in the oropharynx are capable of creating nutrients whose utilization are at least partially mediated by MalE.
In addition to directly affecting growth, nutrient acquisition may also influence the production of virulence factors required for optimal colonization/infection of the oropharynx (49). Investigations in S. pneumoniae and Vibrio cholerae have shown that deletions in carbon metabolism genes, including the maltose operon, led to decreased production of such well-known virulence factors as capsular polysaccharide and cholera toxin (15, 26). Similarly, inactivation of a two-component gene regulatory system (sptR/S) affecting carbohydrate metabolism in GAS resulted in lower gene transcript levels of several key virulence factors including sic and speB during interaction with human saliva (49). Analysis of the key GAS transcriptional regulator mga promoter region shows that a cre site is present, suggesting a direct mechanism by which carbohydrate levels might influence a broad array of virulence factors (10). Therefore, nutrient uptake mediated by MalE may be critical not only for growth but also for adequate production of classical virulence factors needed for pharyngeal colonization.
Recently there has been increased appreciation of the role of basic metabolic processes in bacterial pathogenesis. The ability of Mycobacterium tuberculosis to create latent infection was shown to be affected by inactivation of isocitrate lyase, an enzyme involved in the glyoxylate shunt pathway (32). In an organism more closely related to GAS, two independent investigations have shown that CcpA was key to the virulence of S. pneumoniae in a bacteremia and a nasopharyngeal colonization model (15, 23). The importance of CcpA is thought to be due to its contribution to the preferential utilization of carbon sources (59). Importantly, the S. pneumoniae MalE homologue was one of the eight proteins identified as differentially regulated by CcpA, suggesting that differences in virulence seen in a CcpA-deficient S. pneumoniae strain might be at least partially secondary to effects on MalE (23).
In conclusion, we used results of transcriptome analysis to focus on the putative maltodextrin-binding protein MalE. Our data demonstrate that MalE is central to the ability of GAS to utilize maltose and participates in GAS colonization of the oropharynx. These findings lay the groundwork for a new line of investigation into GAS pathogenesis, namely, how carbohydrate utilization contributes to the ability of GAS to infect humans. As many of the nutrient acquisition mechanisms utilized by GAS appear to be shared by other human mucosal pathogens, such studies may yield broadly applicable insights into microbial host-pathogen interaction.
ACKNOWLEDGMENTS
This work was supported by American Heart Association grant 0565133Y (S.A.S.) and National Institutes of Health grant K12 RR 17655-04 (S.A.S.).
FOOTNOTES
Corresponding author. Mailing address: Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, TX 77025. Phone: (713) 441-5890. Fax: (713) 790-6460. E-mail: jmmusser@tmh.tmc.edu.
REFERENCES
1. Amerongen, A. V., and E. C. Veerman. 2002. Saliva—the defender of the oral cavity. Oral Dis. 8:12-22.
2. Banks, D. J., S. F. Porcella, K. D. Barbian, S. B. Beres, L. E. Philips, J. M. Voyich, F. R. DeLeo, J. M. Martin, G. A. Somerville, and J. M. Musser. 2004. Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain. J. Infect. Dis. 190:727-738.
3. Beres, S. B., E. W. Richter, M. J. Nagiec, P. Sumby, S. F. Porcella, F. R. DeLeo, and J. M. Musser. 2006. Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus. Proc. Natl. Acad. Sci. USA 103:7059-7064.
4. Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M. Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, D. S. Campbell, T. M. Smith, J. K. McCormick, D. Y. Leung, P. M. Schlievert, and J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99:10078-10083.
5. Boos, W., and H. Shuman. 1998. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol. Mol. Biol. Rev. 62:204-229.
6. Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685-694.
7. Chaussee, M. S., R. O. Watson, J. C. Smoot, and J. M. Musser. 2001. Identification of Rgg-regulated exoproteins of Streptococcus pyogenes. Infect. Immun. 69:822-831.
8. Courtney, H. S., and D. L. Hasty. 1991. Aggregation of group A streptococci by human saliva and effect of saliva on streptococcal adherence to host cells. Infect. Immun. 59:1661-1666.
9. Dalton, T. L., and J. R. Scott. 2004. CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J. Bacteriol. 186:3928-3937.
10. Deutscher, J., R. Herro, A. Bourand, I. Mijakovic, and S. Poncet. 2005. P-Ser-HPr—a link between carbon metabolism and the virulence of some pathogenic bacteria. Biochim. Biophys. Acta 1754:118-125.
11. Dippel, R., and W. Boos. 2005. The maltodextrin system of Escherichia coli: metabolism and transport. J. Bacteriol. 187:8322-8331.
12. Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658-4663.
13. Frick, I. M., P. Akesson, M. Rasmussen, A. Schmidtchen, and L. Bjorck. 2003. SIC, a secreted protein of Streptococcus pyogenes that inactivates antibacterial peptides. J. Biol. Chem. 278:16561-16566.
14. Gering, M., and R. Bruckner. 1996. Transcriptional regulation of the sucrase gene of Staphylococcus xylosus by the repressor ScrR. J. Bacteriol. 178:462-469.
15. Giammarinaro, P., and J. C. Paton. 2002. Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae. Infect. Immun. 70:5454-5461.
16. Graham, M. R., K. Virtaneva, S. F. Porcella, W. T. Barry, B. B. Gowen, C. R. Johnson, F. A. Wright, and J. M. Musser. 2005. Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies. Am. J. Pathol. 166:455-465.
17. Green, N. M., S. Zhang, S. F. Porcella, M. J. Nagiec, K. D. Barbian, S. B. Beres, R. B. Lefebvre, and J. M. Musser. 2005. Genome sequence of a serotype M28 strain of group A Streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity. J. Infect. Dis. 192:760-770.
18. Holland, I. B., and M. A. Blight. 1999. ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. J. Mol. Biol. 293:381-399.
19. Hueck, C. J., and W. Hillen. 1995. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria Mol. Microbiol. 15:395-401.
20. Hufnagel, M., S. Koch, R. Creti, L. Baldassarri, and J. Huebner. 2004. A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice. J. Infect. Dis. 189:420-430.
21. Humphrey, S. P., and R. T. Williamson. 2001. A review of saliva: normal composition, flow, and function. J. Prosthet. Dent. 85:162-169.
22. Hytonen, J., S. Haataja, and J. Finne. 2003. Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase. Infect. Immun. 71:784-793.
23. Iyer, R., N. S. Baliga, and A. Camilli. 2005. Catabolite control protein A (CcpA) contributes to virulence and regulation of sugar metabolism in Streptococcus pneumoniae. J. Bacteriol. 187:8340-8349.
24. Jones, A. L., K. M. Knoll, and C. E. Rubens. 2000. Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis. Mol. Microbiol. 37:1444-1455.
25. Kuwayama, H., S. Obara, T. Morio, M. Katoh, H. Urushihara, and Y. Tanaka. 2002. PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res. 30:e2.
26. Lang, H., G. Jonson, J. Holmgren, and E. T. Palva. 1994. The maltose regulon of Vibrio cholerae affects production and secretion of virulence factors. Infect. Immun. 62:4781-4788.
27. Lee, M. H., A. Nittayajarn, R. P. Ross, C. B. Rothschild, D. Parsonage, A. Claiborne, and C. E. Rubens. 1999. Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins. J. Bacteriol. 181:5790-5799.
28. Lei, B., M. Liu, G. L. Chesney, and J. M. Musser. 2004. Identification of new candidate vaccine antigens made by Streptococcus pyogenes: purification and characterization of 16 putative extracellular lipoproteins. J. Infect. Dis. 189:79-89.
29. Lei, B., S. Mackie, S. Lukomski, and J. M. Musser. 2000. Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68:6807-6818.
30. Lukomski, S., N. P. Hoe, I. Abdi, J. Rurangirwa, P. Kordari, M. Liu, S. J. Dou, G. G. Adams, and J. M. Musser. 2000. Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Infect. Immun. 68:535-542.
31. Matsumoto, M., N. P. Hoe, M. Liu, S. B. Beres, G. L. Sylva, C. M. Brandt, G. Haase, and J. M. Musser. 2003. Intrahost sequence variation in the streptococcal inhibitor of complement gene in patients with human pharyngitis. J. Infect. Dis. 187:604-612.
32. McKinney, J. D., K. Honer zu Bentrup, E. J. Munoz-Elias, A. Miczak, B. Chen, W. T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs, Jr., and D. G. Russell. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735-738.
33. Munoz-Elias, E. J., and J. D. McKinney. 2005. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11:638-644.
34. Nakagawa, I., K. Kurokawa, A. Yamashita, M. Nakata, Y. Tomiyasu, N. Okahashi, S. Kawabata, K. Yamazaki, T. Shiba, T. Yasunaga, H. Hayashi, M. Hattori, and S. Hamada. 2003. Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution. Genome Res. 13:1042-1055.
35. Nelson, D., L. Loomis, and V. A. Fischetti. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc. Natl. Acad. Sci. USA 98:4107-4112.
36. Orihuela, C. J., J. N. Radin, J. E. Sublett, G. Gao, D. Kaushal, and E. I. Tuomanen. 2004. Microarray analysis of pneumococcal gene expression during invasive disease. Infect. Immun. 72:5582-5596.
37. Parkhill, J., G. Dougan, K. D. James, N. R. Thomson, D. Pickard, J. Wain, C. Churcher, K. L. Mungall, S. D. Bentley, M. T. Holden, M. Sebaihia, S. Baker, D. Basham, K. Brooks, T. Chillingworth, P. Connerton, A. Cronin, P. Davis, R. M. Davies, L. Dowd, N. White, J. Farrar, T. Feltwell, N. Hamlin, A. Haque, T. T. Hien, S. Holroyd, K. Jagels, A. Krogh, T. S. Larsen, S. Leather, S. Moule, P. O'Gaora, C. Parry, M. Quail, K. Rutherford, M. Simmonds, J. Skelton, K. Stevens, S. Whitehead, and B. G. Barrell. 2001. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413:848-852.
38. Peter, G., and A. L. Smith. 1977. Group A streptococcal infections of the skin and pharynx (second of two parts). N. Engl. J. Med. 297:365-370.
39. Puyet, A., and M. Espinosa. 1993. Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae. Correlation to the Escherichia coli maltose regulon. J. Mol. Biol. 230:800-811.
40. Puyet, A., A. M. Ibanez, and M. Espinosa. 1993. Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features. J. Biol. Chem. 268:25402-25408.
41. Reda, K. B., V. Kapur, J. A. Mollick, J. G. Lamphear, J. M. Musser, and R. R. Rich. 1994. Molecular characterization and phylogenetic distribution of the streptococcal superantigen gene (ssa) from Streptococcus pyogenes. Infect. Immun. 62:1867-1874.
42. Reid, S. D., N. M. Green, G. L. Sylva, J. M. Voyich, E. T. Stenseth, F. R. DeLeo, T. Palzkill, D. E. Low, H. R. Hill, and J. M. Musser. 2002. Postgenomic analysis of four novel antigens of group A Streptococcus: growth phase-dependent gene transcription and human serologic response. J. Bacteriol. 184:6316-6324.
43. Sabharwal, H., F. Michon, D. Nelson, W. Dong, K. Fuchs, R. C. Manjarrez, A. Sarkar, C. Uitz, A. Viteri-Jackson, R. S. R. Suarez, M. Blake, and J. B. Zabriskie. 2006. Group A Streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection. J. Infect. Dis. 193:129-135.
44. Saier, M. H., Jr., S. Chauvaux, G. M. Cook, J. Deutscher, I. T. Paulsen, J. Reizer, and J. J. Ye. 1996. Catabolite repression and inducer control in gram-positive bacteria. Microbiology 142:217-230.
45. Scheffel, F., R. Fleischer, and E. Schneider. 2004. Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius. Biochim. Biophys. Acta 1656:57-65.
46. Schmidtchen, A., I. M. Frick, E. Andersson, H. Tapper, and L. Bjorck. 2002. Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Mol. Microbiol. 46:157-168.
47. Schumann, G. B. 1994. Clinical utility of body fluid analyses: an overview. Clin. Lab. Sci. 7:28-31.
48. Shelburne, S. A., III, C. Granville, M. Tokuyama, I. Sitkiewicz, P. Patel, and J. M. Musser. 2005. Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva. Infect. Immun. 73:4723-4731.
49. Shelburne, S. A., P. Sumby, I. Sitkiewicz, C. N. Granville, F. R. DeLeo, and J. M. Musser. 2005. Central role of a two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva. Proc. Natl. Acad. Sci. USA 102:16037-16042.
50. Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee, G. L. Sylva, D. E. Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins, S. B. Beres, D. S. Campbell, T. M. Smith, Q. Zhang, V. Kapur, J. A. Daly, L. G. Veasy, and J. M. Musser. 2002. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc. Natl. Acad. Sci. USA 99:4668-4673.
51. Sumby, P., K. D. Barbian, D. J. Gardner, A. R. Whitney, D. M. Welty, R. D. Long, J. R. Bailey, M. J. Parnell, N. P. Hoe, G. G. Adams, F. R. Deleo, and J. M. Musser. 2005. Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc. Natl. Acad. Sci. USA 102:1679-1684.
52. Sumby, P., S. F. Porcella, A. G. Madrigal, K. D. Barbian, K. Virtaneva, S. M. Ricklefs, D. E. Sturdevant, M. R. Graham, J. Vuopio-Varkila, N. P. Hoe, and J. M. Musser. 2005. Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events. J. Infect. Dis. 192:771-782.
53. Sutcliffe, I. C., and D. J. Harrington. 2002. Pattern searches for the identification of putative lipoprotein genes in gram-positive bacterial genomes. Microbiology 148:2065-2077.
54. Tchawa Yimga, M., M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen. 2006. Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. Infect. Immun. 74:1130-1140.
55. Tenovuo, J. 2002. Antimicrobial agents in saliva—protection for the whole body. J. Dent. Res. 81:807-809.
56. Tettelin, H., V. Masignani, M. J. Cieslewicz, C. Donati, D. Medini, N. L. Ward, S. V. Angiuoli, J. Crabtree, A. L. Jones, A. S. Durkin, R. T. Deboy, T. M. Davidsen, M. Mora, M. Scarselli, I. Margarit y Ros, J. D. Peterson, C. R. Hauser, J. P. Sundaram, W. C. Nelson, R. Madupu, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, S. A. Sullivan, S. C. Daugherty, D. H. Haft, J. Selengut, M. L. Gwinn, L. Zhou, N. Zafar, H. Khouri, D. Radune, G. Dimitrov, K. Watkins, K. J. O'Connor, S. Smith, T. R. Utterback, O. White, C. E. Rubens, G. Grandi, L. C. Madoff, D. L. Kasper, J. L. Telford, M. R. Wessels, R. Rappuoli, and C. M. Fraser. 2005. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome". Proc. Natl. Acad. Sci. USA 102:13950-13955.
57. Tettelin, H., V. Masignani, M. J. Cieslewicz, J. A. Eisen, S. Peterson, M. R. Wessels, I. T. Paulsen, K. E. Nelson, I. Margarit, T. D. Read, L. C. Madoff, A. M. Wolf, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. T. DeBoy, A. S. Durkin, J. F. Kolonay, R. Madupu, M. R. Lewis, D. Radune, N. B. Fedorova, D. Scanlan, H. Khouri, S. Mulligan, H. A. Carty, R. T. Cline, S. E. Van Aken, J. Gill, M. Scarselli, M. Mora, E. T. Iacobini, C. Brettoni, G. Galli, M. Mariani, F. Vegni, D. Maione, D. Rinaudo, R. Rappuoli, J. L. Telford, D. L. Kasper, G. Grandi, and C. M. Fraser. 2002. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl. Acad. Sci. USA 99:12391-12396.
58. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson, J. Heidelberg, R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M. Gwinn, J. F. Kolonay, W. C. Nelson, J. D. Peterson, L. A. Umayam, O. White, S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H. Khouri, A. M. Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V. Feldblyum, S. Angiuoli, T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus, F. Yang, H. O. Smith, J. C. Venter, B. A. Dougherty, D. A. Morrison, S. K. Hollingshead, and C. M. Fraser. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498-506.
59. Titgemeyer, F., and W. Hillen. 2002. Global control of sugar metabolism: a gram-positive solution. Antonie Leeuwenhoek 82:59-71.
60. van Wezel, G. P., J. White, P. Young, P. W. Postma, and M. J. Bibb. 1997. Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes. Mol. Microbiol. 23:537-549.
61. Virtaneva, K., S. F. Porcella, M. R. Graham, R. M. Ireland, C. A. Johnson, S. M. Ricklefs, I. Babar, L. D. Parkins, R. A. Romero, G. J. Corn, D. J. Gardner, J. R. Bailey, M. J. Parnell, and J. M. Musser. 2005. Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques. Proc. Natl. Acad. Sci. USA 102:9014-9019.
62. Vise, P. D., K. Kodali, N. Hoe, A. Paszczynski, J. M. Musser, and G. W. Daughdrill. 2003. Stable isotope labeling of a group A Streptococcus virulence factor using a chemically defined growth medium. Protein Expr. Purif. 32:232-238.(Samuel A. Shelburne III, Paul Sumby, Iza)