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Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, Montana
Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, Houston, Texas
Department of Pathology, Microbiology, and Immunology, University of CaliforniaDavis, Davis
Puerperal sepsis, a major cause of death of young women in Europe in the 1800s, was due predominantly to the gram-positive pathogen group A Streptococcus. Studies conducted during past decades have shown that serotype M28 strains are the major group A Streptococcus organisms responsible for many of these infections. To begin to increase our understanding of their enrichment in puerperal sepsis, we sequenced the genome of a genetically representative strain. This strain has genes encoding a novel array of prophage virulence factors, cell-surface proteins, and other molecules likely to contribute to host-pathogen interactions. Importantly, genes for 7 inferred extracellular proteins are encoded by a 37.4-kb foreign DNA element that is shared with group B Streptococcus and is present in all serotype M28 strains. Proteins encoded by the 37.4-kb element were expressed extracellularly and in human infections. Acquisition of foreign genes has helped create a disease-specialist clone of this pathogen.
Puerperal sepsis, also known as "childbed fever," killed as many as 1 in every 6 mothers who gave birth in some hospitals in Europe in the mid-1880s [1]. The study of puerperal sepsis has an unusually rich history. Clinical investigation conducted in Austria in the 1840s by Semmelweis determined that puerperal sepsis was transmitted to pregnant women in labor by attending doctors [2]. These studies led to widespread implementation of infection-control measures such as hand washing, thereby changing the course of medical history. Subsequent studies [37] resulted in the understanding that puerperal sepsis is frequently caused by the human pathogenic bacterium group A Streptococcus (GAS).
Strains of GAS are categorized into serotypes on the basis of variation in the M protein, a highly polymorphic cell-surface molecule that is antiphagocytic and, hence, is a virulence factor [8, 9]. More than 125 distinct M protein types and emm gene types are known [10]. Epidemiologic studies conducted over many decades have repeatedly identified significant associations between certain M protein types and human infections [1114]. For example, serotype M28 GAS strains are overrepresented among cases of puerperal sepsis and neonatal GAS infections [11, 12, 1518]. Type M28 strains also are common causes of other types of invasive infections and pharyngitis in many countries [11, 12, 15, 1729].
The molecular mechanisms responsible for the enrichment of serotype M28 strains in these infections are not known because of a general lack of understanding of the pathogenic processes underlying intraspecies disease specificity. As a first step toward gaining new insight into the microbial factors that contribute to infection specificity in GAS, we sequenced the genome of a serotype M28 strain. Our findings suggest that the acquisition of genes encoding novel extracellular proteins has helped create a disease specialist clone of GAS, thereby broadening the ecological niche of this pathogen and modifying characteristics of infection.
MATERIALS AND METHODS
Genome sequencing.
The genome of emm28 strain MGAS6180 was sequenced to a minimum consensus base quality of Q40 by methods used for several other bacteria, including GAS [3033]. This strain was isolated from a case of invasive infection in Texas in the 1990s. This strain is genetically representative of common serotype M28 strains, as assessed by the emm28 allele and the spectrum of prophage-associated virulence genes. The genome was tiled by polymerase chain reaction (PCR) after closure to validate the assembly. Open-reading frames (ORFs) were identified with proprietary software (Integrated Genomics), annotated, and analyzed with the ERGO bioinformatics suite [34]. Strain MGAS6180 has been deposited in the American Type Culture Collection (BAA-1064), and the genome sequence has been deposited in GenBank (accession number SP_MGAS6180 CP000056).
Analysis of a novel foreign genetic element by PCR tiling.
PCR was used to screen diverse GAS strains for a 37.4-kb novel foreign genetic element, designated "region of difference 2" (RD2) (see Results), identified in the genome of strain MGAS6180 (table 1). The presence and genome context of RD2 was assessed by use of 12 pairs of PCR primers (table 2) that amplify a 40-kb region encompassing RD2 and flanking chromosomal genes in strain MGAS6180.
Analysis of prophage elements in natural populations of serotype M28 GAS from different localities and diseases.
A 2-step PCR-based method was used to determine the presence and genome location of prophages and prophage-associated virulence factor genes, as described elsewhere [3638]. A random sample of 114 strains cultured from patients with pharyngitis and invasive infections in Texas, Ontario, and Finland was analyzed. The combination of prophage-associated virulence factor genes was defined as the virulence factor gene profile.
Overexpression and purification of recombinant proteins.
The gene segments encoding the inferred mature forms of novel extracellular proteins were cloned by standard techniques. Five segments of the gene encoding M28_Spy1325 also were cloned by standard techniques. All cloned gene segments were sequenced to rule out the presence of spurious mutations. The recombinant proteins were purified to apparent homogeneity. Detailed protocols can be obtained from the corresponding author by request.
Western immunoblot analysis of serum samples.
Serum samples were obtained from patients with pharyngitis infections caused by serotype M28 strains of GAS. Acute serum samples were obtained at the time of presentation with illness; convalescent serum samples were obtained 3 weeks after treatment.
RESULTS
Overview of the Genome Sequence of Strain MGAS6180 and Comparison with Other Published GAS Genome Sequences
The sequenced genome of strain MGAS6180 is a circular chromosome of 1,897,573 bp with a guanine and cytosine (GC) content of 38.4% (figure 1). The GC content of the genome is essentially identical to that of all other GAS strains whose genomes have been sequenced (range, 38.5% 38.7%) [33, 3943]. There are 1903 predicted coding sequences, which make up 1,675,787 kb (88.3%) of the genome. The gene context and content of the "core" chromosome (i.e., the part of the genome that does not include prophage-like and obvious foreign elements) is very similar to those described for strains SF370 and MGAS5005 (serotype M1), MGAS10394 (serotype M6), MGAS8232 (serotype M18), and MGAS315 (serotype M3) and SSI-1 (serotype M3) [33, 3942]. The genome contains genes that encode many proven or putative secreted virulence factors, including streptolysin O, streptolysin S, pyrogenic toxin superantigens (streptococcal pyrogenic exotoxin C , SpeK, SpeJ, and SmeZ), a secreted phospholipase A2 (SlaA), collagen-like proteins (SclA and SclB), and proteases (SpeB, Mac, and ScpA) [9, 4446].
Putative Prophages or Prophage-Like Elements
Four regions of the genome contain prophage-like elements, or apparent remnants of prophage-like elements (figure 1), which vary in size from 11.8 to 46.3 kb (for ease of description and discussion, these elements will be referred to as "prophages," with the understanding that none of them has been shown to be a bona fide prophage). The 4 prophages are integrated in the half of the chromosome distal to the origin of replication, similar to the great majority of GAS prophages (figure 2). Prophage element DNA makes up 114,704 bp (6.0%) of the chromosome, which is less than in strains SF370 (7.1%), MGAS8232 (10.8%), and MGAS315 and SSI-1 (12.4%) [45].
Prophage 6180.1.
Prophage 6180.1 (46.2 kb) has contiguous genes encoding SpeC and an extracellular DNase (Spd1) and is inserted at the predicted T12att site, the same location reported for prophages 315.2 (serotype M3), 8232.3 (serotype M18), and 10394.3 (serotype M6). However, each of these 4 prophage elements (6180.1, 315.2, 8232.3, and 10394.3) encodes different proven or putative extracellular virulence factors (figures 2 and 3).
Prophage 6180.2.
Prophage 6180.2 (42.3 kb) is inserted at a site analogous to the location of 315.4 in serotype M3 strain MGAS315; like 315.4, it has contiguous genes encoding SpeK and the recently discovered SlaA. Prophages 6180.2 and 315.4 are closely related in overall gene content and nucleotide sequence (figure 3)evidence that these prophages have shared a very recent common ancestor.
Prophage remnant 6180.3.
Prophage remnant 6180.3 (14.3 kb) is closely similar in size and nucleotide sequence to 370.4 in strain SF370 and to 10394.8 in serotype M6 strain MGAS10394 (figure 3), and it is located at the analogous integration site. These 3 elements lack a gene encoding a proven or putative virulence factor [33, 39]. Together, these characteristics indicate that 10394.8, 370.4, and 6180.3 have undoubtedly shared a common ancestor.
Prophage remnant 6180.4.
Prophage remnant 6180.4 (11.7 kb) has no close relationship, overall, with prophages found in previously sequenced GAS genomes. 6180.4 does not encode apparent virulence factors.
Serum Opacity Factor (SOF) Gene Region
SOF is an extracellular lipoproteinase that makes human serum opaque, binds to fibronectin and fibrinogen, and is a virulence factor in mice infected with serotype M2 GAS [4749]. GAS strains historically have been divided into 2 groups (SOF-positive and SOF-negative strains) on the basis of their ability to make human serum opaque. The sof gene (3018 bp) in strain MGAS6180 is located on an 5.3-kb chromosomal segment between homologues of the spy2034 and spy2037 genes of serotype M1 strain SF370 [50]. This genome segment also includes a 1947-bp gene (sfbX) that encodes a fibronectin-binding protein. The location and orientation of the sof gene region in strain MGAS6180 is analogous to that described in emm12, emm49, emm75, emm87, emm92, and emm114 GAS strains [50].
Streptin Gene Cluster Region
Streptin is a type A1 lantibiotic produced by certain GAS strains, including type emm28 organisms [51, 52]. The genome of strain MGAS6180 has a 10-ORF locus (figure 4) that is closely similar in nucleotide sequence to an analogous region that has recently been characterized in a GAS strain of undescribed emm type [53]. In contrast to serotype M1 strain SF370, all 10 ORFs appear to be intact, which is consistent with the observation that emm28 strains express lantibiotic activity.
Two Nonprophage Regions with a Unique Sequence
The genome of strain MGAS6180 has 2 regions of >10 kb with unique, largely nonprophage DNA not found in other sequenced GAS genomes. We will refer to these chromosomal segments as "RD1" and "RD2." Like the 4 prophages, RD1 and RD2 are integrated in the half of the chromosome distal to the origin of replication (figure 1).
RD1.
RD1 (11.1 kb) is integrated into a uracil-methyltransferase tRNA and appears to be a chimeric element composed of genes that are related to those associated with plasmids and phages. This region does not encode apparent secreted or virulence-related proteins. RD1 is flanked by 8-bp direct repeat sequences (ACGTGATG), and the GC content of this element is 30.4%, well below the whole-genome value of 38.4%.
RD2.
RD2 (37.4 kb) is integrated in tRNA-Thr, is flanked by 16-bp direct repeats, and has 7 ORFs predicted to encode proteins with gram-positive secretion signal sequences (figures 1 and 5). Importantly, RD2 is similar in overall gene composition and sequence to regions described in the genome of serotype III and V group B Streptococcus (GBS) (table 3) [54, 55], a pathogen that is the primary cause of sterile-site neonatal infections. The GC content of RD2 (35.1%) is considerably less then the whole-genome value of 38.35% and closely approximates that of GBS (35.6% and 35.7%) [54, 55].
Four of these proteins associated with RD2 (M28_Spy1306, M28_Spy1326, M28_Spy1325, and M28_Spy1336) have an LPXTG motif located at the carboxy terminus that covalently anchors many proteins in gram-positive pathogens to the cell wall. M28_Spy1336 is the R28 protein that has been studied previously in GAS and GBS [5659], whereas the other 3 proteins with LPXTGE motifs have not been found previously in GAS. M28_Spy1306, an inferred 254-aa protein, is 98% identical to a protein of unknown function made by the 2 GBS strains whose genomes have been sequenced [54, 55]. The gene encoding this protein has a GC content of 40.1%, a value that exceeds that of the core genome of strain MGAS6180 (38.4%) and those of other GAS genomes (range, 38.538.7). M28_Spy1325 has a region rich in segments with Pro-Ala-Gly motifs and an LDV integrin-binding motif (figure 6). This protein is related to structurally complex members of the antigen I//II polypeptides produced by oral streptococci [60]. For example, M28_Spy1325 is 29% identical and 45% similar to extracellular protein SspB made by the oral organisms S. gordonii and S. sanguis. SspB, a multifunctional adhesin involved in bacterial binding and aggregation, has been studied extensively in these oral streptococci [6062]. The gene encoding SspB is differentially regulated in response to environmental cues, such as growth in human saliva, and is the subject of human vaccine interest [63]. M28_Spy1326 has an alanine-rich region and an inferred coil-coil motif, similar to regions of the M protein. This molecule may function as an adhesin.
Three inferred proteins (M28_Spy1332, M28_Spy1308, and M28_Spy1307) have a secretion signal sequence but lack an LPXTG motif, which suggests that they are located extracellularly. M28_Spy1332 encodes a hypothetical lipoprotein that is 100% identical to an inferred lipoprotein, designated "GBS0473," described in serotype III GBS [54, 64]. M28_Spy1308 encodes a putative extracellular protein with a conserved CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain. Members of the CHAP superfamily of proteins have been reported to be surface-exposed and important antigens in pathogenic bacteria [65, 66]. M28_Spy1307 encodes a protein that lacks similarity to known conserved functional domains.
Distribution of RD2 in Natural Populations of GAS
Strains of serotype M28 are abundant causes of maternal-neonatal invasive infections [11, 12, 1518, 27]. If the extracellular proteins encoded by RD2 contribute to this abundance, we hypothesized that RD2 would be widespread in natural populations of emm28 strains from diverse localities. To test this hypothesis, we used PCR to analyze 95 emm28 strains from the United States, Canada, and Finland for the presence of 6 of the genes encoding inferred extracellular proteins encoded by RD2 (table 1). All 95 strains tested were PCR positive for these 6 amplicons. To determine whether the entire 37.4-kb RD2 segment was present, 9 randomly chosen serotype M28 strains were studied by PCR tiling across the entire region. All 9 strains were PCR positive for these 12 amplicons, which indicates that all strains had RD2 and that it was located in the same chromosomal location (data not shown). Hence, RD2 is common in M28 strains of GAS.
Inasmuch as RD2 has characteristics that indicate it might be transferred horizontally, we next tested the hypothesis that this region was present in other GAS strains. A sample of 248 strains representing 76 non-emm28 types was studied (table 1). Sixty-four of 248 strains representing 14 emm types were PCR positive for 1 of the 6 genes associated with RD2 that encode inferred extracellular proteins. Furthermore, PCR tiling of a subset of these 64 strains revealed the entire RD2 region to be present in 14 strains representing types emm2, emm4, emm48, and emm77 (data not shown). In the case of emm53 and emm87, an RD2-like element was located at a different position in the genome than in the other emm types, although the exact location was not determined. Thus, although our survey was not exhaustive, we found that RD2 was associated with a distinct subset of GAS strains that includes emm types epidemiologically associated with maternal-fetal infections.
Diversity of Prophage-Associated Virulence Gene Profiles and Elements in Natural Populations of Serotype M28 GAS from Different Localities and Diseases
Strains of serotype M28 GAS have been reported to be genetically diverse [24, 25, 67]. Inasmuch as variation in prophage content and chromosomal site of integration are key contributors to genomic diversity among GAS of the same serotypes [45], we next tested the hypothesis that many distinct combinations of prophages would be present in M28 strains. A sample of 114 M28 strains cultured from patients with pharyngitis or invasive infections in the United States, Canada, and Finland was tested for 14 prophage-associated genes that encode extracellular proven or putative virulence factors. Consistent with the hypothesis, 21 distinct virulence gene profiles were identified among the 114 emm28 strains (table 4). The majority of strains (54%) had a profile that included only the speC and spd1 genes. The profile that included speC, spd1, and sdn was the second most abundant (11%), and the third most abundant profile included speC, spd1, slaA, and speK (8%). Strains with unique virulence factor gene profiles accounted for 11% of the 114 strains.
Characterization of Novel Extracellular Proteins Encoded by RD2
Given the importance of many extracellular proteins in GAS-host interactions [9, 46], we elected to clone the genes for inferred novel extracellular proteins encoded by RD2, purify the proteins, and assess evidence that some of them were produced in vivo. We were unable to clone M28_Spy1325 in its entirety, so 5 gene segments were cloned, and the recombinant proteins were purified to apparent homogeneity (figures 6 and 7). Sequence analysis of the amino terminus of each of the 8 recombinant proteins confirmed that the correct protein had been purified (data not shown).
Western immunoblotting was used to infer whether M28_Spy1332, M28_Spy1306, M28_Spy1326, and M28_Spy1325 were produced in vivo during human GAS infections. Convalescent (but not acute) serum samples obtained from patients with GAS infection were reactive to the proteins (figure 7), which indicates that these extracellular proteins were made in humans with GAS disease.
DISCUSSION
Comparative genomics of human bacterial pathogens.
Comparative genome sequencing of medically important pathogens has become an area of considerable interest [6870]. Six GAS genome sequences are available publicly, which makes this one of the more deeply sequenced pathogenic bacterial pathogens. One critical theme that has emerged is that each genome has genes encoding previously undescribed extracellular proteins that influence human-GAS interaction. For example, several novel exotoxin genes were identified in the genome sequences of serotype M1, M3, and M18 strains, and the genome sequence of the serotype M6 strain revealed a new extracellular cell-wallanchored protein [33, 3943]. Similarly, the serotype M28 strain that we sequenced had at least 6 genes for novel inferred extracellular proteins that may participate in host-pathogen interaction. Discovery of these many new accessory virulence genes in GAS provides an important motivation for ongoing, in-depth analysis of the population genomics of this pathogen. Inasmuch as there are >125 emm types of GAS, largely reflecting distinct clones, we believe that much remains to be learned about novel extracellular proteins used by GAS to cause human disease [10, 71].
Strain variation in prophage-associated virulence gene content.
We identified 21 distinct combinations of prophage-associated virulence genes among the 114 emm28 strains studied (table 4). These results are consistent with data from serotype M1, M3, M6, and M18 strains that have shown that prophages are responsible for the majority of variation in gene content among strains of the same M protein serotype [33, 38, 40, 41, 45].
Potential new insights into puerperal sepsis: RD2, lateral gene transfer, and the molecular genetic basis of bacterial disease specificity.
A key discovery of our research was the identification of a 37.4-kb DNA segment that encodes 7 inferred extracellular proteins, including 4 with an LPXTG carboxy terminal motif characteristic of cell-surface anchored proteins. This region was not present in previously sequenced GAS genomes. Importantly, RD2 is related to regions found in the genomes of GBS (table 3), a pathogen that is the primary cause of invasive infections in neonates [54, 55].
One of the extracellular proteins encoded by RD2 (the R28 protein) promotes the adhesion of GAS to human epithelial cells grown in vitro and confers protective immunity in a mouse model of invasive diseasethese findings have led to speculation that the R28 protein participates in the pathogenesis of puerperal sepsis [5659]. Our discovery of RD2 and the multiple extracellular proteins encoded by it potentially adds a substantial new perspective to our view of the molecular factors that may contribute to the enrichment of emm28 strains in neonatal-maternal, urogenital, and perineal infections. Although the R28 protein functions as an epithelial cell adhesin in vitro, there was no significant difference in mouse virulence between the wild-type M28 strain and an isogenic mutant in which the R28 gene was genetically inactivated [58, 59]. It will be important to dissect the contribution to GAS pathogenesis of each of the extracellular proteins encoded by RD2. Our findings also provide motivation for enhanced study of the homologous proteins produced by GBS. For example, it may be that these extracellular proteins contribute to enhanced colonization of the female urogenital tract.
Expression of prophage-encoded virulence genes can differ significantly as a function of variation in growth environment [72, 73]. This observation led to the proposal that GAS can respond to variable environments with condition-dependent expression of prophage-encoded virulence factors [72]. It is possible that the RD2 genes encoding extracellular proteins are contingency genes, analogous to prophage-encoded virulence genes. We hypothesize that production of the extracellular proteins encoded by genes in RD2 also is differentially regulated in response to changes in the host environment. For example, it may be that transcription of certain RD2 genes is stimulated by exposure to and growth in the human upper respiratory tract, whereas other genes are differentially expressed in response to growth in blood, the female urogenital tract, amniotic fluid, and other normally sterile sites. If the differential expression of RD2 genes occurs, it may contribute to the ability of emm28 strains to survive and thrive in an unusually broad array of anatomic niches for GAS, including the throat, female urogenital tract, and bowel [11, 12, 1518, 26]. Studies are under way to test this hypothesis.
Genome sequencing and molecular population-genomic analysis of GAS strains have shown that prophages are the major source of strain-to-strain variation in gene content [45]. Our discovery that RD2 is distributed among strains of many distinct emm types that have not recently diverged from a single common ancestor suggests that the lateral transfer of RD2 is relatively frequentthat is, the element is fairly promiscuous. Aside from prophages, RD2 is the most widely distributed large, exogenous genetic element that has been described thus far in GAS. Importantly, with the exception of emm28 strains, the presence of RD2 was a variable trait among strains of other emm types that we studied. This observation raises the possibility that RD2 contributes to infection specificity within strains of the same emm type. For example, it is possible that RD2 is overrepresented among strains of the same emm type that cause maternal-neonatal invasive infections (vs. pharyngitis). Regardless, our findings suggest that the acquisition of foreign genes has led to broadening of the ecological niche of GAS, thereby creating a disease-specialist clone of this pathogen.
Acknowledgments
We thank A. Henion and A. Mora, for assistance with statistical analysis and graphics, respectively; A. McGeer, D. Low, S. Shulman, and J. Vuopio-Varkila, for providing group A Streptococcus strains; and N. P. Hoe and F. DeLeo, for critical reading of the manuscript.
References
1. Nuland SB. The doctors' plague: germs, childbed fever, and the strange story of Ignac Semmelweis. New York: W.W. Norton, 2003. First citation in article
2. Semmelweis I. Die Aetiologie, der Begriff und die Prophylaxis des Kindbettfiebers. Pest: C. A. Hartleben, 1861. Translated by Carter KC. The etiology, concept, and prophylaxis of childbed fever. Madison: University of Wisconsin Press, 1983. First citation in article
3. Colebrook L. Infection by anaerobic streptococci in puerperal fever. BMJ 1930; ii:1347. First citation in article
4. Lancefield RC, Hare R. The serological differentiation of pathogenic and non-pathogenic strains of hemolytic streptococci from parturient women. J Exp Med 1935; 61:33549. First citation in article
5. Fromme F. Klinische und bakteriologische studien zum puerperalfieber. Archiv Gynakol 1908; 85:15492. First citation in article
6. Kanter AE, Pilot I. Haemolytic streptococci and their relation to pregnancy and the puerperium. Surg Gynecol Obstet 1924; 38:969. First citation in article
7. Fitzgibbon G, Bigger JW. A clinical and bacteriological investigation of puerperal fever in the Rotunda Hospital, Dublin. BMJ 1925; i:7734. First citation in article
8. Fischetti VA. Streptococcal M protein: molecular design and biological behavior. Clin Microbiol Rev 1989; 2:285314. First citation in article
9. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000; 13:470511. First citation in article
10. Facklam RF, Marin DR, Lovgren M, et al. Extension of the Lancefield classification for group A streptococci by addition of 22 new M protein gene sequences from the clinical isolates: emm103 to emm124. Clin Infect Dis 2002; 34:2838. First citation in article
11. Gaworzewska E, Colman G. Changes in the pattern of infections caused by Streptococcus pyogenes. Epidemiol Infect 1988; 100:25769. First citation in article
12. Colman G, Tanna A, Efstratiou A, Gaworzewska ET. The serotypes of Streptococcus pyogenes present in Britain during 19801990 and their association with disease. J Med Microbiol 1993; 39:16578. First citation in article
13. Bisno AL. Group A streptococcal infections and acute rheumatic fever. N Engl J Med 1991; 325:78393. First citation in article
14. Bisno AL, Stevens DL. Streptococcal infections of skin and soft tissues. N Engl J Med 1996; 334:2405. First citation in article
15. Eriksson BK, Norgren M, McGregor K, Spratt BG, Normark BH. Group A streptococcal infections in Sweden: a comparative study of invasive and noninvasive infections and analysis of dominant T28 emm28 isolates. Clin Infect Dis 2003; 37:118993. First citation in article
16. Chuang I, Van Beneden C, Beall B, Schuchat A, and the Active Bacterial Core Surveillance/Emerging Infections Program Network. Population-based surveillance for postpartum invasive group A Streptococcus infections, 19952000. Clin Infect Dis 2002; 35:66570. First citation in article
17. Tyrrell GJ, Lovgren M, Forwick B, Hoe NP, Musser JM, Talbot JA. M type of group A streptococci isolates submitted to the National Centre for StreptococcusCanada: 19931999. J Clin Microbiol 2002; 40:446671. First citation in article
18. Vlaminckx BJ, van Pelt W, Schouls L, et al. Epidemiological features of invasive and noninvasive group A streptococcal disease in the Netherlands, 19921996. Eur J Clin Microbiol Infect Dis 2004; 23:43444. First citation in article
19. Espinosa LE, Li Z, Gomez Barreto D, et al. M protein gene type distribution among group A streptococcal clinical isolates recovered in Mexico City, Mexico, from 1991 to 2000, and Durango, Mexico, from 1998 to 1999: overlap with type distribution within the United States. J Clin Microbiol 2003; 41:3738. First citation in article
20. Adam D, Scholz H, Helmerking M. Short-course antibiotic treatment of 4782 culture-proven cases of group A streptococcal tonsillopharyngitis and incidence of poststreptococcal sequelae. J Infect Dis 2000; 182:50916. First citation in article
21. Moses AE, Goldberg S, Korenman Z, et al. Invasive group A streptococcal infections, Israel. Emerg Infect Dis 2002; 8:4216. First citation in article
22. Murakami J, Kawabata S, Tereo Y, et al. Distribution of emm genotypes and superantigen genes of Streptococcus pyogenes isolated in Japan, 19949. Epidemiol Infect 2002; 128:397404. First citation in article
23. Shulman ST, Tanz RR, Kabat W, et al. Group A streptococcal pharyngitis serotype surveillance in North America, 20002002. Clin Infect Dis 2004; 39:32532. First citation in article
24. Vlaminckx BJ, Mascini EM, Schellekens J, et al. Site-specific manifestations of invasive group A streptococcal disease: type distribution and corresponding patterns of virulence determinants. J Clin Microbiol 2003; 41:49419. First citation in article
25. Schmitz F-J, Beyer A, Charpentier E, et al. Toxin-gene profile heterogeneity among endemic invasive European group A streptococcal isolates. J Infect Dis 2003; 188:157886. First citation in article
26. Kolmos HJ, Svendsen RN, Nielsen SV. The surgical team as a source of postoperative wound infections caused by Streptococcus pyogenes. J Hosp Infect 1997; 35:20714. First citation in article
27. O'Brien KL, Beall B, Barrett NL, et al. Epidemiology of invasive group A Streptococcus disease in the United States, 19951999. Clin Infect Dis 2002; 35:26876. First citation in article
28. Li Z, Sakota V, Jackson D, Franklin AR, Beall B, for the Active Bacterial Core Surveillance/Emerging Infections Program Network. Array of M protein gene subtypes in 1064 recent group A streptococcus isolates recovered from the active bacterial core surveillance. J Infect Dis 2003; 188:158792. First citation in article
29. Moses, AE, Hidaldo-Grass C, Dan-Goor M, et al. emm typing of M nontypeable invasive group A streptococcal isolates in Israel. J Clin Microbiol 2003; 41:46559. First citation in article
30. DelVecchio VG, Kapatral V, Redkar RJ, et al. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA 2002; 99:4438. First citation in article
31. Kapatral V, Anderson I, Ivanova N, et al. Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J Bacteriol 2002; 184:200518. First citation in article
32. Ivanova N, Sorokin A, Galleron N, et al. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 2003; 423:8791. First citation in article
33. Banks DJ, Porcella SF, Barbian KD, et al. Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain. J Infect Dis 2004; 190:72738. First citation in article
34. Overbeek R, Larsen N, Walunas T, et al. The ERGO genome analysis and discovery system. Nucleic Acid Res 2003; 31:16471. First citation in article
35. Whatmore AM, Kapur V, Sullvnan DH, Musser JM, Kehoe MA. Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A Streptococci. Mol Microbiol 1994; 14:61931. First citation in article
36. Matsumoto M, Hoe NP, Liu M, et al. Intrahost sequence variation in the streptococcal inhibitor of complement gene in human patients with pharyngitis. J Infect Dis 2003; 187:60412. First citation in article
37. Banks DJ, Porcella SF, Barbian KD, Martin JM, Musser JM. Structure and distribution of an unusual chimeric genetic element encoding macrolide resistance in phylogenetically diverse clones of group A Streptococcus. J Infect Dis 2003; 188:1899909. First citation in article
38. Beres SB, Sylva GL, Sturdevant, DE, et al. Genome-wide molecular dissection of serotype M3 group A Streptococcus strains causing two epidemics of invasive infections. Proc Natl Acad Sci USA 2004; 101:118338. First citation in article
39. Feretti JJ, McShan WM, Ajdic D, et al. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 2001; 98:465863. First citation in article
40. Beres SB, Sylva GL, Barbian KD, et al. 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 2002; 99:1007883. First citation in article
41. Smoot JC, Barbian KD, van Gompel JJ, et al. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc Natl Acad Sci USA 2002; 99:466873. First citation in article
42. Nakagawa I, Kurokawa K, Yamashita A, et al. 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 2003; 13:104255. First citation in article
43. Sumby P, Porcella SF, Madrigal AG, et al. Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene-transfer events. J Infect Dis 2005; 192:77182 (in this issue). First citation in article
44. Nagiec MJ, Lei B, Parker SK, et al. Analysis of a novel prophage-encoded group A Streptococcus extracellular phospholipase A2. J Biol Chem 2004; 279:4590918. First citation in article
45. Banks DJ, Beres SB, Musser JM. Critical contribution of phages to group A Streptococcus evolution, genome diversification, and strain emergence. Trends Microbiol 2002; 10:51521. First citation in article
46. Bisno AL, Brito MO, Collins CM. Molecular basis of group A streptococcal virulence. Lancet Infect Dis 2003; 3:191200. First citation in article
47. Courtney HS, Hasty DL, Li Y, Chiang HC, Thacker JL, Dale JB. Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes. Mol Microbiol 1999; 32:8998. First citation in article
48. Haanes EJ, Heath DG, Cleary RP. Architecture of the vir regulons of group A streptococci parallels opacity factor phenotype and M protein class. J Bacteriol 1992; 174:496776. First citation in article
49. Hallas G, Widdowson JP. The relationship between opacity factor and M protein in Streptococcus pyogenes. J Med Microbiol 1983; 16:1326. First citation in article
50. Jeng A, Sakota V, Li Z, Datta V, Beall B, Nizet V. Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX. J Bacteriol 2003; 185:120817. First citation in article
51. Tagg JR. Production of bacteriocin-like inhibitors by group A streptococci of nephritogenic M types. J Clin Microbiol 1984; 19:8847. First citation in article
52. Wescombe PA, Tagg JR. Purification and characterization of streptin, a type A1 lantibiotic produced by Streptococcus pyogenes. Appl Environ Microbiol 2003; 69:273747. First citation in article
53. Karaya K, Shimizu T, Taketo A. New gene cluster for lantibiotic streptin possibly involved in streptolysin S formation. J Biochem 2001; 129:76975. First citation in article
54. Glaser P, Rusniok C, Buchrieser C, et al. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol Microbiol 2002; 45:14991513. First citation in article
55. Tettelin H, Masignani V, Cieslewicz MJ, et al. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc Natl Acad Sci USA 2002; 99:123916. First citation in article
56. Lancefield RC, Perlmann GE. Preparation and properties of a protein (R antigen) occurring in streptococci of group A, type 28 and in certain streptococci of other serological groups. J Exp Med 1952; 96:8397. First citation in article
57. Lancefield RC. Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J Exp Med 1957; 106:52544. First citation in article
58. Stalhammar-Carlemalm M, Areschoug T, Larsson C, Lindahl G. The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells. Mol Microbiol 1999; 33:20819. First citation in article
59. Stalhammar-Carlemalm M, Areschoug T, Larsson C, Lindahl G. Cross-protection between group A and group B streptococci due to cross-reacting surface proteins. J Infect Dis 2000; 182:1429. First citation in article
60. Demuth DR, Duan Y, Brooks W, Holmes AR, McNab R, Jenkinson HF. Tandem genes encode cell-surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors. Mol Microbiol 1996; 20:40313. First citation in article
61. Jenkinson HF, Demuth DR. Structure, function and immunogenicity of streptococcal antigen I/II polypeptides. Mol Microbiol 1997; 23:18390. First citation in article
62. Brady LJ, Cvitkovitch DG, Geric CM, et al. Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule. Infect Immun 1998; 66:427482. First citation in article
63. Du LD, Kolenbrander PE. Identification of saliva-regulated genes of Streptococcus gordonii DL1 by differential display using random arbitrarily primed PCR. Infect Immun 2000; 68:48347. First citation in article
64. Sutcliffe IC, Harrington DJ. Putative lipoproteins of Streptococcus agalactiae identified by bioinformatics genome analysis. Antonie Leeuwenhoek 2004; 85:30515. First citation in article
65. Rigden DJ, Jedrzejas MJ, Galperin MY. Amidase domains from bacterial and phage autolysins define a family of -D,L-glutamate-specific amidohydrolase. Trends Biochem Sci 2003; 28:2304. First citation in article
66. Bateman B, Rawlings ND. The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases. Trends Biochem Sci 2003; 28:2347. First citation in article
67. Johnson DR, Wooten JT, Shet A. Kaplan EL. A comparison of group A streptococci from invasive and uncomplicated infections: are virulent clones responsible for serious streptococcal infections J Infect Dis 2002; 185:158695. First citation in article
68. Fitzgerald JR, Musser JM. Evolutionary genomics of pathogenic bacteria. Trends Microbiol 2001; 9:54753. First citation in article
69. Parkhill J, Sebaihia M, Preston A, et al. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Nat Genet 2003; 35:3240. First citation in article
70. Holden MT, Feil EJ, Lindsay JA, et al. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci USA 2004; 101:978691. First citation in article
71. Enright MC, Spratt BG, Kalia A, Cross JH, Bessen DE. Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect Immun 2001; 69:241627. First citation in article
72. Banks DJ, Lei B, Musser JM. Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315. Infect Immun 2003; 71:707986. First citation in article
73. Broudy TB, Pancholi V, Fischetti VA. The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase. Infect Immun 2002; 70:280511. First citation in article