Skip Navigation

Evolutionary Origin and Emergence of a Highly Successful Clone of Serotype M1 Group A Streptococcus Involved Multiple Horizontal Gene Transfer Events

  1. Paul Sumby1,2,
  2. Steve F. Porcella1,
  3. Andres G. Madrigal1,a,
  4. Kent D. Barbian1,
  5. Kimmo Virtaneva1,
  6. Stacy M. Ricklefs1,
  7. Daniel E. Sturdevant1,
  8. Morag R. Graham1,a,
  9. Jaana Vuopio-Varkila3,
  10. Nancy P. Hoe1 and
  11. James M. Musser1,2
  1. 1Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana;
  2. 2Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, Houston, Texas;
  3. 3National Public Health Institute, Helsinki, Finland
  1. Reprints or correspondence: Dr. James M. Musser, Center for Human Bacterial Pathogenesis Research, Dept. of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (musser{at}bcm.tmc.edu)
 
Next Section

Abstract

To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS

The molecular events involved in the emergence of unusually virulent pathogens are poorly understood. Bacteria can evolve slowly through the accumulation of point mutations or rapidly in an “evolutionary quantum leap” by acquisition of new genetic material through horizontal gene transfer events [1]. New combinations of genes or allelic variants can increase bacterial fitness by conferring unique properties, such as the ability to colonize a previously unexploitable ecological niche or to resist components of the immune system. A number of well-described processes are known to promote horizontal gene transfer events, including prophage transduction, plasmid conjugation, and transformation

Group A Streptococcus (GAS) is responsible for many serious human infections, such as necrotizing fasciitis (NF; also called “the flesh-eating syndrome”) and streptococcal toxic shock syndrome (STSS) [2, 3]. However, the most common forms of streptococcal infection are noninvasive, non–life-threatening infections, such as pharyngitis and impetigo. Host and bacterial factors contribute to the outcome of an infection [4]. The importance of GAS factors in disease manifestation was, in part, identified because of the nonrandom distribution of GAS serotypes among particular diseases. For example, in population-based case series, serotype M1 GAS is usually the most common serotype recovered during episodes of invasive disease [5 –7]

During the mid-1980s, the frequency and severity of invasive infections caused by serotype M1 GAS (e.g., septicemia, STSS, and, especially, NF) suddenly and precipitously increased (reviewed in Musser and Krause [3]). However, little is known about the molecular processes associated with this phenotypic shift. In the present study, we sought to understand the molecular events that contribute to the evolutionary origin and emergence of serotype M1 GAS strains through genomic, single-nucleotide polymorphism (SNP), transcriptome, and protein analyses

Previous SectionNext Section

Materials and Methods

Genome sequencing The genome of strain MGAS5005 was sequenced by methods previously used to sequence the genome of serotype M6 and M28 GAS strains [8, 9]. Directed sequencing was performed, to increase the minimum consensus-base quality to Q40 for regions of low sequence quality in the assembled genome. The entire genome was tiled by polymerase chain reaction (PCR) after closure, to ascertain the validity of the assembly. Open reading frames (ORFs) were identified by use of proprietary software (Integrated Genomics) and were entered into the ERGO bioinformatics suite for annotation [10]. The genome sequence has been deposited in GenBank (accession number CP000017). Strain MGAS5005 has been deposited in the American Type Culture Collection (number BAA-947)

Bacterial strains Sixty-five serotype M1 GAS strains were studied (table 1). The sampling of these organisms was based on the genetic analysis of ∼2000 serotype M1 strains isolated from patients on several continents. These studies have been conducted during the last 13 years in the laboratory of the senior author (J.M.M.). Importantly, these ∼2000 serotype M1 strains include organisms from comprehensive population-based studies conducted over the course of many years in the United States, Canada, Germany, and Finland [11 –16]. Sampling of strains for molecular analysis was done randomly on the basis of our knowledge of the distribution of serotype M1 subclones present in the populations studied. Additional strains studied were selected at random from the senior author’s strain collection, which includes >12,000 isolates from 36 countries worldwide. The strains studied were not subject to systematic sampling bias. The inclusion criteria were designed to provide a temporally and spatially diverse collection of isolates to study. MGAS5005 was isolated in 1996 from the cerebrospinal fluid of an infected patient in Ontario, Canada, and has been used extensively in studies of GAS pathogenesis [17 –19]

GAS DNA-DNA microarray analysis Two spotted microarrays were used for the DNA-DNA hybridizations, to compare old and contemporary serotype M1 GAS isolates. Twenty-nine isolates, of which 21 had been isolated in Finland as part of a population-based study, were analyzed by use of a DNA microarray containing 1702 ORFs that represented 97% of the genome of serotype M1 strain SF370 [20]. In addition, these isolates were analyzed by use of a GAS microarray composed of the above 1702 M1 ORFs supplemented with ORFs uniquely present (relative to strain SF370) in the serotype M18 strain MGAS8232 [21] and the serotype M3 strain MGAS315 [22]. Most of the supplemental ORFs were encoded by prophages present in the genome of the serotype M3 and M18 strains sequenced. SF370 was isolated in 1985 from the infected wound of a patient, and this isolate produces low NAD+-glycohydrolase (NADase) activity, similar to all pre-1988 serotype M1 GAS isolates [20, 23]

Bacteria were grown overnight in Todd-Hewitt broth (Difco Laboratories) supplemented with 0.2% yeast extract (THY broth). Chromosomal DNA was isolated by use of the Puregene DNA isolation kit (Gentra Systems). Probe preparation and microarray hybridization experiments were performed as described elsewhere [21, 24]. Differentially hybridizing spots were identified for each strain by use of QuantArray analysis software (Packard BioScience). ORFs defined as “absent” were either not present in the test strain genome or had sequences divergent enough to prevent hybridization with strain SF370 DNA under highly stringent conditions. DNA-DNA microarray results were verified by PCR mapping and targeted DNA sequencing

SNP analysis PCR primers were designed to flank regions containing putative SNPs (table 2). PCR amplifications were purified by use of the Qiagen PCR purification kit and then sequenced with both forward and reverse primers. The SNP data for each strain were concatenated, resulting in a single character string (nucleotide sequence). Phylogenetic analysis was performed by use of MEGA (version 2.1; available at: http://www.megasoftware.net/) with the neighbor-joining method, in which 1000 bootstrap replicates are used and distance is calculated on the basis of the number of different SNPs

Western immunoblot analysis of culture supernatant proteins  Bacteria were grown to exponential phase (OD600, 0.25) or stationary phase (overnight), and culture supernatants were obtained by centrifugation and filtering through a 0.45-μm filter. Protein was concentrated by ethanol precipitation and was assayed for presence of streptolysin O (SLO) and NADase by techniques described elsewhere [19]

NADase enzyme-activity assay The NADase activity secreted from old and contemporary isolates was assayed as described elsewhere [25]. Briefly, supernatants from overnight cultures were centrifuged and filtered through a 0.45-μm filter. Two-fold serial dilutions of supernatants were performed in microtiter plates with PBS. NAD+ (Sigma), diluted in PBS, was added to a concentration of 0.67 mmol/L before incubation for 1 h at 37°C. Reactions were developed by the addition of NaOH to 2N, were incubated for 1 h in the dark, and were read macroscopically by exciting the samples with 360-nm light. Results are reported as the highest 2-fold dilution capable of hydrolyzing 100 nmol of exogenous NAD+

SLO enzyme-activity assay The SLO activity secreted from old and contemporary isolates was assayed as described elsewhere [26], with slight modifications. Briefly, strains were grown to an OD600 of 0.25±0.05, and supernatants were clarified by centrifugation and filtration (pore size, 0.22 μm). The cleared supernatant was incubated with 20 mmol/L dithiothreitol for 10 min at room temperature and was aliquoted (500 μL) into 2 tubes. Water-soluble cholesterol (25 μg) (cholesterol/methyl-β-cyclodextrin; Sigma-Aldrich), a specific SLO inhibitor, was added to one sample, and the samples were incubated at 37°C for 30 min. A 2% sheep erythrocyte/PBS suspension (250 μL) was added to each sample, was mixed by inversion, and was incubated for 30 min at 37°C. PBS (500 μL) was added to each sample, and unlysed erythrocytes were removed by centrifugation at 3000 g for 5 min. The amount of hemoglobin present in the supernatant was measured by analysis of the optical density at 541 nm. Erythrocytes incubated in water acted as a positive control (100%) for lysis, and fresh THY broth was used as a negative control. Controls were treated exactly as experimental samples, except that medium rather than PBS was added to the water sample for the final step

Affymetrix GeneChip expression microarray analysis A recently described custom-made Affymetrix GeneChip was used for expression microarray studies, to compare old and contemporary genotype M1 GAS isolates [27]. The chip consisted of an antisense oligonucleotide array (18-micron size) that represented >400,000 25-mer probes (16 pairs/probe set). It contained probe sets (42,351 features) for 2662 predicted GAS ORFs that represented a composite superset of 6 GAS genome sequences (M1, M3, M5, M12, M18, and M49). The chip also contained 1925 redundant probe sets that together represented >95% of the nonredundant predicted coding regions in the genome of strain SF370

The transcriptomes of 6 serotype M1 GAS isolates with wild-type covRS genes, which encode a 2-component signal transduction system that regulates ∼15% of the GAS genome [28], were compared by expression microarray analysis. Four of these isolates (MGAS2221, MGAS5322, MGAS1284, and MGAS5087) had a phylogenetic lineage genotype similar to that of strain MGAS5005, whereas the other 2 isolates (MGAS1264 and MGAS1508) had a phylogenetic lineage genotype similar to that of strain SF370 (see Results). Isolates were grown overnight in THY broth at 37°C in 5% CO2. The next morning, 2 cultures of each isolate were seeded with a 1:100 dilution of the overnight cultures in fresh, prewarmed THY broth and were incubated at 37°C in 5% CO2. RNA was obtained from each of the 12 cultures, which were grown to an OD600 of 0.14 (early exponential phase). RNA isolation, cDNA synthesis, labeling, and hybridization was performed as described elsewhere [27]. Gene expression estimates were calculated for each chip by use of GCOS (version 1.1.1; Affymetrix). GAS-specific chip data were normalized across samples, to minimize discrepancies due to experimental variables (e.g., probe preparation and hybridization). A 2-sample Student’s ttest (unequal variance) was applied to the data by use of Partek Pro (version 5.1; Partek), followed by a false-discovery-rate correction (P<.05), to account for multiple testing [27]

Promoter comparisons Promoter regions from genes of interest in MGAS5005 and SF370 were aligned by use of ClustalW, available at the Network Protein Sequence Analysis Web site [29]

Previous SectionNext Section

Results

Gene content variation in serotype M1 GAS strains DNA-DNA microarray analysis revealed restricted variation in gene content among 30 serotype M1 isolates from 6 countries (figure 1). All strains had very similar core genomes corresponding to 93% of the ORFs present in the genome of the reference serotype M1 strain SF370 [20]. A maximum of 113 ORFs (7%) were absent in any one test strain, compared with those in the reference strain, and virtually all variably present ORFs were associated with prophages (figure 1). The majority of strains lacked 3 of the 4 prophages present in the reference strain. Two additional prophages (ϕ5005.1 and ϕ5005.3; see below) were identified in recent isolates by use of a DNA microarray supplemented with ORFs present in the sequenced genomes of serotypes M3 and M18 [21, 22]. One genomic profile predominated among contemporary isolates (figure 1), consistent with the hypothesis of a recent global spread of a distinct serotype M1 clone [11, 12, 30]

Figure 1
View larger version:
Figure 1

Schematic comparing the gene content of 29 serotype M1 group A Streptococcus (GAS) isolates with that of reference strain SF370. The chromosomal position of each gene of SF370 (1702 open reading frames [ORFs]) is shown from top to bottom. The gene content of the 29 serotype M1 isolates relative to strain SF370 is shown from left to right. Isolates are grouped on the basis of the presence or absence of the speA gene. Genes were identified as present (red) absent (black) or divergent (green) by microarray analysis, polymerase chain reaction mapping, and targeted sequencing. Below each prophage designation, putative virulence factor genes present in the prophage are indicated

Together with the results of previous studies [11 –14], these data suggest that reference strain SF370 is genetically distinct from the serotype M1 strains responsible for most recent human infections. To study this issue in more detail, we sequenced the genome of strain MGAS5005, a serotype M1 organism that is genetically representative of contemporary isolates and that has been used extensively in pathogenesis research [17, 18, 31]. Consistent with the data from the DNA-DNA microarray analysis, the genomes of strains MGAS5005 and SF370 were very similar (figure 2A). However, the genomes differed in terms of prophage content, small insertions and deletions, and many SNPs. Prophages accounted for the majority of strain-specific ORFs. The genome of strain MGAS5005 contained 3 prophages (figure 2B ) encoding a total of 3 proven or putative extracellular virulence factors—a potent pyrogenic toxin superantigen (scarlet fever toxin, SpeA2 variant [32]) and 2 DNases (Spd3 and SdaD2 [19, 33])

Figure 2
View larger version:
Figure 2

A Atlas comparing the chromosomes of strains SF370 and MGAS5005. A BLAST analysis was performed comparing MGAS5005 open reading frames (ORFs) with those of SF370. The colored circle represents all MGAS5005 ORFs, with high-homology ORFs (⩾e −10) being color-coded yellow and low-homology or unique ORFs (<e −10) being color-coded blue. The red blocks outside the circle show the locations of MGAS5005 prophages; the green blocks and arrows inside the circle show the locations and insertion sites, respectively, of SF370 prophages. The location of the 36-kb M12-like region is also marked. B Organization and ORF map of the 3 prophages present in the genome of strain MGAS5005. Putative ORFs are indicated by arrows that show the direction of transcription. Groups of genes whose protein products are functionally related are color coded

SNP analysis of 65 serotype M1 GAS strains The availability of 2 genome sequences facilitated high-resolution analysis of genetic relationships among serotype M1 GAS strains via a genomewide study of SNPs. Genetic relationships among 65 geographically and temporally diverse serotype M1 GAS isolates were investigated by analysis of 37 sequence-confirmed SNPs that are distributed throughout the genome (tables 1 and 2). Isolates recovered before the mid-1980s were genetically heterogeneous, as was shown by variation in SNP genotype (figure 3). In striking contrast, virtually all recent isolates had the same SNP genotype, regardless of their place of origin, thus confirming that they were clonally related as a consequence of recent common descent (figure 3)

Figure 3
View larger version:
Figure 3

Genetic relationships among 65 serotype M1 group A Streptococcus (GAS) isolates, according to single-nucleotide polymorphism (SNP) analysis. Thirty-seven SNPs were analyzed in each of the 65 strains. The 37 SNPs are located, on average, 50 kb from each other on the GAS core chromosome. Serotype M3 strain MGAS315 was used as an outgroup

Comparison of a 36-kb chromosomal region between genotype M1 and M12 isolates Most of the SNPs that differentiate strains SF370 and MGAS5005 were distributed around the genome in apparently random fashion. However, a 36-kb region located between purA and nadC had an excessive number of SNPs (figure 4). This unusual pattern of SNP variability suggests that the 36-kb regions from these 2 strains have an evolutionary history quite different from those of the rest of the core genomes. Comparison of the 36-kb region present in strain MGAS5005 with our in-house GAS genome database revealed its virtual identity with the analogous chromosomal segment present in 2 serotype M12 strains (figure 4 and data not shown). In striking contrast, however, the sequence of the 36-kb region in strain SF370 differed considerably (figure 4). This observation was confirmed by resequencing 10,081 bp arrayed across the 36-kb region present in strains SF370 (M1), MGAS5005 (M1), and MGAS9429 (M12). Moreover, analysis of 45 SNPs in the 36-kb region in each of 65 serotype M1 strains from diverse geographic sources confirmed the key finding that old (pre-1988) and more recent (post-1988) serotype M1 strains differ in this chromosomal segment. Together, the data strongly suggest that the 36-kb region in contemporary M1 strains was very recently acquired from a serotype M12 GAS strain by horizontal gene transfer and recombinational replacement

Figure 4
View larger version:
Figure 4

Open reading frame (ORF) map of a 51-kb region in the genomes of strains MGAS9429 (serotype M12), MGAS5005 (serotype M1), and SF370 (serotype M1). The schematic shows the boundaries of a presumed inter-M–type horizontal gene transfer involving ∼36 kb of DNA. The red shading between ORFs indicates 100% identity at the nucleotide level. The 36-kb regions of MGAS5005 and MGAS9429 are identical with the exception of 6 single-nucleotide polymorphisms (SNPs; indicated by asterisks) and 1 in-frame deletion (Δ). In comparison, the 36-kb regions of MGAS5005 and SF370 differ by 435 SNPs and 5 deletions. Select genes of interest are labeled. Transposase ORFs are shown in gray

Comparison of SLO and NADase activity between old and contemporary M1 isolates Two prominent extracellular toxins are encoded by genes present in this 36-kb region: SLO and NADase. SLO is a membrane-lytic toxin [26]. NADase inhibits internalization of GAS by host cells induces apoptosis, and has potent detrimental effects on human polymorphonuclear leukocytes, thereby enhancing GAS survival [23, 25, 34]. It is important to note that serotype M1 strains isolated before the mid-1980s reportedly do not produce NADase, although they do contain an intact gene (nga) for this enzyme [23, 34 –38]. In contrast, serotype M12 strains (regardless of year of isolation) and serotype M1 strains isolated after the mid-1980s do produce NADase [23, 26, 34 –37]. Furthermore, patients infected by serotype M12 strains are known to seroconvert to NADase, which indicates in vivo production in humans [34, 39, 40]. Therefore, we hypothesized that contemporary serotype M1 isolates (MGAS5005-like SNP profile with an M12-like 36-kb region) could produce extracellular NADase. This hypothesis was confirmed by Western immunoblot analysis (figure 5A ). Immunologically reactive protein also was present in the supernatants of older isolates (SF370-like), albeit at substantially lower levels. A concomitant increase in NADase activity in the supernatants of contemporary isolates versus older isolates also was observed (P<.01) (figure 5B ). SLO had a similar pattern of increased protein and activity levels in the supernatants of contemporary isolates (P<.01) (figure 5A and 5C )

Figure 5
View larger version:
Figure 5

Detection of streptolysin O (SLO) and NAD+-glycohydrolase (NADase) in the culture supernatant of disease-matched old (pre-1988) and contemporary (post-1988) serotype M1 group A Streptococcus (GAS) isolates. A SDS-PAGE of concentrated culture supernatants from MGAS5005-like (red) or SF370-like (black) GAS probed with anti-SLO antibody. This antibody is known to cross-react with both SLO and NADase, because of the physical association of these proteins in the immunizing antigen preparation. B NADase enzyme-activity assay, with activity reported as the highest 2-fold dilution capable of hydrolyzing 100 nmol of exogenous NAD+. The experiment was performed in triplicate, with results identical to those shown obtained on each occasion. C SLO enzyme-activity assay, with activity reported as percentage of activity relative to MGAS5005. The experiment was performed in triplicate; shown are mean values, with error bars indicating SDs

Comparison of global gene expression between old and contemporary M1 isolates Our genetic data indicated that serotype M1 strains with a clonal genotype similar to that of strain MGAS5005 were responsible for the unusually severe infections occurring after the mid-1980s. This was in keeping with our previous finding that, in mice, strain MGAS5005 is more virulent than strain SF370 [31]. Although we considered it to be likely that differences in expression of SLO, NADase, and prophage-encoded virulence factors contributed to the high-virulence phenotype, it was also possible that members of the more recent M1 subclone differed from the older strains in their expression of many other genes. To test this hypothesis, the transcriptomes of 4 strains with a phylogenetic lineage genotype similar to that of strain MGAS5005 and 2 strains with a phylogenetic lineage genotype similar to that of strain SF370 GAS were compared by use of an Affymetrix GeneChip. These 6 strains all have a wild-type allele of covR and covS. Only 8 core chromosomal genes were found to be differentially transcribed (⩾2-fold change in transcript level) between the SF370-like and MGAS5005-like isolates (figure 6). All 8 genes were more highly expressed in the MGAS5005-like isolates, with 5 of these 8 genes present in the 36-kb region of DNA involved in the horizontal gene transfer event (figure 6). Consistent with the findings from the extracellular protein analysis (figure 5), 2 of these genes were slo and nga

Figure 6
View larger version:
Figure 6

Expression microarray analysis of the transcriptomes of SF370-like and MGAS5005-like isolates. Shown are log2 values of the fold change in the mean transcript levels of core chromosomal genes that are significantly different (P<.05, Student’s t test followed by a false-discovery-rate correction, to account for multiple comparisons), with at least a 2-fold change in expression between SF370-like and MGAS5005-like isolates. Red and blue data points relate to the presence or absence of these genes within the horizontally transferred 36-kb region, respectively

Previous SectionNext Section

Discussion

Taken together, our genomic, SNP, transcriptomal, and protein analyses suggest that clonal replacement was a key factor associated with the recent increase in the frequency and severity of invasive infections caused by serotype M1 GAS. A genetically distinct serotype M1 clone, apparently more fit than other serotype M1 isolates, emerged during the mid-1980s and rapidly rose to dominance among disease isolates [11, 12, 30]. Moreover, our aggregate data suggest that a clear series of molecular changes account for the major evolutionary events that created the newer M1 clone, which is now highly abundant (figure 7). In this scenario, at least 3 molecular processes are implicated: acquisition of prophages encoding the putative or proven virulence factors SpeA and DNase SdaD2; reciprocal recombination of a chromosomal segment encoding 2 extracellular toxins; and accumulation of 1 or more mutations that increase the expression of a small number of other chromosomal genes. Evidence in support of this order of events is the knowledge that speA2 and sdaD2 have been identified in M1 GAS strains isolated during the 1970s (table 1 and authors’ unpublished data) and that acquisition of the M12-like 36-kb region appears to have occurred during the mid-1980s

Figure 7
View larger version:
Figure 7

Reconstruction of the molecular evolutionary events that resulted in an abundant clone of group A Streptococcus. The hypothesis takes into account data presented here and in previous work [3, 11 –13, 30, 41]. Key events include acquisition of prophages encoding SpeA and SdaD2 (an extracellular DNase virulence factor) and a horizontal gene transfer event involving a 36-kb chromosomal region encoding streptolysin O and NAD+-glycohydrolase. On the basis of near identity in DNA sequence, we hypothesize that a serotype M12 strain served as the donor of this chromosomal region. All serotype M1 strains containing the speA1 allele were recovered before 1988, suggesting that this allele was ancestral to the speA2 allele characteristic of post-1988 M1 isolates. These 2 alleles differ by 1 nucleotide change [3]

Table 1
View larger version:
Table 1

Serotype M1 group A Streptococcus isolates studied

Table 2
View larger version:
Table 2

Polymerase chain reaction primers used for single-nucleotide polymorphism (SNP) analysis

The prophage-encoded genes speA and sdaD2 are associated with the great majority of contemporary genotype M1 isolates (table 1 and figure 1) [11, 12, 41]. Although the exact contribution of SpeA to disease in mouse models of GAS infection is not clear, production of SpeA in humans, coupled with its well-studied superantigen activity, suggests that this protein participates in GAS pathogenesis [17, 42 –45]. Recently, we discovered that SdaD2 increases virulence through enhanced evasion of the innate immune system, likely through degradation of neutrophil extracellular traps, structures that are composed of chromatin and granule proteins and that are released from polymorphonuclear leukocytes [46]

Although it is clear that lateral gene transfer was involved in the acquisition of the M12-like 36-kb region, our experiments here did not address the molecular mechanism responsible. The size of this element and the polylysogenic nature of GAS strains lead us to favor the view that the transfer was due to generalized transduction from a serotype M12 strain to a serotype M1 strain. This hypothesis is strongly supported by the observation that prophage ϕ5005.3, present in the genome of serotype M1 strain MGAS5005, is virtually identical to prophage ϕ9429.3, present in the genome of a serotype M12 strain that has been sequenced (authors’ unpublished data)

The finding that only 8 core chromosomal genes were differentially expressed (⩾2-fold) between genetically representative old and contemporary genotype M1 isolates was unexpected (figure 6). Five of the 8 differentially expressed genes are located within the 36-kb region that distinguishes pre- and post-1988 serotype M1 GAS isolates (figure 4), providing important circumstantial evidence supporting that this horizontal gene transfer event contributed to the emergence of the contemporary M1 clone. The increased slo and nga transcription in contemporary isolates correlates with the increased protein and activity levels observed in the supernatants of contemporary strains (figures 5 and 6)

In principle, the differences in the levels of gene transcripts between strains of the 2 serotype M1 phylogenetic lineages may be explained by polymorphisms that affect promoter activity. Hence, we compared the promoter-region sequences of the 8 differentially expressed genes in strains MGAS5005 and SF370. The −10 and −35 promoter regions of nusG (which encodes the putative transcriptional antitermination protein NusG) and nga described by Kimoto et al. [47] in the homologous genes of group C Streptococcus were used. We found that both nusG and nga had 2 nucleotide changes within the spacer region separating the −10 and −35 promoter sequences. The 6 other differentially expressed genes had identical upstream putative regulatory sequences in strains SF370 and MGAS5005

Nucleotide polymorphisms located within the spacer region can alter promoter activity [48, 49], providing support for the hypothesis that the increased number of nusG and nga transcripts made by strains of the MGAS5005-like M1 lineage is due to enhanced promoter activity. Inasmuch as nga, M5005_Spy0140, and slo are cotranscribed [47], transcription of all 3 genes would be increased in strains containing a stronger nga promoter, which is consistent with our expression microarray data (figure 6)

An inverse relationship between disease severity and SpeB expression by contemporary genotype M1 isolates has been postulated [50]. This raises the possibility that the increase in the severity of invasive infections since the mid-1980s may be due, in part, to down-regulation of SpeB production by the contemporary M1 clone. Our expression microarray data were generated with GAS cells harvested during the exponential phase, and it is possible that additional genes, such as speB are differentially expressed during the stationary phase. However, in this regard we note that Western immunoblot analysis of SpeB in stationary-phase culture supernatants failed to identify a uniform difference in the level of immunoreactive SpeB between old and contemporary genotype M1 isolates (data not shown)

In conclusion, our findings have implications for understanding the molecular events that underlie the emergence of other bacterial clones that are highly virulent or have other characteristic disease phenotypes. Our results stress the importance of deploying integrated genomewide analyses in such endeavors

Previous SectionNext Section

Acknowledgments

We thank M. Gutacker, for help with single-nucleotide polymorphism analysis; K. Pflughoeft, for technical assistance; S. Shelburne, for critical comments; and J. Richard, for editorial assistance

Previous SectionNext Section

Footnotes

  • ↵Data depositions: GenBank accession number for MGAS5005 genome, CP000017

    Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grant UO1-AI-060595)

  • ↵Present affiliations: Boston University School of Medicine, Boston, Massachusetts (A.G.M.); Canadian Centre for Human and Animal Health, Winnipeg, Canada (M.R.G.)

  • Received January 26, 2005.
  • Accepted April 1, 2005.
Previous Section
 

References

  1. 1.↵
    1. Ochman H,
    2. Lawrence JG,
    3. Groisman EA
    . Lateral gene transfer and the nature of bacterial innovation. Nature 2000;405:299-304.
    CrossRefMedline
  2. 2.↵
    1. Cunningham MW
    . Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470-511.
    Abstract/FREE Full Text
  3. 3.↵
    1. Krause RM
    1. Musser JM,
    2. Krause RM
    . The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. In: Krause RM, editor. Emerging infections. New York: Academic Press; 1998. p. 185-218.
  4. 4.↵
    1. Kotb M,
    2. Norrby‐Teglund A,
    3. McGeer A,
    4. et al
    . An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat Med 2002;8:1398-404.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Eriksson BK,
    2. Andersson J,
    3. Holm SE,
    4. Norgren M
    . Epidemiological and clinical aspects of invasive group A streptococcal infections and the streptococcal toxic shock syndrome. Clin Infect Dis 1998;27:1428-36.
    Abstract/FREE Full Text
  6. 6.↵
    1. Tyrrell GJ,
    2. Lovgren M,
    3. Forwick B,
    4. Hoe NP,
    5. Musser JM,
    6. Talbot JA
    . M types of group A streptococcal isolates submitted to the National Centre for Streptococcus (Canada) from 1993 to 1999. J Clin Microbiol 2002;40:4466-71.
    Abstract/FREE Full Text
  7. 7.↵
    1. Zurawski CA,
    2. Bardsley M,
    3. Beall B,
    4. et al
    . Invasive group A streptococcal disease in metropolitan Atlanta: a population‐based assessment. Clin Infect Dis 1998;27:150-7.
    Abstract/FREE Full Text
  8. 8.↵
    1. Banks DJ,
    2. Porcella SF,
    3. Barbian KD,
    4. 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;190:727-38.
    Abstract/FREE Full Text
  9. 9.↵
    1. Green NM,
    2. Zhang S,
    3. Porcella SF,
    4. et al
    . Genome sequence of a serotype M28 strain of group A Streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity. J Infect Dis 2005;192:000-00. (in this issue).
  10. 10.↵
    1. Overbeek R,
    2. Larsen N,
    3. Walunas T,
    4. et al
    . The ERGO genome analysis and discovery system. Nucleic Acids Res 2003;31:164-71.
    Abstract/FREE Full Text
  11. 11.↵
    1. Musser JM,
    2. Hauser AR,
    3. Kim MH,
    4. Schlievert PM,
    5. Nelson K,
    6. Selander RK
    . Streptococcus pyogenes causing toxic‐shock‐like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA 1991;88:2668-72.
    Abstract/FREE Full Text
  12. 12.↵
    1. Musser JM,
    2. Kapur V,
    3. Szeto J,
    4. Pan X,
    5. Swanson DS,
    6. Martin DR
    . Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes of invasive disease. Infect Immun 1995;63:994-1003.
    Abstract/FREE Full Text
  13. 13.↵
    1. Hoe NP,
    2. Nakashima K,
    3. Lukomski S,
    4. et al
    . Rapid selection of complement‐inhibiting protein variants in group A Streptococcus epidemic waves. Nat Med 1999;5:924-9.
    CrossRefMedlineWeb of Science
  14. 14.↵
    1. Hoe NP,
    2. Nakashima K,
    3. Grigsby D,
    4. et al
    . Rapid molecular genetic subtyping of serotype M1 group A Streptococcus strains. Emerg Infect Dis 1999;5:254-63.
    MedlineWeb of Science
  15. 15.↵
    1. Hoe NP,
    2. Vuopio‐Varkila J,
    3. Vaara M,
    4. et al
    . Distribution of streptococcal inhibitor of complement variants in pharyngitis and invasive isolates in an epidemic of serotype M1 group A Streptococcus infection. J Infect Dis 2001;183:633-9.
    Abstract/FREE Full Text
  16. 16.↵
    1. Muotiala A,
    2. Seppala H,
    3. Huovinen P,
    4. Vuopio‐Varkila J
    . Molecular comparison of group A streptococci of T1M1 serotype from invasive and noninvasive infections in Finland. J Infect Dis 1997;175:392-9.
    Abstract/FREE Full Text
  17. 17.↵
    1. Virtaneva K,
    2. Graham MR,
    3. Porcella SF,
    4. et al
    . Group A Streptococcus gene expression in humans and cynomolgus macaques with acute pharyngitis. Infect Immun 2003;71:2199-207.
    Abstract/FREE Full Text
  18. 18.↵
    1. Voyich JM,
    2. Sturdevant DE,
    3. Braughton KR,
    4. et al
    . Genome‐wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 2003;100:1996-2001.
    Abstract/FREE Full Text
  19. 19.↵
    1. Sumby P,
    2. Barbian KD,
    3. Gardner DJ,
    4. et al
    . Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci USA 2005;102:1679-84.
    Abstract/FREE Full Text
  20. 20.↵
    1. Ferretti JJ,
    2. McShan WM,
    3. Ajdic D,
    4. et al
    . Complete genome sequence of an M1 strain of Streptococcus pyogenes . Proc Natl Acad Sci USA 2001;98:4658-63.
    Abstract/FREE Full Text
  21. 21.↵
    1. Smoot JC,
    2. Barbian KD,
    3. Van Gompel JJ,
    4. 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;99:4668-73.
    Abstract/FREE Full Text
  22. 22.↵
    1. Beres SB,
    2. Sylva GL,
    3. Barbian KD,
    4. 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;99:10078-83.
    Abstract/FREE Full Text
  23. 23.↵
    1. Stevens DL,
    2. Salmi DB,
    3. McIndoo ER,
    4. Bryant AE
    . Molecular epidemiology of nga and NAD glycohydrolase/ADP‐ribosyltransferase activity among Streptococcus pyogenes causing streptococcal toxic shock syndrome. J Infect Dis 2000;182:1117-28.
    Abstract/FREE Full Text
  24. 24.↵
    1. Fitzgerald JR,
    2. Sturdevant DE,
    3. Mackie SM,
    4. Gill SR,
    5. Musser JM
    . Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin‐resistant strains and the toxic shock syndrome epidemic. Proc Natl Acad Sci USA 2001;98:8821-6.
    Abstract/FREE Full Text
  25. 25.↵
    1. Bricker AL,
    2. Cywes C,
    3. Ashbaugh CD,
    4. Wessels MR
    . NAD+‐glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci. Mol Microbiol 2002;44:257-69.
    CrossRefMedlineWeb of Science
  26. 26.↵
    1. Limbago B,
    2. Penumalli V,
    3. Weinrick B,
    4. Scott JR
    . Role of streptolysin O in a mouse model of invasive group A streptococcal disease. Infect Immun 2000;68:6384-90.
    Abstract/FREE Full Text
  27. 27.↵
    1. Graham MR,
    2. Virtaneva K,
    3. Porcella SF,
    4. et al
    . Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies. Am J Pathol 2005;166:455-65.
    MedlineWeb of Science
  28. 28.↵
    1. Graham MR,
    2. Smoot LM,
    3. Migliaccio CA,
    4. et al
    . Virulence control in group A Streptococcus by a two‐component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci USA 2002;99:13855-60.
    Abstract/FREE Full Text
  29. 29.↵
    1. Combet C,
    2. Blanchet C,
    3. Geourjon C,
    4. Deléage G
    . NPS@: Network Protein Sequence Analysis. Trends Biochem Sci 2000;25:147-150.
    CrossRefMedlineWeb of Science
  30. 30.↵
    1. Cleary PP,
    2. Kaplan EL,
    3. Handley JP,
    4. et al
    . Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 1992;339:518-21.
    CrossRefMedlineWeb of Science
  31. 31.↵
    1. Lukomski S,
    2. Nakashima K,
    3. Abdi I,
    4. et al
    . Identification and characterization of the scl gene encoding a group A Streptococcus extracellular protein virulence factor with similarity to human collagen. Infect Immun 2000;68:6542-53.
    Abstract/FREE Full Text
  32. 32.↵
    1. Weeks CR,
    2. Ferretti JJ
    . The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12. Infect Immun 1984;46:531-6.
    Abstract/FREE Full Text
  33. 33.↵
    1. Aziz RK,
    2. Ismail SA,
    3. Park HW,
    4. Kotb M
    . Post‐proteomic identification of a novel phage‐encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes . Mol Microbiol 2004;54:184-97.
    CrossRefMedlineWeb of Science
  34. 34.↵
    1. Bernheimer AW,
    2. Lazarides PD,
    3. Wilson AT
    . Diphosphopyridine nucleotidase as an extracellular product of streptococcal growth and its possible relationship to leukotoxicity. J Exp Med 1957;106:27-37.
    Abstract/FREE Full Text
  35. 35.↵
    1. Lazarides PD,
    2. Bernheimer AW
    . Association of production of diphosphopyridine nucleotidase with serological type of group A Streptococcus . J Bacteriol 1957;74:412-3.
    FREE Full Text
  36. 36.↵
    1. Lutticken R,
    2. Lutticken D,
    3. Johnson DR,
    4. Wannamaker LW
    . Application of a new method for detecting streptococcal nicotinamide adenine dinucleotide glycohydrolase to various M types of Streptococcus pyog enes . J Clin Microbiol 1976;3:533-6.
    Abstract/FREE Full Text
  37. 37.↵
    1. Ajdic D,
    2. McShan WM,
    3. Savic DJ,
    4. Gerlach D,
    5. Ferretti JJ
    . The NAD‐glycohydrolase (nga) gene of Streptococcus pyogenes . FEMS Microbiol Lett 2000;191:235-41.
    CrossRefMedlineWeb of Science
  38. 38.↵
    1. Karasawa T,
    2. Yamakawa K,
    3. Tanaka D,
    4. Gyobu Y,
    5. Nakamura S
    . NAD+‐glycohydrolase productivity of haemolytic streptococci assayed by a simple fluorescent method and its relation to T serotype. FEMS Microbiol Lett 1995;128:289-92.
    CrossRefMedlineWeb of Science
  39. 39.↵
    1. Bernhard GC,
    2. Stollerman GH
    . Serum inhibition of streptococcal diphosphopyridine nucleotidase in uncomplicated streptococcal pharyngitis and in rheumatic fever. J Clin Invest 1959;38:1942-9.
    CrossRefMedlineWeb of Science
  40. 40.↵
    1. Kellner A,
    2. Freeman EB,
    3. Carlson AS
    . Neutralizing antibodies to streptococcal diphosphopyridine nucleotidase in the serum of experimental animals and human beings. J Exp Med 1958;108:299-309.
    Abstract
  41. 41.↵
    1. Cleary PP,
    2. LaPenta D,
    3. Vessela R,
    4. Lam H,
    5. Cue D
    . A globally disseminated M1 subclone of group A streptococci differs from other subclones by 70 kilobases of prophage DNA and capacity for high‐frequency intracellular invasion. Infect Immun 1998;66:5592-7.
    Abstract/FREE Full Text
  42. 42.↵
    1. Sriskandan S,
    2. Unnikrishnan M,
    3. Krausz T,
    4. Cohen J
    . Molecular analysis of the role of streptococcal pyrogenic exotoxin A (SPEA) in invasive soft‐tissue infection resulting from Streptococcus pyogenes . Mol Microbiol 1999;33:778-90.
    CrossRefMedlineWeb of Science
  43. 43.↵
    1. Unnikrishnan M,
    2. Cohen J,
    3. Sriskandan S
    . Complementation of a speA negative Streptococcus pyogenes with speA: effects on virulence and production of streptococcal pyrogenic exotoxin A. Microb Pathog 2001;31:109-14.
    CrossRefMedlineWeb of Science
  44. 44.↵
    1. Sriskandan S,
    2. Unnikrishnan M,
    3. Krausz T,
    4. et al
    . Enhanced susceptibility to superantigen‐associated streptococcal sepsis in human leukocyte antigen–DQ transgenic mice. J Infect Dis 2001;184:166-73.
    Abstract/FREE Full Text
  45. 45.↵
    1. Llewelyn M,
    2. Sriskandan S,
    3. Peakman M,
    4. et al
    . HLA class II polymorphisms determine responses to bacterial superantigens. J Immunol 2004;172:1719-26.
    Abstract/FREE Full Text
  46. 46.↵
    1. Brinkmann V,
    2. Reichard U,
    3. Goosmann C,
    4. et al
    . Neutrophil extracellular traps kill bacteria. Science 2004;303:1532-5.
    Abstract/FREE Full Text
  47. 47.↵
    1. Kimoto H,
    2. Fujii Y,
    3. Yokota Y,
    4. Taketo A
    . Molecular characterization of NADase‐streptolysin O operon of hemolytic streptococci. Biochim Biophys Acta 2005;1681:134-49.
    Medline
  48. 48.↵
    1. Liu M,
    2. Tolstorukov M,
    3. Zhurkin V,
    4. Garges S,
    5. Adhya S
    . A mutant spacer sequence between −35 and −10 elements makes the P lac promoter hyperactive and cAMP receptor protein‐independent. Proc Natl Acad Sci USA 2004;101:6911-6.
    Abstract/FREE Full Text
  49. 49.↵
    1. Borst DW,
    2. Betley MJ
    . Mutations in the promoter spacer region and early transcribed region increase expression of staphylococcal enterotoxin A. Infect Immun 1993;61:5421-5.
    Abstract/FREE Full Text
  50. 50.↵
    1. Kansal RG,
    2. McGeer A,
    3. Low DE,
    4. Norrby‐Teglund A,
    5. Kotb M
    . Inverse relationship between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases. Infect Immun 2000;68:6362-9.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2005) 192 (5): 771-782. doi: 10.1086/432514
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most