Genome-wide molecular dissection of serotype M3 group A Streptococcus strains causing two epidemics of invasive infections

Abstract

Molecular factors that contribute to the emergence of new virulent bacterial subclones and epidemics are poorly understood. We hypothesized that analysis of a population-based strain sample of serotype M3 group A Streptococcus (GAS) recovered from patients with invasive infection by using genome-wide investigative methods would provide new insight into this fundamental infectious disease problem. Serotype M3 GAS strains (n = 255) cultured from patients in Ontario, Canada, over 11 years and representing two distinct infection peaks were studied. Genetic diversity was indexed by pulsed-field gel electrophoresis, DNA-DNA microarray, whole-genome PCR scanning, prophage genotyping, targeted gene sequencing, and single-nucleotide polymorphism genotyping. All variation in gene content was attributable to acquisition or loss of prophages, a molecular process that generated unique combinations of proven or putative virulence genes. Distinct serotype M3 genotypes experienced rapid population expansion and caused infections that differed significantly in character and severity. Molecular genetic analysis, combined with immunologic studies, implicated a 4-aa duplication in the extreme N terminus of M protein as a factor contributing to an epidemic wave of serotype M3 invasive infections. This finding has implications for GAS vaccine research. Genome-wide analysis of population-based strain samples cultured from clinically well defined patients is crucial for understanding the molecular events underlying bacterial epidemics.

Fig. 1.

Characteristics of M3 strains studied and infection type. (A) Epidemiologic curve of GAS serotype M3 invasive infections in Ontario, Canada. Stacked columns are color-coded to indicate prophage genotype and emm3 allele. (B) Occurrence of invasive disease types. Illustrated is the percent of the most abundant disease types in the epidemic peaks centered around 1995 and 2000. Significantly more necrotizing fasciitis infections occurred in the 1995 peak than in the 2000 peak (P = 0.008). (C) Prophage and prophage-encoded virulence factor gene content of the isolates. Indicated is the arbitrarily designated prophage genotype (on the top), number of isolates in each prophage genotype (on the bottom), and the prophage content (on the left) and corresponding prophage-encoded virulence factor genes (on the right).

Fig. 2.

M3 protein variants. (A) The inferred N-terminal amino acid sequences of the 18 emm3 alleles found in this study are shown aligned with the prototype Emm3.0 sequence. The designation of the M3 protein variants (on the left), variants identified in this study (red), and the number of isolates comprising each variant (blue) are indicated. (B) Relationships among emm3 alleles. Phylogenetic reconstruction by the method of neighbor joining was used to generate an unrooted tree by using the emm3 nucleotide sequence encoding amino acids 1–98 of the mature M3 protein. Only alleles encoding Emm3.2-like variants with the D-A-R-S duplication diverged as a genetically related group.

Fig. 3.

Distribution of sclB CAAAA nucleotide repeats in the 255 isolates. The 5′ end of the sclB gene was sequenced in all 255 isolates, and the number of CAAAA pentanucleotide repeats was determined.

Fig. 4.

SNP genotypes identified among the 255 M3 isolates. SNP genotypes (SGs) based on nucleotides present at 20 sites are shown. 315 refers to strain MGAS315, and SSI-1 refers to strain SSI-1.

Fig. 5.

Immunologic analysis of Emm3.1 and Emm3.2. (A) Emm3 synthetic peptides used to immunize rabbits. M3.1 and M3.2 peptides correspond to the first 24 and 28 aa of mature Emm3.1 and Emm3.2, respectively. The first four amino acids of mature Emm3.1, which are duplicated in Emm3.2, are shown in red. (B) ELISA reactivity of rabbit antibodies with Emm3.1 and Emm3.2 peptides. Affinity-purified rabbit anti-Emm3 peptide antibodies were diluted 1:80,000. Underlined amino acids correspond to the peptides used to immunize rabbits. (C) Human PMN phagocytosis studies. Strains MGAS3392 (Emm3.1) and MGAS9887 (Emm3.2) were opsonized with either rabbit anti-M3.1 or anti-M3.2 antibodies at the indicated concentrations and incubated with human PMNs. Values are the mean of five to six independent assays using PMNs obtained from different donors. Error bars show the standard error.

Fig. 6.

Schematic showing summary of temporal changes in serotype M3 subclones. The six major serotype M3 subclones defined by difference in distribution of SNPs, prophage content, and/or emm3 allele, identified among the isolates are shown (large font). Colored arrow length reflects the temporal distribution, and colored arrow height reflects the relative abundance of the subclones. The number of isolates of each subclone in the 1995 and 2000 epidemic peaks are given, and the total annual number of isolates is shown above the time line (on the bottom).