Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus

Abstract

In recent years we have studied the relationship between strain genotypes and patient phenotypes in group A Streptococcus (GAS), a model human bacterial pathogen that causes extensive morbidity and mortality worldwide. We have concentrated our efforts on serotype M3 organisms because these strains are common causes of pharyngeal and invasive infections, produce unusually severe invasive infections, and can exhibit epidemic behavior. Our studies have been hindered by the lack of genome-scale phylogenies of multiple GAS strains and whole-genome sequences of multiple serotype M3 strains recovered from individuals with defined clinical phenotypes. To remove some of these impediments, we sequenced to closure the genome of four additional GAS strains and conducted comparative genomic resequencing of 12 contemporary serotype M3 strains representing distinct genotypes and phenotypes. Serotype M3 strains are a single phylogenetic lineage. Strains from asymptomatic throat carriers were significantly less virulent for mice than sterile-site isolates and evolved to a less virulent phenotype by multiple genetic pathways. Strain persistence or extinction between epidemics was strongly associated with presence or absence, respectively, of the prophage encoding streptococcal pyrogenic exotoxin A. A serotype M3 clone significantly underrepresented among necrotizing fasciitis cases has a unique frameshift mutation that truncates MtsR, a transcriptional regulator controlling expression of genes encoding iron-acquisition proteins. Expression microarray analysis of this clone confirmed significant alteration in expression of genes encoding iron metabolism proteins. Our analysis provided unprecedented detail about the molecular anatomy of bacterial strain genotype-patient phenotype relationships.

Fig. 1.

Interserotype genetic relationships. (A) Relationships inferred from 43,356 core SNP loci concatenated nucleotides. (B) Relationships inferred from 3,134 MLST internal gene segment concatenated nucleotides.

Fig. 2.

Invasive and carrier isolate virulence comparison. (A) Comparison of mean LD₉₀ values at 48 h after IP injection. (B) Comparison of mean survival time for ≈5 × 10⁸ CFU IP dose. All speA⁺ strains are ΦG3.01. Invasive groups that differ significantly from the carriers are marked with an asterisk. Bars are the SEM.

Fig. 3.

Intraserotype M3 core SNP matrix. Invasive isolate strain designations are color-coded to indicate SNP genotypes as previously defined (4), and carrier strains are colored blue. SNP numbers are color-coded to indicate a comparison between strains of the same SNP genotype or between carriers. Strain SSI-1 is given in gray to indicate that its SNPs were determined by in silico comparison and not by CGR. The average number of core SNPs strain-to-strain is 54.

Fig. 4.

Intraserotype M3 genetic relationships. (A) Relationships among contemporary M3 strains inferred from 248 core SNP loci concatenated nucleotides. (B) Relationships among invasive isolates inferred from 167 core SNP loci concatenated nucleotides. Invasive strain designations are color coded to indicate SNP genotypes as previously defined (4). Asymptomatic carrier isolates are colored aqua, and the high-virulence reference strain MGAS315 is colored red.

Fig. 5.

Mts regulon and genes exhibiting altered expression. (A) Illustrated is the region of the M3 genome encoding the Mts regulon. Boxed in red is the frame-shifted translation product. (B) Iron acquisition and oxidative stress response genes exhibiting altered expression. Expression of each of these genes was significantly different (P < 0.0001) under conditions of exponential aerobic growth in metal replete rich media. Bars are the standard error of the mean (n = 6). In strain MGAS9887, expression of mtsR was decreased, whereas mtsABC was increased ≈1.5 fold. Expression of shr (streptococcal heme receptor) and htsABC was increased ≈3.5-fold. Expression of ribonucleotide-diphosphate reductase, nrdFIE, and alkyl hydroperoxide reductase, ahpCA, was increased ≈20- and ≈1.5-fold, respectively. Findings are consistent with the loss of MtsR repression of iron acquisition systems leading to intracellular iron accumulation, increased formation of reactive oxygen species, and induction of an oxidative stress response.