Distinct signatures of diversifying selection revealed by genome analysis of respiratory tract and invasive bacterial populations

Abstract

Many pathogens colonize different anatomical sites, but the selective pressures contributing to survival in the diverse niches are poorly understood. Group A Streptococcus (GAS) is a human-adapted bacterium that causes a range of infections. Much effort has been expended to dissect the molecular basis of invasive (sterile-site) infections, but little is known about the genomes of strains causing pharyngitis (streptococcal "sore throat"). Additionally, there is essentially nothing known about the genetic relationships between populations of invasive and pharyngitis strains. In particular, it is unclear if invasive strains represent a distinct genetic subpopulation of strains that cause pharyngitis. We compared the genomes of 86 serotype M3 GAS pharyngitis strains with those of 215 invasive M3 strains from the same geographical location. The pharyngitis and invasive groups were highly related to each other and had virtually identical phylogenetic structures, indicating they belong to the same genetic pool. Despite the overall high degree of genetic similarity, we discovered that strains from different host environments (i.e., throat, normally sterile sites) have distinct patterns of diversifying selection at the nucleotide level. In particular, the pattern of polymorphisms in the hyaluronic acid capsule synthesis operon was especially different between the two strain populations. This finding was mirrored by data obtained from full-genome analysis of strains sequentially cultured from nonhuman primates. Our results answer the long-standing question of the genetic relationship between GAS pharyngitis and invasive strains. The data provide previously undescribed information about the evolutionary history of pathogenic microbes that cause disease in different anatomical sites.

Fig. 1.

Comparison of GAS genes with an excess of polymorphisms in pharyngitis and invasive strains. Shown is the distribution of χ² statistics along with corresponding Bonferroni-adjusted P values to correct for multiple testing. Analysis for pharyngitis (A) and invasive (B) strains is shown.

Fig. 2.

Phenotypic impact of genetic variation in the has operon. Schematic of polymorphisms within the has operon promoter (A), hasB (B), and covS (C) genes. Polymorphisms found in invasive strains are shown above the diagram, whereas polymorphisms in pharyngitis strains are indicated below. Regions involved in CovR binding are indicated in black boxes, and the −35/−10 regions are shown in green. Insertions shown in the red box were identified in strains collected from the nonhuman primate experimental pharyngitis protocol. Nucleotide positions in A are labeled according to their distance from the translation GTG start codon. In B and C, labels refer to amino acid positions and asterisks indicate insertions or deletions that result in early protein truncation. (D) Photographs showing colony morphology differences in has promoter mutants. (Scale bar: 1 cm.) (E) Hyaluronic acid quantification for invasive and pharyngitis strains. (F) Quantitative PCR measurement of hasA transcript levels for pharyngitis and invasive mutants shown in pharyngitis appear in black. Asterisks in E and F indicate statistically significant differences relative to control calculated using the Student's t test assuming unequal variances. Values for invasive strains are shown in red, and those for pharyngitis strains are shown in black.

Fig. 3.

Unrooted neighbor-joining phylogenetic trees assembled from the complete list of all core biallelic SNPs. The trees for the 86 pharyngitis strains (A) and for 100 temporally matched invasive strains (B) are shown. Despite being assembled completely independently from each other, both phylogenetic trees show a remarkably similar overall structure, suggesting common evolutionary histories.

Fig. 4.

Combined phylogenetic tree of invasive and pharyngitis strains. Unrooted neighbor-joining phylogenetic trees were assembled from the complete list of all core biallelic SNPs.