Trading Capsule for Increased Cytotoxin Production: Contribution to Virulence of a Newly Emerged Clade of emm89 Streptococcus pyogenes

Abstract

Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes.

Importance: S. pyogenes (group A streptococcus [GAS]) causes pharyngitis ("strep throat"), necrotizing fasciitis, and other human infections. Serious infections caused by emm89 S. pyogenes strains have recently increased in frequency in many countries. Based on whole-genome sequence analysis of 1,125 strains recovered from patients on two continents, we discovered that a new emm89 clone, termed clade 3, has two distinct genetic features compared to its predecessors: (i) absence of the genes encoding antiphagocytic hyaluronic acid capsule virulence factor and (ii) increased production of the secreted cytolytic toxins SPN and SLO. emm89 S. pyogenes strains with the clade 3 phenotype (absence of capsule and high expression of SPN and SLO) are highly virulent in mice. These findings provide new understanding of how new virulent clones emerge and cause severe infections worldwide. This newfound knowledge of S. pyogenes virulence can be used to help understand future epidemics and conduct new translational research.

FIG 1

In vitro characteristics of 27 emm89 isolates. (A) NADase activities of emm89 isolates in the culture supernatant. (B) Production of hyaluronic acid capsule by emm89 isolates. Asterisks indicate that strains 11027, 26934, 26963, 27065, 26938, and 26957, which produce higher levels of HA capsule, also have a 38-bp deletion in the hasA upstream region (Fig. 3). NADase assays and capsule assays were performed in triplicate on three separate occasions. Replicate data are expressed as the mean ± SD.

FIG 2

In vitro characteristics of emm89 reference strains and isogenic mutant strains with different nga promoter region sequences. (A) Schematic showing the nga promoter region sequences of the strains assayed. (B to D) qRT-PCR analysis of nga and slo transcript levels and Western immunoblot analysis of secreted SLO and SPN in the culture supernatant. Transcript analysis was done in triplicate on three separate occasions.

FIG 3

In vitro characteristics of emm89 reference strains and isogenic mutant strains with different hasA upstream promoter region sequences. (A) Schematic showing the hasA upstream promoter region sequences of the assayed strains. The relative position of the “region of deletion” relative to P1 and P2 was graphed according to Falaleeva et al. (27). (B and C) hasA transcript level and hyaluronic acid capsule production of assayed strains. Transcript analysis and capsule assays were done in triplicate on three separate occasions.

FIG 4

In vitro characteristics of nine emm89 strains with three nga promoter patterns that produce different levels of hyaluronic acid capsule. (A) Relative transcript levels of nga and slo and enzymatic activities of SPN (NADase) and SLO in the culture supernatant. (B) Western immunoblot analysis of SLO and SPN in the culture supernatant. (C) Transcript level of hasA and hyaluronic acid capsule production of strains analyzed.

FIG 5

Acapsular emm89 strain with a variant 3 nga promoter is highly virulent in a mouse model of necrotizing fasciitis. (A) Kaplan-Meier near-survival curves of mice infected with strain MGAS23530 (low toxin, low capsule), 23530-V1 (low toxin, low capsule), 23530-V3 (high toxin, low capsule), 23530-CapN (low toxin, no capsule), 23530-V1-CapN (low toxin, no capsule), and 23530-V3-CapN (high toxin, no capsule). (B to D) Subsections of panel A that show comparisons between capsule-positive strains and capsule-negative strains. (E to J) Gross pathology analysis of strain virulence in a mouse model of necrotizing fasciitis. The boxed region in panels I and J marks the comparatively larger lesion caused by variant 3 strains expressing large amounts of SPN and SLO toxins.

FIG 6

Capsule production is dispensable for emm89 virulence among strains that produce high levels of SPN and SLO. (A to C) Kaplan-Meier near-survival curves of mice infected with strains that produce different levels of capsule and toxins. (D) Histopathology scores of mouse limb tissue infected with each strain. Data are expressed as means ± SEM. (E to M) Histopathologic analysis of the virulence of isogenic mutant strains in a mouse model of necrotizing fasciitis. Original magnification, 4×.