Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates

Abstract

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

Figure 1. More SLO is secreted in STSS isolates than in isolates from non-invasive infections.

The supernatants from an overnight culture (OD₆₀₀ = 1.0) of emm3 S. pyogenes clinical isolates (non-STSS: C500, OT22, and K33; STSS: NIH1, NIH3, NIH8, NIH34, NIH152, NIH249, NIH327, and NIH352; non-STSS isolates with the mutated rgg gene: OT22rgg and K33rgg; STSS isolates complemented with the intact rgg gene: NIH8rgg⁺ and NIH34rgg⁺) were concentrated with trichloroacetic acid, and 5 µl of each sample was analyzed by western blotting with rabbit anti-SLO polyclonal antibody. Representative data of two independent experiments are shown.

Figure 2. Mutation of the rgg gene influences expression of virulence-associated genes.

The expression of virulence-associated genes in non-STSS, STSS GAS isolates, and strains into which an intact gene or mutant rgg or mutant csrS gene had been introduced was analyzed by RT-PCR; columns represent the relative mRNA expression levels of virulence-associated genes of each strain: nicotine adenine dinucleotide glycohydrolase (nga), streptolysin O (slo), streptokinase (ska), protein G-related alpha2-macroglobulin-binding protein (grab), streptodornase (phage-associated) (sdn), streptolysin S (sagA), streptococcal pyrogenic endotoxin (speB), and IL-8 protease (scpC). The expression level of K33 strain is shown as 1. Values are means ± SD (n = 4).

Figure 3. Mutation of rgg gene enhances the lethality and histopathology of GAS in mouse in vivo infection models.

(A) Survival curves of mice infected with each strain. Mice were intraperitoneally inoculated with 1×10⁷ CFU of each GAS, and mouse survival was observed for 7 days post-infection. Mortality differences were statistically significant (P<0.01), as determined by a log-rank test. Survival curves were generated from 3 independent experiments using a total of 10–16 ddY mice for each strain. (B) Histopathological changes in the kidneys of mice infected with GAS. Tissue was extracted at 24 h after the intraperitoneal injection of GAS (1×10⁷ CFU). The black arrows indicate clusters of bacteria with filtrated inflammatory cells. The triangle heads indicate fibrous debris. (C) Lesion areas of subcutaneous infection in hairless mice injected with GAS. 1×10⁷ CFU in 100 µL suspension of GAS in PBS was injected subcutaneously, and the lesion area and body weight were measured each day after infection. The peak values are shown as means ± SD (n = 5). *The skin-lesion area in rgg mutant strains-infected mice was significantly larger than that in rgg intact strains (p<0.05), as estimated by ANOVA.

Figure 4. Effect of rgg and other mutations on migration and survival of human neutrophils.

(A) The effect of human neutrophil migration in response to IL-8 by various GAS strains (K33, NIH34, and their rgg, slo, scpC, nga, and csrS mutants) was analyzed using a Transwell system and flow cytometry. About 62% of applied human neutrophils migrated through the Transwell, under the conditions of IL-8 addition. Values shown are means ± SD (n = 3). *p<0.05, as estimated by ANOVA. The results shown are representative of one of five individual experiments, all of which had similar results. (B) The viability of human neutrophils in the lower wells of a Transwell system, after migration in response to IL-8. The migrated human neutrophils were bought into contact with various GAS strains (K33, NIH34, and their rgg, slo, scpC, nga, and csrS mutants), and the remaining viable neutrophils were counted. Values shown are means ± SD (n = 3). *p<0.05, as estimated by ANOVA.

Figure 5. Schema of regulatory network and its dysfunction in STSS isolates leading to host evasion.

CsrS phosphorylates CsrR, and the CsrR represses expression of a number of virulence genes including rgg and scpC . CsrS also positively regulates the expression of rgg , which suppresses slo gene expression (Figure 2). The rgg mutation causes an overexpression of SLO, which kills neutrophils, but has no influence on ScpC expression. In the csrR mutant, overproduced ScpC inhibits the migration of neutrophils, and upregulated Rgg reduces the slo gene expression. In the csrS mutant, inactive form of CsrR leads to the overproduction of ScpC, which inhibits the migration of neutrophils, and decrease of Rgg leads to the overproduction of SLO, which kills neutrophils.