Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

Abstract

Background Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. Methods Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. Results Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. Conclusions Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes

“…To assess the pro-inflammatory response by moDCs, cells were infected with four GAS strains harboring functional CovR/S (CovR/S + ; 2006, 5448, 5626, 8157) or four covS mutant strains carrying non-functional CovR/S (CovR/S − ; 2002, MGAS5005, 5448AP, 8003) and IL1β, IL-8 as well as IL-18 release in response to infections was measured. The sequences as well as CovR/S functionality of the respective strains were previously assessed [ 54 ] (Additional file 2 : Table S2) and confirmed via SpeB proteolytic activity on casein agar plates (Additional file 1 : Fig. S2).…”

“…The following GAS strains were used: NSTI/STSS strains 5448, 5626, 8003, and 8157 (provided by Donald E. Low, Mount Sinai Hospital, Toronto, Canada) [ 28 , 34 ], 2002 and 2006 from the INFECT cohort [ 54 ], MGAS5005 [ 60 ], 5448AP [ 69 ], 5448Δ emm1 [ 39 ], and 5448Δ slo [ 64 ]. All strains were cultured in Todd-Hewitt broth (Carl Roth) supplemented with 1.5% (w/v) yeast extract (Carl Roth) at 37 °C.…”

“…Hemolytic activity of GAS stationary culture supernatants was assessed as previously described [ 36 ]. To assess CovR/S functionality, SpeB secretion and activity were tested via casein digestion assay [ 54 ]. Serial dilutions of bacteria were plated on modified Columbia agar containing 3% (w/v) skim milk (both Sigma-Aldrich) following incubation under 37 °C and 5% CO 2 atmosphere for 24 h. SpeB producers, which harbor functional CovR/S, were characterized by a clearance zone around the colonies, whereas non-producers (dysfunctional CovR/S) had no zone of clearance.…”

“…Analyses of CovR and CovS sequences were performed as described previously [ 54 ]. In brief, genes were identified via BLAST (v. 2.9.0 +) using a custom Python script.…”