Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection

Abstract

Neutrophil chemoattractant interleukin (IL)-8 is cleaved and inactivated by the Streptococcus pyogenes cell envelope protease SpyCEP. A range of clinical S. pyogenes strains of differing emm type demonstrated SpyCEP activity, although transcription of the SpyCEP gene cepA differed 1000-fold between isolates. Disruption of the 2-component regulatory system covR/S in pharyngeal isolates increased cepA transcription 100-fold; this finding is consistent with endogenous CovR/S-mediated repression of cepA being responsible for low SpyCEP expression in some S. pyogenes strains associated with pharyngitis. Among patients with invasive S. pyogenes infection, disease severity and outcome were associated with the SpyCEP activity of the isolate. Lethal invasive isolate H292 (emm81) expressed more cepA than did other tested isolates. This strain carried a unique covR mutation that impaired binding to the cepA promoter. CovR/S sequence comparison in other clinical isolates revealed community-wide dissemination of covS mutations but not covR mutations. The results highlight a potential hazard and underline the importance of continuing molecular epidemiological surveillance for community-wide dissemination of CovR/S mutant hyperinvasive strains.

Figure 1

Inhibition of interleukin(IL)-8 degrading activity by anti-Streptococcus pyogenes cell envelope proteinase (SpyCEP) serum. IL-8 degrading activity was measured in a range of isolates of different emm-types (emm 81, 1, 2 3, 5, 12, 22, 28, 44, 75, 78, 89). Bacterial supernatants were co-incubated with 5ng/ml IL-8 and either normal rabbit serum or anti-SpyCEP serum. The percentage of IL-8 degraded represents comparison with IL-8 incubated with serum only. Data are the mean of 3 separate experiments measured in duplicate. Invasive isolates (black squares) had higher cleaving activity compared to non-invasive isolates (white triangles) (P<0.011, Mann-Whitney U test,). Dotted line: reference emm1 strain H305

Figure 2

A, Comparison of cepA transcription between invasive and non-invasive isolates. Target gene transcripts were measured as the no. of copies per 10,000 copies of the housekeeping gene gyrA, with reference to a plasmid of known copy number. Data are grouped by isolate source. Reference strain: H305, M1T1 scarlet fever isolate. Strain H292 is shown as an outlier. Invasive isolates expressed more cepA than did non-invasive isolates, (P<0.046, Mann-Whitney U test). B, Correlation between copies of cepA and copies of hasA. The expression of cepA correlated positively with the expression of hasA (r= 0.67, Spearman rank correlation; P<0.003).

Figure 3

De-repression of cepA in a low producer of Streptococcus pyogenes cell envelope proteinase (SpyCEP), by covR/S mutation. A, Western blot analysis for SpyCEP in culture supernatant and cell wall extract from wild type (WT) emm75 pharyngeal isolate and ΔcovR/S mutant grown to early-log (EL), mid-log (ML) and late-log (LL) phases of growth. B, Coomassie blue-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing IL-8 cleavage after incubation with supernatant from WT or ΔcovR/S mutant. IL-8 incubated in Todd-Hewitt broth alone was used as a control. As a positive control for cleavage, IL-8 was incubated with supernatant from the high SpyCEP producer, H292. C, No. of copies of cepA transcript per 10,000 copies of gyrA transcript measured by real-time polymerase chain reaction comparing WT (black bars) and ΔcovR/S mutant (white bars) at EL, ML and LL phase. Each bar denotes the mean of 3 independent samples measured in triplicate (+standard deviation). The effect of the CovR/S mutation was reproduced in another pharyngeal emm2 isolate.

Figure 4

Association of Streptococcus pyogenes cell envelope proteinase (SpyCEP) activity with disease outcome. A, SpyCEP activity was measured in the culture supernatant of clinical invasive S. pyogenes disease isolates by means of enzyme-linked immunosorbent assay (ELISA) performed to calculate interleukin(IL)-8 degrading activity as a percentage of the control, averaged over 3 separate experiments. Isolates were matched to the disease outcome of the patient from which they originated (survivors or non-survivors). The median value of the data is denoted by a horizontal line. SpyCEP activity was significantly higher in strains from non-survivors (P< 0.016, Mann-Whitney U test). B, Association of patient serum IL-6 levels with the SpyCEP activity of the causative isolate. IL-6 was measured by ELISA performed on patient serum samples obtained at the onset of bacteremia. High IL-6 levels were associated with high SpyCEP activity apparent at SpyCEP activity >90% (dotted line).

Figure 5

Association of CovR substitution with high cepA and hasA transcription. Copies of cepA (A) and hasA (B) transcript expressed by H292, compared to other contemporaneous emm81 isolates (M81a-e) measured as copies per 10,000 copies of gyrA. Each bar denotes the mean (+standard deviation) of 3 independent samples obtained at early log (white bar), mid log (stripe bar) and late log (black bar) phase, measured in triplicate.

Figure 6

Reduction in binding associated with mutant CovR, compared with wild-type CovR. Electromobility shift assays with wild-type CovR or mutant CovR either without phosphorylation (left) or with phosphorylation by preincubation with acetyl phosphate (right). The amount of protein added is expressed in micrograms above each lane. Positions of unbound DNA and CovR-promoter complexes are denoted by arrows. A Binding of wild-type and mutant CovR to a 295bp region of the cepA promoter. B Binding of wild-type or mutant CovR to a 344bp region of the hasA promoter.