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, 25 (19), 4628-37

A Streptococcal Protease That Degrades CXC Chemokines and Impairs Bacterial Clearance From Infected Tissues

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A Streptococcal Protease That Degrades CXC Chemokines and Impairs Bacterial Clearance From Infected Tissues

Carlos Hidalgo-Grass et al. EMBO J.

Abstract

Group A Streptococcus (GAS) causes the life-threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL-8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two-component system-SilA/B. In addition, we demonstrate that in vitro, ScpC degrades the CXC chemokines: IL-8 (human), KC, and MIP-2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC-deficient mutant is innocuous for untreated mice but lethal for PMN-depleted mice. ScpC degrades KC and MIP-2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response.

Figures

Figure 1
Figure 1
Identification of ScpC as a putative IL-8 protease. (A) The effect of SilCR on the transcription of GAS serine peptidase. The abundance of the indicated serine peptidase transcripts relative to that of gyrA was determined by real-time RT–PCR in RNA derived from WT grown to OD of 0.4 at 600 nm in the absence (clear bars) or presence (shaded bars) of SilCR (10 μg/ml). The values are mean obtained from analysis in duplicate of three independent RNA samples. Error bars represent standard deviation (s.d.). SilCR downregulates the transcription of scpC. P<0.001 (Student's test). (B) Genomic arrangement and domain organization of ScpC. Upper panel: The arrows depict the identified ORFs and their direction of transcription. The 5′ and 3′ EcoRV sites at positions 1801 and 2554 bp from the beginning of scpC were used to replace the internal scpA coding sequence with Ωkm2. spy0421 encodes a product of 236 aa with no homology to characterized proteins. Lower panel: The map of the motifs identified in the predicted sequence of ScpC includes the following: the pre-pro (PP) domain (residues 1–123) containing the signal sequence (residues 1–34) depicted as an arrow, protease domain (PR) (residues 124–688) containing Asp, His, and Ser forming the catalytic triad; the A domain (residues 689–1128) and the B/H domain (residues 1129–1560), the cell wall domain (W) (residues 1561–1613), the cell wall anchor domain (AN) (residues 1613–1647), and the LPxTG motif starting at residue 1613. The DNA region removed by the EcoRV digestion contains a segment of 251 aa (hatched) including Ser617 of the catalytic triad.
Figure 2
Figure 2
ScpC significantly contributes to virulence in the murine model of GAS necrotizing soft tissue infections. (A) Inactivation of scpC but not of scpA abolished lethality of GAS. Mice were injected subcutaneously with 1 × 108 CFU of WT (formula image n=32), ΔscpA (formula image n=31), and ΔscpAscpC (formula image n=28) and survival was monitored daily. The Kaplan–Meier analysis performed on five different experiments shows P<0.001 for ΔscpAscpC versus WT or versus ΔscpA and P>0.05 for WT versus ΔscpA (log rank (Mantel–Cox) test). (B) Weight change in control mice (•) and mice challenged with 1 × 108 CFU of WT (♦), ΔscpA (▪), and ΔscpAscpC (▴). Experiments were repeated five times with similar results. (C) Mean total lesion size (cm2). Mice were injected with 1 × 108 CFU of WT (♦ n=9), ΔscpA (▪ n=15), and ΔscpAscpC (▴ n=9), photographed daily, and the area was calculated using ImageJ software. Error bars represent s.d. WT and ΔscpA versus ΔscpAscpC P<0.05 (Student's test) for time points 1 and 2 and P<0.001 (Student's test) for time points 3 and 4. WT versus ΔscpA P>0.05 (Student's test) at all time points.
Figure 3
Figure 3
ScpC is absolutely required for chemokine degradation. (A) ScpC is responsible for IL-8 degradation. The determination of IL-8 after 2 h of proteolysis in control (in the absence of bacterial supernatant, 100%) and in supernatants of WT, ΔscpA, ΔscpAscpC, ΔscpAscpC-pLscpC, and ΔscpAscpC-pLZ was conducted by ELISA. The values are the mean obtained from analysis in duplicate (n=3). Error bars represent s.d. (B) IL-8 is cleaved by WT supernatant. Control represents IL-8 incubated for 16 h in the absence of bacterial supernatant. Samples containing IL-8 and supernatants from WT, ΔscpAscpC, and ΔscpA were incubated for the indicated time points and subjected to 17.5% SDS–PAGE, which was then silver stained. The arrows represent the molecular weights of IL-8 and of the generated 6 kDa form. (C) ScpC is responsible for KC degradation. The determination of KC after 16 h of proteolysis in control (in the absence of bacterial supernatant, 100%) and supernatants of WT, ΔscpA, ΔscpAscpC, and ΔscpAscpC-pLScpC was conducted by ELISA. The values are the mean obtained from analysis in duplicate (n=3). Error bars represent s.d. (D) ScpC is responsible for MIP-2 degradation. The determination of MIP-2 after 16 h of proteolysis in control (in the absence of bacterial supernatant, 100%) and supernatants of WT, ΔscpA, ΔscpAscpC, and ΔscpAscpC-pLScpC was conducted by ELISA. The values are mean obtained from analysis in duplicate (n=3). Error bars represent s.d. (E) Kinetics of chemokine degradation. WT supernatant was incubated with IL-8 (formula image), KC (formula image), MIP-2 (formula image), RANTES (formula image), LIX74 (formula image), or LIX93 (formula image) and the concentration of the chemokines at the indicated time points was determined by ELISA. 100% represents the concentration of chemokines at time zero. Error bars represent s.d.
Figure 4
Figure 4
ScpC is responsible for degradation of KC and MIP-2 in infected skin. (A) KC levels (ng/mg protein) extracted from skin lesions of mice inoculated with WT (formula image), ΔscpA (▵), and ΔscpAscpC (▴). Each point represents mean of the determinations performed on three mice killed at the indicated time points after infection. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P<0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA for each time point. P>0.05 (Student's test) for WT versus ΔscpA. (B) MIP-2 levels (ng/mg protein) extracted from skin lesions of mice inoculated with WT (formula image), ΔscpA (□), and ΔscpAscpC (▪). Each point represents mean of the determinations performed on three mice killed at the indicated time points after infection. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P<0.01 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA. P>0.05 (Student's test) for WT versus ΔscpA for each time point. (C) LIX levels (ng/mg protein) extracted from skin lesions of mice inoculated with WT (formula image), ΔscpA (○), and ΔscpAscpC (•). Each point represents mean of the determinations performed on three mice killed at the indicated time points after infection. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P>0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA. P>0.05 (Student's test) for WT versus ΔscpA for each time point. (D) KC levels (ng/spleen) in spleens of three mice from the same groups of mice described in panel A. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P>0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA for each time point. (E) MIP-2 levels (ng/spleen) in spleens of three mice from the same groups of mice described in panel B. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P>0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA for each time point. (F) KC levels (ng/ml) in sera of three mice from the same groups of mice described in panel A. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P>0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA for each time point. (G) MIP-2 levels (ng/ml) in sera of three mice from the same groups of mice described in panel B. Assays by ELISA were conducted in duplicate. Error bars represent s.d. P>0.05 (Student's test) of ΔscpAscpC versus WT or versus ΔscpA for each time point.
Figure 5
Figure 5
ScpC impairs PMN recruitment. (A) Forty-eight hours after inoculation, lesional (GAS) and control (PBS) 6 mm punch biopsy specimens were taken and the amount of MPO activity (units/mg protein) was determined. Each bar represents the mean±s.d. of two determinations conducted on four specimens. P<0.001 (Student's test) of ΔscpAscpC versus either WT or ΔscpA. (B) Representative photomicrographs of sections labeled with H&E prepared 2 days after inoculation with WT and its derived mutants. The gray arrow indicates presence of bacteria whereas the black arrow indicates presence of PMN. (C) PMN depletion renders mice sensitive to the ΔscpAscpC mutant. Mice were injected subcutaneously with 1 × 108 CFU of ΔscpAscpC and survival was monitored daily in cyclophosphamide-treated (formula image n=7), RB6-8C5-treated (formula image n=10), or PBS-treated (formula image n=17) mice. The Kaplan–Meier analysis shows statistically significant difference (P<0.001) in the survival of the three groups of mice (using the log rank (Mantel–Cox) test).

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