Complement inhibition by Sarcoptes scabiei protects Streptococcus pyogenes - An in vitro study to unravel the molecular mechanisms behind the poorly understood predilection of S. pyogenes to infect mite-induced skin lesions

Abstract

Background: On a global scale scabies is one of the most common dermatological conditions, imposing a considerable economic burden on individuals, communities and health systems. There is substantial epidemiological evidence that in tropical regions scabies is often causing pyoderma and subsequently serious illness due to invasion by opportunistic bacteria. The health burden due to complicated scabies causing cellulitis, bacteraemia and sepsis, heart and kidney diseases in resource-poor communities is extreme. Co-infections of group A streptococcus (GAS) and scabies mites is a common phenomenon in the tropics. Both pathogens produce multiple complement inhibitors to overcome the host innate defence. We investigated the relative role of classical (CP), lectin (LP) and alternative pathways (AP) towards a pyodermic GAS isolate 88/30 in the presence of a scabies mite complement inhibitor, SMSB4.

Methodology/principal findings: Opsonophagocytosis assays in fresh blood showed baseline immunity towards GAS. The role of innate immunity was investigated by deposition of the first complement components of each pathway, specifically C1q, FB and MBL from normal human serum on GAS. C1q deposition was the highest followed by FB deposition while MBL deposition was undetectable, suggesting that CP and AP may be mainly activated by GAS. We confirmed this result using sera depleted of either C1q or FB, and serum deficient in MBL. Recombinant SMSB4 was produced and purified from Pichia pastoris. SMSB4 reduced the baseline immunity against GAS by decreasing the formation of CP- and AP-C3 convertases, subsequently affecting opsonisation and the release of anaphylatoxin.

Conclusions/significance: Our results indicate that the complement-inhibitory function of SMSB4 promotes the survival of GAS in vitro and inferably in the microenvironment of the mite-infested skin. Understanding the tripartite interactions between host, parasite and microbial pathogens at a molecular level may serve as a basis to develop improved intervention strategies targeting scabies and associated bacterial infections.

Fig 1. GAS clinical isolates are naturally resistant to blood killing (A) and deposition of C1q, MBL, FB (B) and C3b, indicative of opsonisation (C) on the cell surface of GAS 88/30.

Skin strains 88/30, PRS30 (emm cluster D), throat strains PRS8, 5448 (emm cluster A-C) and skin/throat strains PRS55, PRS15 (emm cluster E) were harvested from mid-log growth phase culture (OD₆₀₀ 0.35). Suspension of GAS in PBS (1 ×10³ cfu/ml) were added into fresh blood pre-treated with GVB²⁺ buffer. After 3 h incubation samples were plated in duplicate on CBAC agar plates and bacteria were enumerated as cfu/ml. GAS cell in PBS without blood at time 0 (T0) was plated simultaneously and the numbers of bacteria grown served as the baseline for normalisation and for calculating the fold difference of bacteria numbers grown from the experimental samples (A). Maxisorp 96-well plates coated with GAS cells were incubated with increasing concentrations of NHS (B) or with 5% NHS or sera depleted of either C1q (C1^-) or FB (FB^-) and serum naturally deficient in MBL (MBL^d) (C). Complement deposition was detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm (B, C). Data represent the means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using two way ANOVA with Tukey’s multiple comparison test (A, B) and one way ANOVA with Dunnett’s multiple comparison tests (C). **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant.

Fig 2. Level of C1q deposition (A), effect of anti-GAS antibodies on C3b deposition (B), and the role of IgG on the CP-dependent deposition of various complement components (C) on the surface of GAS 88/30.

Complement deposition was detected by ELISA using primary human-specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Depositions of IgG and the complement components C1q, C4b and C3b are shown in panels (left to right) in the presence (white column) and absence (grey column) of IgG (C). Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using one way ANOVA with Dunnett’s multiple comparison tests with a single pooled variance NHS (A, B) and two way ANOVA with Sidak’s multiple comparison test (C). **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant.

Fig 3. Scabies mite complement inhibitor SMSB4 reduces the baseline immunity of GAS clinical isolates in fresh blood (A) and promotes the growth of GAS skin strain 88/30 in a dose-dependent manner (B).

Skin strains 88/30, PRS30 (emm cluster D), throat strains PRS8, 5448 (emm cluster A-C) and skin/throat strains PRS55, PRS15 (emm cluster E) (A) or GAS 88/30 only (B) were harvested from mid-log growth phase culture (OD₆₀₀ 0.35). GAS diluted in PBS (1 ×10³ cfu/ml) were added into fresh blood pre-treated with either 2 μM (A) or a range of concentrations (B) of either SMSB4 or BSA. After 3 h incubation samples were plated in duplicate on CBAC agar plates and bacteria were enumerated as cfu/ml. The challenge dose of GAS cells in PBS without blood (A) or the challenge dose in blood with GVB²⁺ buffer (B) was plated simultaneously and the numbers of bacteria grown served as the baseline for normalisation and for calculating the fold difference of bacteria numbers from the experimental samples. Data represent the means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using two way ANOVA with Tukey’s (A) or Sidak’s (B) multiple comparison test. **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig 4. SMSB4 causes reduction in C4b deposition (A) factor B deposition (B) and properdin deposition (C) on GAS 88/30.

Cells were incubated with 5% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Complement deposition was detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between BSA and SMSB4 treated samples were estimated using two way ANOVA with Sidak’s multiple comparison test. **, p<0.01; ***, p<0.001, ****, p<0.0001.

Fig 5. Effect of SMSB4 on opsonisation measured as C3b deposition (A) and the release of anaphylatoxin measured indirectly by ELISA as deposition of sC5b9 complex (B) on GAS 88/30.

Cells were incubated with 5% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Complement deposition was detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between BSA and SMSB4 treated samples were estimated using two way ANOVA with Sidak’s multiple comparison test. **, p<0.01; ***, p<0.001, ****, p<0.0001.