A scabies mite serpin interferes with complement-mediated neutrophil functions and promotes staphylococcal growth

Abstract

Background: Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus.

Methodology/principal findings: Blood killing of S. aureus was measured in whole blood bactericidal assays, counting viable bacteria recovered after treatment in fresh blood containing active complement and phagocytes, treated with recombinant SMSB4. SMSB4 inhibited the blood killing of various strains of S. aureus including methicillin-resistant and methicillin-sensitive isolates. Staphylococcal growth was promoted in a dose-dependent manner. We investigated the effect of SMSB4 on the complement-mediated neutrophil functions, namely phagocytosis, opsonization and anaphylatoxin release, by flow cytometry and in enzyme linked immuno sorbent assays (ELISA). SMSB4 reduced phagocytosis of S. aureus by neutrophils. It inhibited the deposition of C3b, C4b and properdin on the bacteria surface, but did not affect the depositions of C1q and MBL. SMSB4 also inhibited C5 cleavage as indicated by a reduced C5b-9 deposition.

Conclusions/significance: We postulate that SMSB4 interferes with the activation of all three complement pathways by reducing the amount of C3 convertase formed. We conclude that SMSB4 interferes with the complement-dependent killing function of neutrophils, thereby reducing opsonization, phagocytosis and further recruitment of neutrophils to the site of infection. As a consequence secreted scabies mites complement inhibitors, such as SMSB4, provide favorable conditions for the onset of S. aureus co-infection in the scabies-infected microenvironment by suppressing the immediate host immune response.

Figure 1. SMSB4 reduces the blood killing of S. aureus strain Xen29 in whole blood (A) and pyoderma isolates of S. aureus (B).

SMSB4 promotes bacteria growth in a concentration dependent manner (C) similarly to CVF (D). S. aureus Xen29 or pyoderma isolates MRSA strains (HS16, M34), MSSA strains (HS56, M5) were harvested from mid-log growth phase culture. Bacteria (1×10⁵ cfu/ml) were challenged with whole blood pre-treated with either 100 µg/ml SMSB4, positive control 10 µg/ml CVF, negative controls 100 µg/ml BSA or GVB²⁺ buffer only. S. aureus cells in PBS only without blood was also included to illustrate that the reduction in bacteria number was due to blood killing (A). Numbers of bacteria were counted as cfu/ml at various time points (A) or at 3 h (B, C, D). Bacterial recovery was calculated as a percentage of the challenge dose. Results are shown as 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. **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant (B).

Figure 2. SMSB4 reduces phagocytosis of S. aureus by neutrophils.

FITC-labeled bacteria (10⁵ cfu) was opsonized either with 20% NHS or heated serum and challenged with neutrophils (10⁶ cells). Serum samples were pre-treated for 30 min at 37°C with 50 µg/ml SMSB4, 50 µg/ml BSA (A), or various concentrations of SMSB4 or BSA (B). Uptake of FITC by neutrophils was measured at various time points over 1 h (A) or at 40 min (B). Results are shown as means ± SEM from three independent experiments.

Figure 3. SMSB4 reduces deposition of C3b (A) and MAC complex (SC5b-9) formation (B).

The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×10⁶ cfu/ml of S. aureus. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were 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.

Figure 4. Effect of SMSB4 on the depositions of C4b (A), C1q, MBL and properdin (B) on S. aureus cells.

The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×10⁶ cfu/ml of S. aureus. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were 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; ns, not significant (B).