Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway

Abstract

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.

Figure 1. SMIPP-Ss D1 (A) and I1 (B) inhibit the lectin pathway from the level of MBL.

Microtitre plates coated with 100 µg/ml mannan were incubated with 2% NHS pre-incubated with various concentrations of SMIPP-S or BSA as a negative control. Complement activation was assessed by detection of deposited MBL, C4b, C3b and C9 with specific antibody. The absorbance value obtained in the absence of SMIPP-S was defined as 100%. The means of three independent experiments performed in duplicate +/− standard deviation (SD) are shown. Statistical significance of observed differences was calculated by two-way ANOVA and Bonferroni post test. *, p<0.05, **, p<0.01, ***, p<0.001, ns, p>0.05.

Figure 2. Investigating binding between MBL and MASPs with SMIPP-Ss.

To ascertain if SMIPP-Ss bind to MBL when it is associated with MASPs in a complex, immobilised D1 (A) or I1 (B), mannan as a positive control or BSA as a negative control were incubated with increasing concentrations of MBL in complex with MASPSs. MBL was then detected with specific antibody. To determine if SMIPP-S D1 or I1 bind directly to MASP-1 or MASP-2, immobilised SMIPP-S D1, I1 or BSA as a negative control were incubated with rMASP-1 (C) or rMASP-2 (D). rMASP-1 and rMASP-2 were then detected with specific antibody. The means of three independent experiments performed in duplicate +/− standard deviation (SD) are shown. Statistical significance of observed differences was calculated by two-way ANOVA and Bonferroni post test. *, p<0.05, **, p<0.01, ***, p<0.001, ns, p>0.05.

Figure 3. Investigating the mechanism of inhibition of lectin pathway activation via the MBL complex.

To determine if MASP-1 (A) or MASP-2 (B) remain associated with MBL in the presence of SMIPP-S D1 or I1 increasing concentrations of immobilised SMIPP-S protein or BSA as a negative control were incubated with MBL complexed with MASPs. MASP-1 and MASP-2 were then detected with specific antibody. The means of three independent experiments performed in duplicate +/− standard deviation (SD) are shown. Statistical significance of observed differences was calculated by two-way ANOVA and Bonferroni post test. *, p<0.05, **, p<0.01, ***, p<0.001, ns, p>0.05.

Figure 4. Sequence alignment of the SMIPP-S family.

Sequence alignment of the SMIPP-S family highlighted a cluster of conserved surface exposed residues surrounding K103 and K108. Aligned residues are shaded indicating high (black) to low (grey) identity. D1 and I1 sequences are coloured yellow, conserved surface exposed residues are boxed in red and residues K103 and K108 are coloured green.

Figure 5. SMIPP-S mutants have lost inhibitory function in the lectin pathway.

To determine if D1 mutants (A) and I1 mutants (B) had lost inhibitory function in the lectin pathway deposition assays were performed. Microtitre plates coated with 100 µg/ml mannan were incubated with 2% NHS pre-incubated with 5 µg/ml D1 (positive control) or D1 mutant (A), 25 µg/ml I1 (positive control) or I1 mutant, or BSA as a negative control. Complement activation was assessed by detection of deposited MBL with specific antibody. The means of three independent experiments performed in duplicate +/− standard deviation (SD) are shown. Statistical significance of observed differences was calculated by two-way ANOVA and Bonferroni post test. *, p<0.05, **, p<0.01, ***, p<0.001, ns, p>0.05.