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SystemParalogs Inhibit the Human Complement Scabies Mite Inactivated Serine Protease

Fischer and Anna M. BlomRobert N. Pike, Ashley M. Buckle, David J. Kemp, Katja Frida C. Bergström, Simone Reynolds, Masego Johnstone,

http://www.jimmunol.org/content/182/12/7809doi: 10.4049/jimmunol.0804205

2009; 182:7809-7817; ;J Immunol 

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Scabies Mite Inactivated Serine Protease Paralogs Inhibit theHuman Complement System1

Frida C. Bergstrom,2* Simone Reynolds,2†‡ Masego Johnstone,† Robert N. Pike,§

Ashley M. Buckle,§ David J. Kemp,† Katja Fischer,2† and Anna M. Blom2,3*

Infestation of skin by the parasitic itch mite Sarcoptes scabiei afflicts 300 million people worldwide and there is a need for noveland efficient therapies. We have previously identified a multigene family of serine proteases comprising multiple catalyticallyinactive members (scabies mite-inactivated protease paralogs (SMIPPs)), which are secreted into the gut of S. scabiei. SMIPPs arelocated in the mite gut and in feces excreted into the upper epidermis. Scabies mites feed on epidermal protein, including hostplasma; consequently, they are exposed to host defense mechanisms both internally and externally. We found that two recom-binantly expressed SMIPPs inhibited all three pathways of the human complement system. Both SMIPPs exerted their inhibitoryaction due to binding of three molecules involved in the three different mechanisms which initiate complement: C1q, mannose-binding lectin, and properdin. Both SMIPPs bound to the stalk domains of C1q, possibly displacing or inhibiting C1r/C1s, whichare associated with the same domain. Furthermore, we found that binding of both SMIPPs to properdin resulted in preventionof assembly of the alternative pathway convertases. However, the SMIPPs were not able to dissociate already formed convertases.Immunohistochemical staining demonstrated the presence of C1q in the gut of scabies mites in skin burrows. We propose thatSMIPPs minimize complement-mediated gut damage and thus create a favorable environment for the scabies mites. The Journalof Immunology, 2009, 182: 7809–7817.

S cabies is a parasitic infection of the skin that causes sig-nificant morbidity within socially disadvantaged popula-tions, such as Aboriginal communities in Northern Aus-

tralia, and afflicts 300 million people worldwide. It is caused by theburrowing of the ectoparasitic itch mite Sarcoptes scabiei in thelower stratum corneum layer of the skin. Scabies often results inserious secondary Streptococcus pyogenes infections that may leadto rheumatic fever and heart disease (1, 2). Emerging resistance tothe currently available therapeutics permethrin and ivermectin em-phasizes the need to identify potential targets for chemotherapeuticand/or immunological intervention (3).

Innate immunity, including the complement system found inhuman plasma, is one of the main host defenses against infections.Scabies mites imbibe serum while burrowing (4, 5) and must there-fore have mechanisms to protect against adverse effects of com-plement activation. The complement system may be triggered by

various ligands and its activation can proceed via three enzymaticcascades: the classical, alternative, and lectin pathways. The clas-sical pathway is initiated by the binding of the C1 complex (com-prised of C1q, C1r, and C1s) to immune complexes, while thelectin pathway recognizes particular carbohydrates via mannose-binding lectin (MBL)4 and other plasma lectins. The alternativepathway is triggered due to structural instability of the C3 mole-cule leading to autoactivation, direct binding of properdin (6), or asan amplification loop to the two other pathways. Activation of anyof these three pathways leads to the opsonization of the target withthe complement component C3b, which mediates phagocytosis.Formation of C3b proceeds to the assembly of a membrane attackcomplex and release of the chemoattractant anaphylatoxin C5a.Furthermore, the activated complement cascade affects adaptiveimmunity at the level of both B cells (7) and T cells (8).

Every successful pathogen must have a strategy to attenuatecomplement to establish persistent infection. Some pathogens uti-lize a number of inhibitory molecules to block some of the mul-tiple components of the complement cascade and thereby evadecomplement attack (9). Identification of these molecules and func-tional characterization of their roles at the pathogen-host interfaceare important facets in the emerging field of infection biology. Thediverse complement evasion strategies among many metazoan par-asites (10) are representative of common mechanisms for immuneand complement defense among human pathogens (9). Schisto-somes, for example, are reported to possess a plethora of regula-tory proteins, including molecules acquired from the host, whichimpede the complement cascade (11). As an example, paramyosinof Schistosoma mansoni binds to the human complement proteins

*Department of Laboratory Medicine, Wallenberg Laboratory, University HospitalMalmo, Lund University, Malmo, Sweden; †Infectious Diseases and ImmunologyDivision, Queensland Institute of Medical Research, Brisbane, Australia; ‡School ofVeterinary Sciences, The University of Queensland, St. Lucia, Australia; and §De-partment of Biochemistry & Molecular Biology, Monash University, Clayton, Vic-toria, Australia

Received for publication December 17, 2008. Accepted for publication April16, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by Program Grant 496600 and a fellowship to D.J.K. fromthe Australian National Health and Medical Research Council and by grants from theSwedish Research Council, Swedish Foundation for Strategic Research, Foundationsof Osterlund, Kock, King Gustav V’s 80th Anniversary Foundation, Knut and AliceWallenberg, Inga-Britt and Arne Lundberg, and research grants from the UniversityHospitals in Malmo.2 F.C.B., S.R., K.F., and A.M.B. contributed equally to the study.3 Address correspondence and reprint requests to Dr. Anna M. Blom, Department ofLaboratory Medicine, Medical Protein Chemistry, The Wallenberg Laboratory, Floor4, University Hospital Malmo entrance 46, Lund University, SE-205 02 Malmo, Swe-den. E-mail: anna.blom@med.lu.se

4 Abbreviations used in this paper: MBL, mannose-binding lectin; C4BP, C4b-bind-ing protein; HDM, house dust mite; NHS, normal human serum; SMIPP, scabies miteinactivated protease paralog; RT, room temperature; MES, 2-(N-morpholino)ethane-sulfonic acid.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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C8 and C9 and thereby inhibits complement activation at the ter-minal stage (12). Schistosomes may also capture C2 and C3 (11).In addition, a surface FcR might bind Ig in an unfavorable orien-tation and thereby limit its ability to activate complement. In miceinfested with larval Strongyloides stercoralis, C3 was not requiredfor the development of adaptive immunity, but was necessary forthe larval killing process during both protective innate and adap-tive immune responses (13).

Immune evasion strategies of scabies mites are largely un-known. The scarcity of molecular data on scabies is due to thedifficulty in obtaining mites in sufficient numbers, because the par-asite burden is generally very low and an in vitro culture systemfor propagating mites is not available. Therefore, we constructedcDNA libraries from mRNA extracted from mites in skin shed intothe bedding of crusted scabies patients. An expressed sequence taglibrary of 43,776 sequences has been established from these clonesat the Australian Genome Research Facility (14). Using BLASTsearches, we identified, among others, multiple scabies mite ho-mologs of the group 3 serine protease allergens (15). Astigmatidhouse dust mites (HDM) are closely related to scabies and theirgroup 3 HDM allergens have been implicated as elicitors of theallergic response in asthma. They are serine proteases that are se-creted into the gut of the mites and assumed to play key roles indigestion (16). Hence, homologs of HDM group 3 allergens inscabies mites may be potential targets of immunotherapy.

The 33 sequences identified clustered into three major clades.Remarkably, all but one of the paralogs contain mutations in thecatalytic triad ruling out the possibility that they can act as pro-teases by a known mechanism. We have accordingly named thisfamily scabies mite inactivated protease paralogs (SMIPPs) (14).SMIPPs of each clade are present in the mite gut and excreted infecal pellets (4). We show here that representative SMIPPs fromtwo different subclades inhibit all three complement pathways,which may provide protection against gut damage by complementin serum imbibed by the mites.

Materials and MethodsExpression of SMIPP protein in Pichia pastoris and purification

The nomenclature of the molecules investigated here is based on a phylo-genetic tree illustrating the evolutionary relationships between the SMIPPs(17). Recombinant SMIPP-Ss I1 and D1 were produced in P. pastoris bycloning SMIPP-coding sequences retrieved from the original cDNA clonesYv6023A04 (GenBank accession no. AY333081; www.ncbi.nlm.nih.gov/GenBank) and YvT004A06 (GenBank accession no. AY333085) corre-sponding to the mature sequences predicted (15). To direct secretion of theexpressed protein into the medium, all constructs placed the predicted Nterminus flush with the Kex2 signal peptide cleavage site in the transfervector pPICZ�A (Invitrogen). A stop codon at the 3� end of the SMIPPsequence was introduced to ensure that the recombinant protein did notinclude any of the C-terminal tags of the vector. The plasmid was propa-gated in Escherichia coli strain TOP10F�, linearized with SacI, and elec-troporated into P. pastoris KM71H (Invitrogen). Media for colony growthand biomass or induction cultures were made according to the manufac-turer’s instructions. Cells were initially plated onto supported nitrocellu-lose filters placed on agar plates containing 100 �g/ml Zeocin (Invitrogen).After a 24-h incubation at 29°C, filters were moved onto YPDS mediumcontaining 2 mg/ml Zeocin for selection of transformants with increasednumbers of SMIPP gene copies. To ensure pure clonal isolates, singlecolonies were plated twice on two subsequent YPDS/Zeocin agar plates.Transformants were assessed to select for the highest expressing cloneaccording to the manufacturer’s manual. Biomass cultures were grown for24 h and induction cultures for 60–70 h with shaking at 29oC. SMIPPexpression was detected on a Western blot using mouse antiserum gener-ated against purified recombinant SMIPP molecules produced in E. coli.Correct processing of the recombinant protein at the Kex2 cleavage sitewas confirmed after purification by N-terminal sequencing at the PeptideBiology Laboratory (Monash University, Clayton, Victoria, Australia)(data not shown).

Mature SMIPP protein secreted from P. pastoris was purified as fol-lows: expression culture supernatant was collected following centrifuga-tion at 12,000 � g for 45 min at 4°C and stored at �80°C until required.The supernatant was thawed and prepared for hydrophobic interactionchromatography by the gradual addition of ammonium sulfate to a finalconcentration of 1.5 M and adjustment of pH to 5.0. The preparation wascentrifuged for 30 min at 20,000 � g. The supernatant was filtered througha 0.45-�m filter and the filtrate was loaded onto a 5-ml HiTrap phenyl-Sepharose column (GE Healthcare), pre-equilibrated with 1.5 M ammo-nium sulfate and 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and10% (v/v) glycerol (pH 5.0). Unbound material was removed by washingwith equilibration buffer. Protein was eluted stepwise with 50 ml each of1.0, 0.5, and 0 M ammonium sulfate. Fractions were subjected to dialysisagainst 50 mM MES and 10% (v/v) glycerol (pH 5.0) and those containingSMIPP protein, as identified by electrophoresis on SDS-PAGE, werepooled, loaded onto a 5-ml HiTrap SP- Sepharose FF column (GE Health-care) equilibrated in 50 mM MES (pH 5.0), and washed with 50 ml of thesame buffer. A linear gradient of 0–0.4 M NaCl over 30 ml was applied tothe column and purification was monitored by electrophoresis of fractionson 10% SDS-PAGE and Western blotting analysis. On average, yields of0.9–1.3 mg of pure protein per 200 ml of culture were obtained.

Mutagenesis of N-glycosylation sites

When subjected to SDS-PAGE, each SMIPP produced a major bandslightly larger than the same molecule produced in E. coli plus a smear of

FIGURE 1. Characterization of purified SMIPP proteins. A and B, Thepurified proteins were separated on 10% SDS-PAGE gels under reducingconditions and visualized by staining with Coomassie blue R-250. A,SMIPP-S I1 expressed in P. pastoris: wild-type protein (lane 1), mutantprotein N84Q/N191Q (lane 2), N147Q (lane 3) and N84Q/N147Q/N191Q(lane 4), and E. coli-expressed wild-type protein (lane 5). B, SMIPP-S D1expressed in P. pastoris: wild-type protein (lane 1), mutant protein N145Q(lane 2), and N193Q (lane 3). SMIPP-S D1 protein with both N-glycosyl-ation sites mutated was unstable (data not shown). Lane 4 shows E. coli-expressed wild-type protein. The proteins used for the complement assaysreported here are indicated by arrows. C and D, The proteolytic activity ofthe SMIPPs was tested using degradation assays. Two concentrations ofeach SMIPP (100 and 200 �g/ml) were incubated with trace amountsof 125I-C4b (C) or 125I-C3b (D) for 1.5 h at 37°C. The reaction was ter-minated by addition of SDS-PAGE sample buffer, containing DTT, fol-lowed by heating at 95°C for 3 min. The proteins were separated on a10–15% gradient SDS-PAGE gel and visualized using PhosphorImager.Factor I, along with its cofactors C4BP (for C4b degradation) and factor H(for C3b degradation), was used as a positive control, as was interpain A,a bacterial proteinase. In the negative controls, only 125I-C4b or 125I-C3bwas added. (FI, factor I; FH, factor H).

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larger material (Fig. 1, A and B). The SMIPP-Ss I1 and D1 have three andtwo predicted N-glycosylation sites, respectively. Function can be hinderedby artifactual hyperglycosylation; therefore, we systematically mutated thepredicted glycosylation sites from N to Q at positions 84, 147, and 191 ofthe mature protein of SMIPP-S I1 and at position 145 and 193 of themature protein of SMIPP-S D1 singly and in combination by oligonucle-otide-based site-directed mutagenesis. Among the created Pichia mutants(Fig. 1, A and B), clones that expressed homogenous, nonglycosylatedproteins were chosen for additional experiments. These clones were des-ignated I1 and D1 for SMIPP-S I1 and SMIPP-S D1, respectively.

Proteins

C1, C2, C3b, C4b, C5, C6, C7, C8, C9, factor B, factor D, and properdinwere purchased from Complement Technology. Human MBL was pur-chased from Statens Serum Institutet (Copenhagen, Denmark). C1q (18),factor H (19), C4b-binding protein (C4BP) (20), and factor I (21) werepurified from human plasma as described previously. The stalk and headregions of C1q were prepared by partial proteolytic digestion of intact C1qas described previously (22, 23). Pepsin (Worthington Biochemical) wasused for preparation of the stalk region and purified collagenase (Clostrid-ium histolyticum, code CLSPA) for preparation of the head region waspurchased from the same company. C3b and C4b were labeled with 125Iusing the chloramine-T method (24). The specific activity was 0.4–0.5MBq/�g of protein. Interpain A was expressed and purified as describedpreviously (25).

C3b/C4b degradation assay

To confirm that the two SMIPPs tested in this study were indeed inactiveproteases and not able to cleave complement proteins, such as C3b or C4b,a degradation assay was performed. Two concentrations (100 and 200 �g/ml) of SMIPPs were mixed with trace amounts of 125I-labeled C3b or C4bin TBS in a total volume of 40 �l. As positive controls, 15 �g/ml factor I,along with 200 �g/ml of a cofactor (C4BP for the degradation of C4b orfactor H for the degradation of C3b), were added to the 125I-C3b or 125I-C4b. Recently, interpain A, a cysteine proteinase from Prevotella interme-dia, has been shown to inhibit complement by degrading C3 and C4 (25).Therefore, interpain A was also tested here as a positive control at a con-centration of 1 �M. The samples were incubated for 1.5 h at 37°C and thereaction was terminated by the addition of SDS-PAGE sample buffer withreducing agent (DTT). The samples were then incubated at 95°C for 3 minand separated on a 10–15% gradient SDS-PAGE gel. The separated pro-teins were visualized by using a PhosphorImager (Molecular Dynamics/GEHealthcare).

Hemolytic assay

To assess the activity of the classical pathway of complement, sheep eryth-rocytes (Swedish National Veterinary Institute) were washed three timeswith DGVB2� buffer (2.5 mM Veronal buffer (pH 7.3), 70 mM NaCl, 140mM glucose, 0.1% gelatin, 1 mM MgCl2, and 0.15 mM CaCl2), resus-pended to a concentration of 109 cells/ml, and incubated, with gentle shak-ing for 20 min at 37°C, with an equal volume of amboceptor (Dade Be-hring) diluted 1/3000 in DGVB2� buffer. Amboceptor is an Ab againstsheep erythrocytes, which activates the classical pathway of complement.After two washes with DGVB2�, 8 � 107 cells/ml were incubated for 1 hat 37°C with 0.125% normal human serum (NHS), diluted in DGVB2�

buffer, in a total volume of 150 �l. Before incubation with erythrocytes,NHS was preincubated with various concentrations of SMIPPs or BSA, asa negative control, for 15 min at room temperature (RT). NHS was pre-pared from blood of six healthy volunteers after informed consent andfollowing review by the local ethical board at Lund University. The sam-ples were centrifuged and the hemolytic activity (i.e., the amount of lysederythrocytes) was determined by spectrophotometric measurement of theamount of released hemoglobin (405 nm). The lysis obtained in the absenceof SMIPPs was defined as 100% hemolytic activity.

To assess the activity of the alternative pathway, rabbit erythrocytes(obtained from a local farm) were washed three times with Mg2� EGTAbuffer (2.5 mM Veronal buffer (pH 7.3) containing 70 mM NaCl, 140 mMglucose, 0.1% gelatin, 7 mM MgCl2, and 10 mM EGTA). Erythrocytes ata concentration of 6 � 107 cells/ml were incubated for 1 h at 37°C with1.3% NHS diluted in Mg2� EGTA buffer in a total volume of 150 �l.Before incubation with erythrocytes, NHS was preincubated with variousconcentrations of SMIPPs or BSA, as a negative control, for 15 min at RT.The samples were centrifuged and the hemolytic activity was determinedspectrophotometrically as described above. The lysis obtained in the ab-sence of SMIPPs was defined as 100% hemolytic activity.

Complement activation assays

Unless stated otherwise, all incubation steps were done at RT, in 50 �l ofsolution, followed by extensive washing with 50 mM Tris-HCl (pH 8.0),150 mM NaCl, and 0.1% (v/v) Tween 20. Microtiter plates (Maxisorp;

FIGURE 2. SMIPPs inhibit the hemolytic activity of human serum.Sheep erythrocytes sensitized with Abs (A, classical pathway) or rabbiterythrocytes (B, alternative pathway) were incubated with 0.125% or 1.3%NHS, respectively. Serum was preincubated for 15 min at RT with variousconcentrations of SMIPPs or BSA as a negative control. After 1 h of in-cubation of NHS with erythrocytes at 37°C, the degree of lysis was esti-mated by the measurement of released hemoglobin (absorbance at 405 nm).The lysis obtained in the absence of SMIPPs was defined as 100% hemo-lytic activity. C, The hemolytic activity of the fully glycosylated counter-parts of D1 and I1 was tested in both the classical and alternative pathwaysas described above. One concentration of each protein was tested: 75 �g/mlfor the classical pathway and 200 �g/ml for the alternative pathway. Anaverage of three independent experiments performed in duplicate is pre-sented with bars indicating SD. C, The statistical significance of the dif-ferences between BSA and all tested SMIPPs were estimated for the twoassays using a one-way ANOVA with Tukey’s multiple comparison test;���, p � 0.001.

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Nunc) were incubated overnight at 4°C with 75 mM sodium carbonate (pH9.6) containing the following: for the classical pathway, 2.5 �g/ml aggre-gated human IgG (Immuno); for the lectin pathway, 100 �g/ml mannan(Sigma-Aldrich); and for the alternative pathway, 20 �g/ml zymosan(Sigma-Aldrich). The wells were blocked with 200 �l of blocking solution(1% (w/v) BSA in PBS) for 2 h. For the classical and lectin pathways,respectively, 3% NHS was diluted in DGVB2� buffer and incubated for 20min (for detection of C4b and C3b) or 45 min (for detection of C1q and C9)at 37°C. For the alternative pathway, 5% NHS was diluted in Mg2� EGTAbuffer and incubated for 35 min for detection of C3b or for 1 h for detectionof C9. NHS was preincubated for 15 min at RT with various concentrationsof SMIPPs or BSA, as a negative control, before addition to the microtiterplate. Complement activation was assessed by detection of deposited com-plement proteins using rabbit polyclonal Abs against C1q (DakoCytoma-tion), C4b (DakoCytomation), C3d (DakoCytomation), or goat polyclonalAbs against MBL (R&D Systems) and C9 (Complement Technology) di-luted in blocking solution. After a 1-h incubation, HRP-conjugated sec-ondary Abs against rabbit or goat Igs (DakoCytomation), diluted in block-ing solution, were added for 30 min. Bound enzyme was detected using1,2-phenylenediamine dihydrochloride tablets (DakoCytomation) and theabsorbance was measured at 490 nm. The absorbance obtained in the ab-sence of SMIPPs was defined as 100%.

Direct binding assay

Unless stated otherwise, all incubation steps were done in 50 �l of solution,followed by extensive washing, as described for the complement activationassays. Various complement proteins were coated onto microtiter plates asdescribed above at a concentration of 10 �g/ml. BSA (1% (w/v)) was used

as a negative control. The wells were blocked with 200 �l of blockingsolution and incubated at RT for 2 h. When screening for binding of var-ious complement proteins and examining binding of C1, C1q, C1q head,and C1q stalk, 200 �g/ml SMIPP preparations were added to the wells. Atitration was also performed for the binding of C1q, MBL, and properdinusing concentrations ranging between 12.5 and 200 �g/ml. The SMIPPswere diluted in 50 mM HEPES (pH 7.4), 100 mM NaCl, and 2 mM CaCl2and were allowed to bind overnight at 4°C. Specific murine polyclonal Absagainst SMIPPs were then added to the wells, followed by a HRP-conju-gated goat anti-mouse IgG Ab (DakoCytomation). All Abs were diluted inblocking buffer and incubated at RT for 1 h. Bound enzyme was detectedas described above.

Assembly and decay of the alternative C3 convertase

To measure the assembly and decay of the alternative C3 convertase inNHS and in the presence of SMIPPs, a microtiter plate-based assay wasperformed as described previously (26). Briefly, microtiter plates werecoated with 100 �l of 0.1% agarose in water for 48 h at 37°C. To study theassembly of the alternative C3 convertase, in the presence of SMIPPs, 5%NHS in Mg2� EGTA buffer was preincubated with 0–200 �g/ml D1, I1,or BSA, as a negative control, for 15 min at RT in a total volume of 50 �l.Factor H, at a concentration of 100 �g/ml, was used as a positive control.After preincubation, the samples were added to the agarose- coated plates,incubated for 1 h at 37°C, and then washed three times with 250 �l of TBScontaining 10 mg/ml BSA and 2 mM MgCl2. The amount of assembled C3convertases, i.e., the amount of deposited factor B and properdin, was

FIGURE 3. SMIPPs inhibit theclassical and the lectin pathways. Ag-gregated human IgG (classical path-way, left panel) and mannan (lectinpathway, right panel) were immobi-lized on microtiter plates and allowedto activate 2% or 3% NHS, respec-tively, containing various concentra-tions of SMIPPs or BSA as a negativecontrol (preincubated for 15 min atRT). After 20 min (for C4b and C3b)or 45 min (for C1q, MBL, and C9) ofincubation at 37°C, the plates werewashed and the deposited C1q, MBL,C4b, C3b, and C9 was detected withspecific polyclonal Abs. The absor-bance obtained in the absence ofSMIPPs was defined as 100%. An av-erage of three independent experi-ments performed in duplicate is pre-sented with bars indicating SD.

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detected by addition of 100 �l of either goat anti-human factor B (Com-plement Technology) or goat anti-human properdin (Complement Tech-nology) diluted 1/1000 in TBS containing 10 mg/ml BSA and 2 mMMgCl2. After a 1-h incubation at 37°C, the plates were washed as aboveand 100 �l of 1/1000 diluted HRP-conjugated rabbit anti-goat IgG (Dako-Cytomation) was added and incubated for 1 h at 37°C. The plates were thenwashed twice with TBS containing 10 mg/ml BSA, once with TBS con-taining 10 mg/ml BSA and 0.1% (v/v) Tween 20, and finally once withnormal TBS. The amount of remaining factor B and properdin on the platewas detected as above.

To measure the decay of the alternative C3 convertase, in the presenceof SMIPPs, the assay was mainly performed as above, but instead of pre-incubating serum and proteins and adding them together to the plate, 5%NHS was first added alone to the plate and incubated for 1 h at 37°C. Afterwashing three times with TBS containing 10 mg/ml BSA and 2 mMMgCl2, the SMIPPs and factor H were added and incubated for 1 h at 37°C.The amount of remaining factor B and properdin was then detected asabove.

Localization of human C1q and IgG by immunohistochemistry

Human skin biopsies infested with scabies mites were formalin-fixed over-night, paraffin embedded, and cut into 4-�m serial sections. For localiza-tion studies, slides were prepared as described previously (4, 5) and serialsections underwent an Ag heat-retrieval procedure in a solution containing1% (w/v) Revealit-Ag in 20 mM Tris (pH 7.5; ImmunoSolution) at 85°Cfor 5 min. The slides were cooled and washed with distilled water for 10min followed by 15 min washes in PBS at pH 7.2. Serial sections wereblocked with 10% donkey serum in 1% (w/v) BSA in PBS. Endogenousperoxidase activity was blocked with 3% (v/v) H2O2 in blocking buffer.The sections were incubated overnight with rabbit anti-human C1q poly-clonal Ab (DakoCytomation) at a 1/200 dilution. The primary Ab wasprobed with the secondary Ab Dako-EnVision anti-rabbit-HRP conjugate(DakoCytomation) for 20 min. As a positive control, to identify the mitegut, adjacent sections were incubated with anti-human IgG-HRP poly-clonal Ab (Abcam) in 1/500 dilutions. As a negative control, preimmuneserum in 1/500 dilutions was incubated on sections of the same series. Allslides were washed in PBS and the Vector NovaRed peroxidase substratekit (Vector Laboratories) was used for staining according to the manufac-turer’s recommendations. Counterstaining with hematoxylin was done aspreviously described (4, 5). Ethical approval for the use of shed skin crustsfrom the bedding of a patient with recurrent crusted scabies was obtainedfrom the Human Research Ethics Committee of the Northern TerritoryDepartment of Health and Community Services and the Menzies School ofHealth Research.

Statistical analysis

All experiments were done at least three times in duplicates, if not statedotherwise. Statistical significance was determined using one-way ANOVAwith Tukey’s multiple comparison test (GraphPad Software). Values ofp � 0.05 were considered significant (�, p � 0.05; ��, p � 0.01; and ���,p � 0.001).

ResultsSMIPPs show no proteolytic activity

Despite their structural similarity to proteases, SMIPPs were pre-dicted to be inactive due to the structure of their putative activesites (17). To confirm this hypothesis, we tested whether theSMIPPs could degrade the activated complement components C3band C4b similarly to what we observed for bacterial proteases suchas interpain A (24). The two SMIPPs D1 and I1 showed no pro-teolytic activity, since no degradation products of either C4b (Fig.1C) or C3b (Fig. 1D) could be detected in contrast to what wasobserved for positive controls.

SMIPPs inhibit the hemolytic activity of human serum

To determine whether the two SMIPPs D1 and I1 had any inhib-itory effect on the human complement system, two hemolytic as-says were performed measuring activity of the classical and thealternative pathway, respectively. Human serum, as a source of allcomplement proteins, was pretreated with various concentrationsof SMIPPs before incubation with erythrocytes. Both SMIPPswere able to fully inhibit both the classical pathway (Fig. 2A) and

alternative pathway (Fig. 2B). Higher concentrations of SMIPPs(200 �g/ml) were required to inhibit the alternative pathway com-pared with the classical pathway (75 �g/ml). This is probably dueto differences in serum concentration used in the two assays: 1.3%serum in the alternative hemolytic assay vs 0.125% serum in theclassical hemolytic assay. These serum concentrations were cho-sen on the basis of an initial titration and represent conditions inwhich each assay is most sensitive to changes. The alternativepathway is known to require higher concentrations of serum tofunction properly.

To elucidate whether the nonglycosylated proteins used in thisstudy behaved in the same manner as the fully glycosylated coun-terparts, we also tested fully glycosylated D1 and I1 in hemolyticassays. One concentration of each of the SMIPP proteins wastested for the classical pathway (75 �g/ml) and alternative path-way (200 �g/ml). Fully glycosylated D1 and I1 inhibited bothcomplement pathways to a similar extent as their nonglycosylatedcounterparts (Fig. 2C). However, inhibition of the classical path-way was somewhat less pronounced for glycosylated I1 comparedwith the nonglycosylated molecule ( p � 0.001). We also testeddifferent concentrations of the fully glycosylated D1 and I1 in thecomplement activation assays for the three pathways, measuringC3b deposition (data not shown). We found that the nonglycosy-lated and the fully glycosylated D1 and I1 inhibited all three path-ways in a very similar manner. The small difference seen for I1 inthe classical pathway hemolytic assay could, however, also be no-ticed in the classical pathway complement activation assay, but notin the assays measuring activity of the lectin and alternative path-ways. Based on the similar results obtained for the non- and fullyglycosylated proteins, we continued testing only the nonglycosy-lated variants in this study.

FIGURE 4. SMIPPs inhibit the alternative pathway. Zymosan was im-mobilized on microtiter plates and allowed to activate 5% NHS containingvarious concentrations of SMIPPs or BSA as a negative control (preincu-bated for 15 min at RT). After 35 min (for C3b) or 1 h (for C9) of incu-bation, the plates at 37°C were washed and the deposited C3b and C9 weredetected with specific polyclonal Abs. The absorbance obtained in the ab-sence of SMIPPs was defined as 100%. An average of three independentexperiments performed in duplicate is presented with bars indicating SD.

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SMIPPs inhibit all three complement pathways

The complement system works in a cascade-like manner with con-secutive proteolytic activation of a number of proteins. To assessat what level of the cascade the SMIPPs inhibit complement, amicrotiter plate-based deposition assay was performed in whichcomplement activation was initiated by specific ligands for eachpathway. After addition of human serum, pretreated withSMIPPs, the deposited complement proteins were detected us-ing specific Abs.

In the case of the classical pathway, complement was activatedby aggregated IgG deposited on the plate. When adding serumtogether with either D1 or I1, deposition of all four tested com-plement factors, C1q, C4b, C3b, and C9, was decreased (Fig. 3, leftpanel). The same pattern was observed for the lectin pathway; afteractivation with mannan, deposition of MBL, C4b, C3b, and C9was inhibited by both D1 and I1 (Fig. 3, right panel). This was alsotrue for the alternative pathway; after initiation with immobilizedzymosan, C3b and C9 deposition was decreased (Fig. 4). Overall,D1 seemed to inhibit the pathways slightly less efficiently than I1,especially for the alternative pathway, where double the amount ofD1 (400 �g/ml for D1 and 200 �g/ml for I1) was needed to com-pletely inhibit deposition of C3b and C9.

SMIPPs bind directly to C1q, MBL, and properdin

Structural and functional analysis of the two SMIPPs analyzed inthe current study found that they are devoid of proteolytic activity(17) and we could not detect any degradation of complementproteins such as C3 and C4 by the SMIPPs (Fig. 1, C and D).To elucidate whether the complement inhibition seen in theprevious assays is due to SMIPPs binding to complement pro-teins, a microtiter plate-based direct binding assay was used. Afirst screening was performed, where the plates were coatedwith various complement proteins, followed by incubation withone concentration (200 �g/ml) of SMIPPs and the binding wasdetected with specific murine polyclonal Abs against D1 and I1.In this direct binding assay, a clear interaction of both D1 (Fig.5A) and I1 (Fig. 5B) with C1q, MBL, and properdin was ob-served under conditions of physiological ionic strength and pH.The strongest binding of D1 and I1 was observed to C1q. Toexamine these interactions further, various concentrations (from0 to 200 �g/ml) of the SMIPPs were allowed to bind to theimmobilized complement proteins and the results showed thatthe binding of C1q, MBL, and properdin was dose dependentfor both D1 (Fig. 5C) and I1 (Fig. 5D). Also, under these ex-perimental conditions, the binding to C1q was shown to be the

FIGURE 5. Direct binding ofSMIPPs to various complement pro-teins. A, B, and E, Microtiter plateswere coated with various complementproteins or BSA as a negative controland incubated with 200 �g/ml D1 orI1 overnight at 4°C. C and D, Micro-titer plates were coated with C1q,MBL, properdin, or BSA and variousconcentrations of D1 (C) and I1 (D)were added. In all of the assays,bound protein was detected using spe-cific polyclonal Abs against D1 andI1. An average of three independentexperiments performed in duplicate ispresented with bars indicating SD.The statistical significance of differ-ences between BSA and the rest ofthe groups was estimated using a one-way ANOVA with Tukey’s multiplecomparison test: �*, p � 0.05; ��,p � 0.01; and ���, p � 0.001.

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strongest interaction. Cross-competition assays in which D1 orI1 was allowed to bind immobilized complement proteins in thepresence of another complement protein as competitor showedthat C1q, MBL, and properdin bind to different sites on SMIPPs(data not shown).

SMIPPs interact mainly with the collagenous stalk of C1q

C1q is part of the C1 complex along with two molecules of eachof the smaller proteolytic subunits C1r and C1s. C1q is the rec-ognition molecule responsible for the activation of the classicalpathway and it is composed of six subunits. Each subunit has aglobular head at the C terminus and a collagenous stalk at the Nterminus. C1q binds many different ligands and most of the ligandsthat activate C1q and lead to activation of the classical pathwaybind to the globular head domains. On the contrary, ligands bind-ing to the collagenous stalk of C1q may inhibit activation via theclassical pathway (27).

To identify the binding site for SMIPPs on C1q, head and stalkregions of C1q were prepared, using partial proteolytic digestion ofintact C1q, with collagenase (for heads) and pepsin (for stalks). Bycoating these isolated parts of C1q on a microtiter plate, we coulddemonstrate that the binding of both D1 and I1 was localized to thecollagenous stalk region of C1q (Fig. 5E). The binding to the glob-ular heads was not significantly different from the binding to thenegative control BSA. We also demonstrated that the binding toC1q is significantly stronger than to the intact C1 complex. This isprobably due to the fact that C1r and C1s also interact with thestalks and may be competing to some extent with SMIPPs.

SMIPPs inhibit assembly of alternative pathway C3 convertasesdue to their ability to bind properdin, but they do not acceleratedecay of preassembled convertases

We observed that the SMIPPs inhibited the alternative pathway ofcomplement, both in hemolytic (Fig. 2B) and complement depo-sition assays (Fig. 4). This could be due to binding of the positiveregulator of this pathway, properdin, which occurs as shown inFig. 5. To investigate whether the binding of the SMIPPs to pro-perdin would inhibit the assembly and/or accelerate the decay ofthe alternative C3 convertase, a microtiter plate-based assay wasperformed. Agarose, a polysaccharide that will activate the alter-native pathway of complement (28), was coated onto microtiterplates, followed by addition of NHS, as a source of complementproteins and the formed C3 convertases were detected by measur-ing the amount of deposited factor B and properdin using specific

FIGURE 6. SMIPPs inhibit assembly of alternative pathway C3 convertases due to their ability to bind properdin, but they do not accelerate decay ofpreassembled convertases. Microtiter plates were coated with agarose, followed by the addition of NHS, and the C3 convertases formed were detected bymeasuring the amount of deposited factor B and properdin with specific polyclonal Abs. The SMIPPs (or BSA as a negative control) were either addedtogether with NHS to study the assembly of the convertase (A) or after NHS incubation to study the decay (B). Factor H was added as a positive controlbut only at one concentration; 100 �g/ml. The absorbance obtained in the absence of SMIPPs was defined as 100% and an average of three independentexperiments performed in duplicate is presented with bars indicating SD. The statistical significance of differences between BSA and the rest of the groupswas estimated using a one-way ANOVA with Tukey’s multiple comparison test: ���, p � 0.001. FH, factor H.

FIGURE 7. Immunohistological colocalization of C1q and host IgG in-dicates that human complement is ingested into the mite gut. A schematicdiagram (A) outlines the features visible in three serial histological sectionsof mite-infested epidermis. The epidermis is shown in blue, the burrows inwhite, the bodies of two mites in gray, and the mite gut in red. B, C, andD. The matching serial histological sections. Red staining indicates bindingof Ab to protein. Section (B) was probed with anti-human IgG, a marker forthe mite gut (4). Section C shows staining of the mite gut when probingwith anti-C1q, while section D, probed with preimmune mouse serum as anegative control, remained unstained. b, Burrow wall; c, cuticle; es, esoph-agus; g, gut; m, mouth parts. Scale bar, 100 �m).

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polyclonal Abs. To assess the assembly of the alternative C3 con-vertase, in the presence of SMIPPs, D1 and I1 were preincubatedwith NHS before being added to the agarose- coated plates. FactorH was used as a positive control, since it is known to bind C3b andhinder the formation of the alternative C3 convertase. Both D1 andI1 significantly hindered the formation of the convertase (Fig. 6A)at both tested concentrations, as indicated by the reduced amountof deposited factor B and properdin. To assess the ability of theSMIPPs to accelerate the decay of the alternative C3 convertase,the proteins were added after NHS incubation, i.e., after the as-sembly of the convertases. Factor H was also in this setup used asa positive control, due to its known ability to accelerate the decayof the alternative convertase, by directly binding C3b and displac-ing Bb from the convertase. This can be seen in Fig. 6B, where theaddition of factor H resulted in a decrease of deposited factor B. How-ever, the SMIPPs were not able, at any tested concentration, to ac-celerate the decay of the alternative C3 convertase (Fig. 6B). To-gether, these results demonstrate that the SMIPPs are able to inhibitthe assembly of the alternative C3 convertase, but once the convertaseis preformed, SMIPPs are not able to accelerate its decay.

SMIPPs colocalize with human complement factor C1q in themite digestive system

The presence of complement factors in the scabies mite gut wasclearly demonstrated by probing serial sections of human mite-infested skin with Abs against human C1q in comparison to stain-ing with human IgG, which for the purpose of this experimentserved as a positive control for gut localization (Fig. 7). HumanIgG was localized to the gut (Fig. 7B) as previously documented(4, 5). This confirmed that, in the adjacent serial section (Fig. 7C),labeling by anti-C1q Abs was indeed in the gut. Staining of the gutwas the most easily seen; however, sections that were in the rightplane also showed staining in the esophagus (Fig. 7, B and C). Thelocalization of SMIPP proteins to the digestive system of the mitehas previously been shown in sections of scabies-infested humanskin (4, 5) probed with polyclonal Abs against individual SMIPPs.In all cases, the sections probed with the preimmune serum onlystained with the counterstain, as demonstrated in Fig. 7D, clearlyexcluding any unwanted background staining.

DiscussionThe complement system has several sensory molecules able torecognize molecular patterns foreign to its host. Furthermore, itcan be strongly activated by both natural, low- affinity IgM Abs aswell as specific, high-affinity IgG Abs. Not surprisingly, manypathogens utilize multiple sets of evasion strategies directed atinhibition of complement, as redundancy and multiplicity are im-portant for immune and complement evasion (9). In the currentstudy, we found that SMIPPs of scabies mites are able to interactwith several complement proteins, which leads to inhibition of allthree pathways of complement. This finding is consistent with he-matophagous parasites having complement escape mechanisms(10). Both Necator americanus and Hemonchus contortus expressa calreticulin-like protein that binds C1q (29, 30). Ticks such asIxodes scapularis and Ixodes ricinus express proteins preventingdeposition of C3b, such as Isac (26) and IRACs/IxACs (31, 32).IRACs and IxACs, along with Salp20, also have the ability to bindproperdin (33). Interestingly, the SMIPPs analyzed in the currentstudy combine the properties of many of the molecules describedabove, as they have the ability to bind C1q, MBL, and properdin.

C1q is a promiscuous molecule able to interact with many li-gands. In general, it appears that ligands binding to the globularheads of C1q, such as Igs, C-reactive protein, amyloid, and someextracellular matrix proteins, such as fibromodulin and osteoad-

herin, are all strong activators of the classical pathway of comple-ment (34). On the other hand, ligands that interact with the stalksof C1q, such as the extracellular matrix proteins decorin and bi-glycan, are able to inhibit the classical pathway (27). This is prob-ably due to competition for binding with the C1r/C1s enzymes thatadhere to the stalk region and perhaps also due to effects on con-formational changes in the C1 complex that are characteristic andrequired for complement activation upon binding to activating li-gands. We found that SMIPPs bind to the stalk region of C1q,similar to what has been previously observed for decorin, whichserves as an explanation as to why SMIPPs are able to inhibit theclassical pathway. A similar mechanism is likely to be applicablefor MBL because it has a similar overall architecture to C1q, par-ticularly in its collagenous stalk and globular head domains, butthese domains of MBL were not available for us to study. How-ever, the inhibitory effect of SMIPPs on the lectin pathway isclearly due to their ability to bind MBL.

Properdin is a positive regulator of the alternative pathway thatexerts its effect by binding C3b and thereby stabilizing the alter-native C3 convertase, resulting in a prolonging of the half-life ofthe convertase of up to 10-fold (35). It also inhibits the binding offactor I to C3b, thereby protecting it from proteolytic cleavage(36). We observed in the present study that SMIPPs inhibit thealternative pathway of complement by binding to properdin andthereby inhibiting the assembly of the alternative pathway C3 con-vertase. However, unlike Salp20 from ticks, the SMIPPs were notable to displace properdin from the previously formed C3 conver-tase and were thereby not able to accelerate the decay of the con-vertase complex. SMIPPs thereby act in the same manner as theIxAC proteins, which also bind properdin and inhibit the assemblyof the alternative pathway C3 convertase, but are not able to dis-place properdin from previously formed convertase complexes.

The x-ray crystal structures of SMIPP-SI1 and SMIPP-SD1have recently been determined (17) and therefore may provideclues to the mechanisms of complement-binding activities reportedhere. Both SMIPP structures show significant structural changeswithin the active site region, providing a structural rationalizationfor their lack of protease activity. However, analysis of the hydro-phobic and electrostatic properties of their molecular surfaces re-veals no apparent common mode of interaction with other proteins(typically protein-protein interaction sites comprise significant re-gions of hydrophobic or charged nature). Inspection of sequencevariability within the SMIPP family, in the context of the struc-tures, suggests that the family is conformationally diverse and thusable to present multiple protein interaction binding sites that couldbind a range of complement proteins. However, the absence ofstructural information for MBL, properdin, and C1q precludesmodeling of such potential interactions. At least for the twoSMIPPs studied here, no common structural mode of complementinhibition is apparent; it is possible that the structural diversitywithin the SMIPP family allows multiple modes of binding thatwould be required to inhibit several structurally unrelated mem-bers of the complement system. Further insights may be obtainedby structural characterization of other members of the SMIPP fam-ily, as well as complement-binding studies of mutants, which cannow be rationally guided by the available structural data.

In some of the assays presented in this study, relatively highconcentrations of SMIPPs were needed to obtain a significant com-plement inhibitory effect, a phenomenon that has been observedpreviously in studies of other known complement inhibitors (27,37). The concentrations of SMIPPs in the mite gut are not knownand the local concentrations of complement proteins at the sites ofmite infection are also not determined. However, 33 SMIPPs havenow been identified and, since both of the SMIPPs tested here

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show complement inhibitory functions, it is possible that many ofthese molecules inhibit complement. This makes it quite plausiblethat high concentrations of SMIPPs will be found in vivo. Obvi-ously, further studies will be needed to assess the in vivo relevanceof SMIPPs in defense against complement.

We have demonstrated that SMIPPs are present in the gut of themite and excreted in feces (4, 5), hence their site(s) of action maybe both internal and external to the mite. Internally, as the gutcontains host plasma (4, 5), complement cascades must be inhib-ited in a milieu in which digestion of protein food can occur. Thisseems to be the obvious role of the SMIPPs. Externally, the inhi-bition of complement may have further consequences for the host.By interfering with host complement, the SMIPPs may effectivelyenhance the survival of pathogenic bacteria that colonize the miteburrows. In line with this hypothesis, we have previously shownthat cysteine proteinases from Porphyromonas gingivalis degradecomplement factors and may provide an advantage to other peri-odontal pathogens residing in the same location (38). Ultimately,the inhibition of the host complement by scabies mite productssuch as SMIPPs may account for much of the associated secondarybacterial skin infections and downstream chronic disease. There isdirect evidence that communities that have received treatment forscabies experience a dramatic reduction in bacterial skin sores (39,40). Hence, elucidating the mechanism of the interaction betweenSMIPPs and the individual complement components is a vital stepin preventing attenuation of the pathways which are pivotal to hostdefense against both the scabies mite and secondary bacterialpathogens. This in turn may lead to development of new treatmentsagainst this important but neglected parasite.

AcknowledgmentsWe thank Kate Mounsey, Deborah Holt, Shelley Walton, and Bart Curriefrom the Tropical and Emerging Infectious Diseases Division of the Men-zies School of Health Research in Darwin, Australia for providing materialfor localization studies.

DisclosuresThe authors have no financial conflict of interest.

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