Identification of a novel PYP-1 gene in Sarcoptes scabiei and its potential as a serodiagnostic candidate by indirect-ELISA.

Scabies is a parasitic disease caused by the ectoparasite Sarcoptes scabiei, affecting different mammalian species, including rabbits, worldwide. In the present study, we cloned and expressed a novel inorganic pyrophosphatase, Ssc-PYP-1, from S. scabiei var. cuniculi. Immunofluorescence staining showed that native Ssc-PYP-1 was localized in the tegument around the mouthparts and the entire legs, as well as in the cuticle of the mites. Interestingly, obvious staining was also observed on the fecal pellets of mites and in the integument of the mites. Based on its good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant Ssc-PYP-1 (rSsc-PYP-1) as the capture antigen was developed to diagnose sarcoptic mange in naturally infected rabbits; the assay had a sensitivity of 92·0% and specificity of 93·6%. Finally, using the rSsc-PYP-1-ELISA, the Ssc-PYP-1 antibody from 10 experimentally infected rabbits could be detected from 1 week post-infection. This is the first report of S. scabiei inorganic pyrophosphatase and the protein could serve as a potential serodiagnostic candidate for sarcoptic mange in rabbits.

Pseudoproteases: mechanisms and function.

Catalytically inactive enzymes (also known as pseudoproteases, protease homologues or paralogues, non-peptidase homologues, non-enzymes and pseudoenzymes) have traditionally been hypothesized to act as regulators of their active homologues. However, those that have been characterized demonstrate that inactive enzymes have an extensive and expanding role in biological processes, including regulation, inhibition and immune modulation. With the emergence of each new genome, more inactive enzymes are being identified, and their abundance and potential as therapeutic targets has been realized. In the light of the growing interest in this emerging field the present review focuses on the classification, structure, function and mechanism of inactive enzymes. Examples of how inactivity is defined, how this is reflected in the structure, functions of inactive enzymes in biological processes and their mode of action are discussed.

Characterisation and analysis of thioredoxin peroxidase as a potential antigen for the serodiagnosis of sarcoptic mange in rabbits by dot-ELISA.

BACKGROUND: Scabies caused by Sarcoptes scabiei is a widespread but a neglected tropical zoonosis. In this study, we characterised a S. scabiei thioredoxin peroxidase (SsTPx) and evaluated a recombinant SsTPx as a diagnostic antigen in rabbits. METHODS: The open reading frame of the gene encoding SsTPx-2 was amplified and the recombinant protein was expressed in Escherichia coli cells and purified. SsTPx was localized in mite tissue by immunolocalisation using the purified recombinant protein. Serodiagnosis assays were carried out in 203 New Zealand White rabbit serum samples by dot-ELISA. RESULT: The open reading frame (489 bp) of the gene encodes an 18.11 kDa protein, which showed highly homology to that of Psoroptes cuniculi (98.77% identity) and belongs to the 2-Cys family of peroxiredoxins. SsTPx was mainly distributed in muscle tissues of mites, integument of the epidermis and the anterior end of S. scabiei. Although SsTPx cross-reactivity with psoroptic mites was observed, the SsTPx dot-ELISA showed excellent diagnostic ability, with 95.3% sensitivity and 93.8% specificity in mange-infected and uninfected groups. CONCLUSIONS: This study showed that the purified SsTPx is a highly sensitive antigen for the diagnosis of mange infection by dot-ELISA. This technique is a rapid and convenient method that can be used worldwide for the clinical diagnosis of sarcoptic mange in rabbits, and is especially useful in developing regions.

Scabies mite inactivated serine protease paralogs inhibit the human complement system.

Infestation of skin by the parasitic itch mite Sarcoptes scabiei afflicts 300 million people worldwide and there is a need for novel and efficient therapies. We have previously identified a multigene family of serine proteases comprising multiple catalytically inactive members (scabies mite-inactivated protease paralogs (SMIPPs)), which are secreted into the gut of S. scabiei. SMIPPs are located in the mite gut and in feces excreted into the upper epidermis. Scabies mites feed on epidermal protein, including host plasma; consequently, they are exposed to host defense mechanisms both internally and externally. We found that two recombinantly expressed SMIPPs inhibited all three pathways of the human complement system. Both SMIPPs exerted their inhibitory action 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, which are associated with the same domain. Furthermore, we found that binding of both SMIPPs to properdin resulted in prevention of 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 that SMIPPs minimize complement-mediated gut damage and thus create a favorable environment for the scabies mites.

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis.

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.

Diagnostic value of the molecular detection of Sarcoptes scabiei from a skin scraping in patients with suspected scabies.

Scabies is a highly contagious parasitic disease associated with long-term residence in nursing homes, and it is a public health burden worldwide. However, atypical skin manifestations are frequent and the widely used diagnostic test based on microscopic examinations has limited sensitivity. We evaluated the diagnostic value of polymerase chain reaction (PCR) from skin scraping in patients with suspected scabies. Adult patients with suspected scabies, unrelated diseases or healthy volunteers were enrolled at a tertiary hospital, in Seoul, South Korea, from December 2017 through October 2018. We classified participants based on the consensus criteria established by the International Alliance for the Control of Scabies in 2018; confirmed (microscopic mite detection), clinical (scabies burrow or typical lesions with two history features including itch and close contact with scabies patients), suspected scabies (typical lesion with one history feature or atypical lesion with two history features), or no scabies. PCR was performed on the skin scrapings to target the cytochrome c oxidase subunit 1 (cox1) gene of Sarcoptes scabiei. A total of 47 participants, 33 with suspected scabies, 10 with unrelated diseases, and 4 healthy volunteers were enrolled. Of the 33 patients, 22 were classified as confirmed scabies, 2 as clinical scabies, 6 as suspected scabies, and 3 as no scabies. The sensitivities of the microscopic examination were 100%, 92%, and 73% in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.006). The sensitivities of PCR were 86%, 83%, and 80% in confirmed scabies; confirmed and clinical scabies; and confirmed, clinical, and suspected scabies, respectively (p = 0.59). The specificity of the scabies PCR in the no scabies control was 100% (95% CI = 80-100).PCR testing for scabies may be helpful in the improvement of sensitivity for the diagnosis of scabies by clinical criteria.

Phylogenetic relationships, stage-specific expression and localisation of a unique family of inactive cysteine proteases in Sarcoptes scabiei.

BACKGROUND: Scabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites. We postulated that the SMIPP-Cs have evolved as an adaptation to the parasitic lifestyle of the scabies mite. To formulate testable hypotheses for their functions and to propose possible strategies for translational research we investigated whether the SMIPP-Cs are common to all scabies mite varieties and where within the mite body as well as when throughout the parasitic life-cycle they are expressed. RESULTS: SMIPP-C sequences from human, pig and dog mites were analysed bioinformatically and the phylogenetic relationships between the SMIPP-C multi-copy gene families of human, pig and dog mites were established. Results suggest that amplification of the SMIPP-C genes occurred in a common ancestor and individual genes evolved independently in the different mite varieties. Recombinant human mite SMIPP-C proteins were produced and used for murine polyclonal antibody production. Immunohistology on skin sections from human patients localised the SMIPP-Cs in the mite gut and in mite faeces within in the epidermal skin burrows. SMIPP-C transcription into mRNA in different life stages was assessed in human and pig mites by reverse transcription followed by droplet digital PCR (ddPCR). High transcription levels of SMIPP-C genes were detected in the adult female life stage in comparison to all other life stages. CONCLUSIONS: The fact that the SMIPP-Cs are unique to three Sarcoptes varieties, present in all burrowing life stages and highly expressed in the digestive system of the infective adult female life stage may highlight an essential role in parasitism. As they are excreted from the gut in scybala they presumably are able to interact or interfere with host proteins present in the epidermis.

Molecular characterization and allergenicity potential of triosephosphate isomerase from Sarcoptes scabiei.

Scabies is an allergic skin disease that affects millions of mammals worldwide, including humans. It is a neglected tropical disease that represents a significant public health threat, particularly in economically disadvantaged populations. An effective vaccine is not currently available, and the exact mode of pathogenesis remains unclear. Herein, we identified, cloned and recombinantly expressed triosephosphate isomerase from Sarcoptes scabiei (S. scabiei). Immunohistochemical analyses showed that S. scabiei triosephosphate isomerase (Ss-TIM) is localized in the legs and chewing mouthparts of mites, and in infected rabbit skin (keratinized skin and embedded mites). Intradermal skin tests of rabbits injected with recombinant S. scabiei triosephosphate isomerase (rSs-TIM) revealed a flare, erythema and wheal reaction. These findings suggest that Ss-TIM may contribute to host invasion and induce an allergic response in the host.

A chitinase-like protein from Sarcoptes scabiei as a candidate anti-mite vaccine that contributes to immune protection in rabbits.

BACKGROUND: Scabies is caused by Sarcoptes scabiei burrowing into the stratum corneum of the host's skin and is detrimental to the health of humans and animals. Vaccines are an attractive alternative to replace the acaricides currently used in their control. METHODS: In the present study, the S. scabiei chitinase-like protein 5 (SsCLP5) was characterized and recombinant SsCLP5 (rSsCLP5) was evaluated as a candidate vaccine protein for anti-mite protection in rabbits. The expression, characterization and immunolocalization of SsCLP5 were examined. Vaccination experiments were performed on three test groups (n = 12 per group) immunized with purified rSsCLP5. Control groups (n = 12 per group) were immunized with PBS, QuilA saponin or empty vector protein. After challenge, the inflammatory reaction and skin lesions were graded and rSsCLP5 indirect ELISA was used to detect antibody IgG levels in serum samples at the time of vaccination and post-challenge. RESULTS: The results showed that rSsCLP5 had high immunoreactivity and immunogenicity. In S. scabiei, SsCLP5 had a wide distribution in the chewing mouthpart, legs and exoskeleton, especially the outer layer of the exoskeleton. Vaccination with rSsCLP5 resulted in 74.3% (26/35) of rabbits showing no detectable lesions after challenge with S. scabiei. CONCLUSIONS: Our data demonstrate that rSsCLP5 is a promising candidate for a recombinant protein-based vaccine against S. scabiei. This study also provides a method for studying scabies vaccine using rabbit as an animal model and a basis for screening more effective candidate proteins.

Expression and characterisation of a Sarcoptes scabiei protein tyrosine kinase as a potential antigen for scabies diagnosis.

Scabies is a disease that harms humans and other animals that is caused by the itch mite Sarcoptes scabiei burrowing into the stratum corneum of the skin. In the early stages of scabies, symptoms are often subclinical and there are no effective diagnostic methods. Herein, we cloned, expressed and characterised an S. scabiei protein tyrosine kinase (SsPTK) and evaluated its diagnostic value as a recombinant antigen in rabbit during the early stages of Sarcoptes infestation. The SsPTK protein is ~30 kDa, lacks a signal peptide, and shares high homology with a PTK from the rabbit ear mite Psoroptes ovis cuniculi. The protein was widely distributed at the front end of mites, particularly in the chewing mouthparts and legs. Indirect ELISA using recombinant SsPTK showed good diagnostic value, with 95.2% (40/42) sensitivity and 94.1% (48/51) specificity for detecting anti-PTK antibody in serum samples from naturally-infested rabbits. More importantly, PTK ELISA could diagnose infection in the early stages (infestation for 1 week) with an accuracy of 100% (24/24). SsPTK therefore shows potential as a sensitive antigen for the early diagnosis of parasitic mite infestation.

Scabies mite inactivated serine protease paralogues are present both internally in the mite gut and externally in feces.

The scabies mite, Sarcoptes scabiei, is the causative agent of scabies, a disease that is common among disadvantaged populations and facilitates streptococcal infections with serious sequelae. Previously, we encountered large families of genes encoding paralogues of house dust mite protease allergens with their catalytic sites inactivated by mutation (scabies mite inactivated protease paralogues [SMIPPs]). We postulated that SMIPPs have evolved as an adaptation to the parasitic lifestyle of the scabies mite, functioning as competitive inhibitors of proteases involved in the host-parasite interaction. To propose testable hypotheses for their functions, it is essential to know their locations in the mite. Here we show by immunohistochemistry that SMIPPs exist in two compartments: 1) internal to the mite in the gut and 2) external to the mite after excretion from the gut in scybala (fecal pellets). SMIPPs may well function in both of these compartments to evade host proteases.

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Identification and characterization of Sarcoptes scabiei and Dermatophagoides pteronyssinus glutathione S-transferases: implication as a potential major allergen in crusted scabies.

The astigmatid mite Sarcoptes scabiei is the causative agent of scabies, a highly infectious parasitic disease of the skin. Although the mite causes marked hypersensitivity reactions, particularly in crusted (severe) scabies, little is known about the specific scabies mite molecules involved in such immunologic responses. We have identified six genes encoding scabies mite homologues of mu and delta-like glutathione S-transferases (GSTs) as well as novel house dust mite GSTs. A mu class S. scabiei GST was subcloned into a prokaryotic expression system. The purified recombinant protein rSsGST01 reacted strongly with IgE and IgG4 in sera from crusted scabies patients. This response was not observed with control antigens or with ordinary scabies and uninfested patient sera. In addition, the specific IgE response to rSsGST01 did not correlate with the total IgE level of the patient. These results suggest that GST may play a role in the pathophysiology associated with crusted scabies.

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

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.

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

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.

Studies on the acaricidal mechanism of the active components from neem (Azadirachta indica) oil against Sarcoptes scabiei var. cuniculi.

Octadecanoic acid-3,4-tetrahydrofuran diester, isolated from neem (Azadirachta indica) oil, exhibited potent acaricidal activity against Sarcoptes scabiei var. cuniculi. In this paper, the acaricidal mechanism of octadecanoic acid-3,4-tetrahydrofuran diester against Sarcoptes scabiei var. cuniculi was evaluated based on pathologic histology and enzyme activities. The results showed that after compound treatment for 24h at a concentration of 20mg/mL, the lesions of mites were prominent under transmission electron microscopy. The lesions consisted of the lysis of dermis cell membranes and cell nuclear membranes, mitochondrial morphological abnormalities, the drop of spinal disorders, and mitochondrial vacuolization. The activity of superoxide dismutase (SOD), peroxidase (POD), glutathione-s-transferases (GSTs), and Ca(2+)-ATPase of mites significantly changed after treatment with octadecanoic acid-3,4-tetrahydrofuran diester compared with the control group. The activities of SOD, POD, and Ca(2+)-ATPase were significantly suppressed, whereas that of GSTs was activated. These results indicated that the mechanism of the acaricidal activity of octadecanoic acid-3,4-tetrahydrofuran diester was mainly achieved through interference with the energy metabolism of mites, thus resulting in insect death.

Structural mechanisms of inactivation in scabies mite serine protease paralogues.

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.