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.

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.

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.

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.