Effects of Sarcoptes scabiei Translationally Controlled Tumor Protein (TCTP) on Histamine Release and Degranulation of KU812 Cells.

Scabies is a common parasitic dermatological infection worldwide that is often neglected. Scabies mites stimulate host inflammatory symptoms via secreted and excreted proteins, which induce basophil and mast cell degranulation and host histamine release. However, the mechanism of degranulation and histamine release is unclear. Moreover, the Sarcoptes scabiei translationally controlled tumor protein (TCTP) is predicted as an excreted protein, which may be involved in host inflammatory response regulation. First, we evaluated S. scabiei TCTP gene (SsTCTP) transcription in larvae, nymphs, and adults by qRT-PCR, and SsTCTP transcription was highest in larvae, followed by nymphs. Second, we found that the S. scabiei TCTP recombinant protein (rSsTCTP) promoted mice histamine release in vivo by Evans blue Miles assay. Therefore, to further explore the possible role of S. scabiei TCTP in host inflammatory response regulation, we established a degranulation model of KU812 cells. The results of the degranulation model suggested that rSsTCTP could induce enhanced degranulation of KU812 cells and increase the secretion of histamine and the expression of IL-4, IL-6, and IL-13 in vitro. In conclusion, we speculate that scabies mites could stimulate host histamine release and Th2 response by excreting S. scabiei TCTP.

Comparative genomics of Sarcoptes scabiei provide new insights into adaptation to permanent parasitism and within-host species divergence.

Sarcoptes scabiei is the causative agent of a highly contagious skin disease in humans and more than 100 mammals. Here, we reported the first chromosome-level reference genome of S. scabiei isolated from rabbits, with a contig N50 size of 5.92 Mb, a total assembled length of 57.30 Mb, ∼12.65% repetitive sequences and 9333 predicted protein-coding genes. The phylogenetic tree based on 1338 shared high-confidence single-copy orthologous genes estimated that the mammalian ectoparasite S. scabiei and the plant-feeding mite Tetranychus urticae separated ∼340 million years ago. Both neighbour-joining tree and principal component analysis of 20 mite populations isolated from four hosts (humans, pigs, dogs and rabbits) distributed in three countries (China, Australia and the USA) consistently supported genetic subdivisions according to host species rather than geographical location. The demographic history of S. scabiei reconstructed by multiple sequentially Markovian coalescent analysis suggested that S. scabiei isolated from rabbits, humans, dogs and pigs diverged ∼5000 years ago. Investigation of the homeobox (Hox) genes revealed that S. scabiei contains 8 of 10 canonical Hox genes that are present in the arthropod ancestor, and the absence of the Abd-A gene may correlate with the long gap between their front and back legs. Comparative genomics demonstrated that genes specific to scabies mites were mainly enriched in nutrition digestive systems, whereas genes in the families that involved detoxification (cytochrome P450, carboxyl/cholinesterases and the ATP-binding cassette transporter C group) were extremely contracted compared with that of other mites analysed in this study. Selective sweep analysis of mite populations from various pairs of two out of the four host species revealed that the strongest selective sweep signals were mainly enriched in cysteine-type peptidase activity and apoptosis. The results provided clues for the mechanisms of S. scabiei adaptation to a permanent parasitic lifestyle and knowledge that would enable further control of this highly contagious skin disease.

Scabies in a 14-year-old girl with superficial epidermolytic ichthyosis.

A 14-year-old girl who reported generalized scaling and hyperkeratosis since age 1 year presented with severe pruritus of several months' duration. Scabies mites were detected, and molecular genetic analysis subsequently revealed a rare pathogenic variant in the keratin 2 (KRT2) gene, confirming a diagnosis of superficial epidermolytic ichthyosis. Treatment with oral ivermectin led to complete remission of symptoms. Disorders of keratinization can mimic clinical signs of scabies, leading to a delay in diagnosis.

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 of calmodulin from Sarcoptes scabiei.

Scabies, caused by the mite Sarcoptes scabiei, is a highly contagious parasitic disease that affects millions of people and other mammals worldwide. Calmodulin (CaM) is an important calcium sensor that participates in various critical physiological processes. In this study, the CaM of Sarcoptes scabiei (SsCaM) was cloned and expressed, and sequence analyses were performed using bioinformatics tools. Recombinant SsCaM (rSsCaM) was used to detect antigenicity using immunoblotting assays, and the serodiagnostic potential of rSsCaM was assessed by indirect enzyme-linked immuno-sorbent assay (ELISA). The calcium binding properties and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence of rSsCaM were also measured. The results indicated that SsCaM contains a 450-bp open reading frame that encodes for a polypeptide with 149 amino acids, and SsCaM was expressed as a soluble protein. Multiple sequence alignment and phylogenetic analyses indicated similarity and genetic distance between SsCaM and other species. The calcium binding properties and ANS fluorescence of rSsCaM indicated typical calcium binding characteristics. Immunolocalizaton assay showed that SsCaM was widespread in S. scabiei. SsCaM-based ELISA exhibited a sensitivity of 87.5% (28/32) and a specificity of 22.5% (9/40) for detecting anti-CaM antibodies in the sera of naturally infected rabbits. The findings of this study provide a comprehensive molecular characterization of SsCaM and suggest that rSsCaM is inappropriate for detecting S. scabiei. The results may also contribute to future studies on the molecular characteristics of the CaM of parasites.

Vaccination of rabbits with immunodominant antigens from Sarcoptes scabiei induced high levels of humoral responses and pro-inflammatory cytokines but confers limited protection.

BACKGROUND: Vaccination is an attractive ecological alternative to the use of acaricides for parasite control. However, effective anti-parasite vaccines against sarcoptic mange have not yet been developed. The purpose of this study was first to identify Sarcoptes scabiei immunodominant antigens and second to evaluate them as vaccine candidates in a rabbit/S. scabiei var. cuniculi model. METHODS: The S. scabiei Ssλ15 immunodominant antigen was selected by immunoscreening of a S. scabiei var. hominis cDNA. The full-length cDNA was sequenced and cloned into the pGEX vector and the recombinant protein expressed in BL21 (DE3) cells and purified. A vaccination trial was performed consisting of a test group (n = 8) immunised with recAgs (a mix of two recombinant antigens, Ssλ15 and the previously described Ssλ20∆B3) and a control group (n = 8) immunised with PBS. All analyses were performed with R Statistical Environment with α set at 0.050. RESULTS: The full-length open reading frame of the 1,821 nt cloned cDNA encodes a 64 kDa polypeptide, the sequence of which had 96 % identity with a hypothetical protein of S. scabiei. Ssλ15 was localised by immunostaining of skin sections in the tegument surrounding the mouthparts and the coxa in the legs of mites. Rabbit immunisation with recAgs induced high levels of specific IgG (P < 0.010) and increased levels of total IgEs. However, no significant clinical protection against S. scabiei challenge was detected. Unexpectedly, the group immunised with the recAgs mix had significantly higher lesion scores (P = 0.050) although lower mean mite densities than those observed in the control group. These results might indicate that the lesions in the recAgs group were due not only to the mites density but also to an exacerbated immunological response after challenge, which is in agreement with the specific high levels of pro-inflammatory cytokines (IL-1 and TNFα) detected after challenge in this group. CONCLUSIONS: The selected antigens delivered as recombinant proteins had no clinical protective efficacy against S. scabiei infestation although immunisation reduced mite density. However, these results pave the way for future studies on alternative production systems, adjuvants, delivery methods and combinations of antigens in order to manage stimulation of clinical protective immune responses.

Draft genome of the scabies mite.

BACKGROUND: The disease scabies, caused by the ectoparasitic mite, Sarcoptes scabiei, causes significant morbidity in humans and other mammals worldwide. However, there is limited data available regarding the molecular basis of host specificity and host-parasite interactions. Therefore, we sought to produce a draft genome for S. scabiei and use this to identify molecular markers that will be useful for phylogenetic population studies and to identify candidate protein-coding genes that are critical to the unique biology of the parasite. METHODS: S. scabiei var. canis DNA was isolated from living mites and sequenced to ultra-deep coverage using paired-end technology. Sequence reads were assembled into gapped contigs using de Bruijn graph based algorithms. The assembled genome was examined for repetitive elements and gene annotation was performed using ab initio, and homology-based methods. RESULTS: The draft genome assembly was about 56.2 Mb and included a mitochondrial genome contig. The predicted proteome contained 10,644 proteins, ~67 % of which appear to have clear orthologs in other species. The genome also contained more than 140,000 simple sequence repeat loci that may be useful for population-level studies. The mitochondrial genome contained 13 protein coding loci and 20 transfer RNAs. Hundreds of candidate salivary gland protein genes were identified by comparing the scabies mite predicted proteome with sialoproteins and transcripts identified in ticks and other hematophagous arthropods. These include serpins, ferritins, reprolysins, apyrases and new members of the macrophage migration inhibitory factor (MIF) gene family. Numerous other genes coding for salivary proteins, metabolic enzymes, structural proteins, proteins that are potentially immune modulating, and vaccine candidates were identified. The genes encoding cysteine and serine protease paralogs as well as mu-type glutathione S-transferases are represented by gene clusters. S. scabiei possessed homologs for most of the 33 dust mite allergens. CONCLUSION: The draft genome is useful for advancing our understanding of the host-parasite interaction, the biology of the mite and its phylogenetic relationship to other Acari. The identification of antigen-producing genes, candidate immune modulating proteins and pathways, and genes responsible for acaricide resistance offers opportunities for developing new methods for diagnosing, treating and preventing this disease.

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.

Temporal stability in the genetic structure of Sarcoptes scabiei under the host-taxon law: empirical evidences from wildlife-derived Sarcoptes mite in Asturias, Spain.

BACKGROUND: Implicitly, parasite molecular studies assume temporal genetic stability. In this study we tested, for the first time to our knowledge, the extent of changes in genetic diversity and structure of Sarcoptes mite populations from Pyrenean chamois (Rupicapra pyrenaica) in Asturias (Spain), using one multiplex of 9 microsatellite markers and Sarcoptes samples from sympatric Pyrenean chamois, red deer (Cervus elaphus), roe deer (Capreolus capreolus) and red fox (Vulpes vulpes). RESULTS: The analysis of an 11-years interval period found little change in the genetic diversity (allelic diversity, and observed and expected heterozygosity). The temporal stability in the genetic diversity was confirmed by population structure analysis, which was not significantly variable over time. Population structure analysis revealed temporal stability in the genetic diversity of Sarcoptes mite under the host-taxon law (herbivore derived- and carnivore derived-Sarcoptes mite) among the sympatric wild animals from Asturias. CONCLUSIONS: The confirmation of parasite temporal genetic stability is of vital interest to allow generalizations to be made, which have further implications regarding the genetic structure, epidemiology and monitoring protocols of the ubiquitous Sarcoptes mite. This could eventually be applied to other parasite species.

Identification and heterologous expression of a Sarcoptes scabiei cDNA encoding a structural antigen with immunodiagnostic potential.

The mite Sarcoptes scabiei causes sarcoptic mange (or scabies), a disease of considerable human and veterinary significance. An S. scabiei cDNA clone of about 2 kb was isolated from a S. scabiei var. hominis expression library by immunological screening using blood serum from a naturally infected chamois (Rupicapra rupicapra). The nucleotide sequence of the identified cDNA contains an open reading frame of 1930 bp that encodes a 642 amino acid polypeptide. This polypeptide shows tandem repeats of a glycine-serine rich 20 residue sequence followed by a unique C-terminal glutamate rich 54 residue sequence. The cDNA or the deduced polypeptide did not show significant similarities to any of the sequences in the databases. A carboxyl-terminal fragment of this polypeptide (residues 380 to 642) was efficiently expressed in Escherichia coli as a fusion with Glutathione S-transferase and then was used to produce a specific antiserum. The antigen encoded by the cDNA was located at the integument of the mite's epidermis and the cavities surrounding its vital organs. Western blot analysis of mite extracts using the specific antiserum against the recombinant protein identified antigens larger that 60 kDa indicating that the isolated cDNA did not contain the full ORF. Moreover, we designed a diagnostic assay based on the carboxyl-terminal fragment of the antigen for the identification of infected animals.

Expressed sequence tag analysis of Sarcoptes scabiei.

Sarcoptes scabiei is an important parasitic mite in both man and animals. Little is known about the molecular interactions between this pathogen and its host. This is in part explained by the paucity of mite-derived material, including antigens. To extend the knowledge of the molecular repertoire in S. scabiei, we have performed a gene survey by an expressed sequence tag (EST) analysis. A total of 1020 ESTs were generated from an S. scabiei cDNA library. The average sequence read was 510 bp after editing and the overall sequencing success was 89%. Clustering of the sequences resulted in 76 clusters, comprising 36% of the ESTs. Sequence similarity searches showed that almost half of the S. scabiei ESTs could be assigned a putative identity. Many of these transcripts shared similarity with genes involved in basic metabolism and cellular organization. In the data set we also identified several proteases and other types of potential allergens implicated in various disease mechanisms. A relatively large fraction of the ESTs coded for different proteins carrying protease inhibitor-like domains. The clones with no similarity to previously identified genes constituted 11% of our transcripts. The EST data generated in this study will be a valuable resource in further studies of the biology of S. scabiei and in the identification of genes that can serve as potential targets in the control of the parasite.