Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

Background

The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.

Methodology/Principal Findings

We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.

Conclusions/Significance

The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.     

The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease

The helminth parasite Fasciola hepatica secretes cathepsin L cysteine proteases to invade its host, migrate through tissues and digest haemoglobin, its main source of amino acids. Here we investigated the importance of pH in regulating the activity and functions of the major cathepsin L protease FheCL1. The slightly acidic pH of the parasite gut facilitates the auto-catalytic activation of FheCL1 from its inactive proFheCL1 zymogen; this process was ∼40-fold faster at pH 4.5 than at pH 7.0. Active mature FheCL1 is very stable at acidic and neutral conditions (the enzyme retained ∼45% activity when incubated at 37°C and pH 4.5 for 10 days) and displayed a broad pH range for activity peptide substrates and the protein ovalbumin, peaking between pH 5.5 and pH 7.0. This pH profile likely reflects the need for FheCL1 to function both in the parasite gut and in the host tissues. FheCL1, however, could not cleave its natural substrate Hb in the pH range pH 5.5 and pH 7.0; digestion occurred only at pH≤4.5, which coincided with pH-induced dissociation of the Hb tetramer. Our studies indicate that the acidic pH of the parasite relaxes the Hb structure, making it susceptible to proteolysis by FheCL1. This process is enhanced by glutathione (GSH), the main reducing agent contained in red blood cells. Using mass spectrometry, we show that FheCL1 can degrade Hb to small peptides, predominantly of 4–14 residues, but cannot release free amino acids. Therefore, we suggest that Hb degradation is not completed in the gut lumen but that the resulting peptides are absorbed by the gut epithelial cells for further processing by intracellular di- and amino-peptidases to free amino acids that are distributed through the parasite tissue for protein anabolism.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.     

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.     

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.     

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches

BackgroundFasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica.Conclusions/SignificanceThe present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.     

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Background

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.

Methods

In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.

Results

The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).

Conclusions

These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.     

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.     

Polymorphism of the ND1 and CO1 Mitochondrial Genes in Populations of Liver Fluke Fasciola hepatica

Polymorphism of fragments of the ND1 and CO1 mitochondrial genes was for the first time found in four liver fluke Fasciola hepatica samples from Ukraine, Belarus, Moscow region, and Mordovia. The ND1 and CO1fragments were respectively 292 and 433 bp in size, with polymorphic sites amounting to 2.7 and 0.9% of the total sequence. Seven haplotypes were found in the four samples; two haplotypes (A and B) were most common (29.1 and 45.8%, respectively) in the pooled sample. The haplotype frequency distribution differed among the four populations. Haplotype B prevailed in the Mordovian and Moscow region samples. In addition, these samples had a higher number of unique haplotypes (A2, A3, B2). The results testify to genetic differences of the four geographically distant populations of F. hepatica.     

Polymorphism of the ND1 and CO1 mitochondrial genes in populations of liver fluke Fasciola hepatica

Polymorphism of fragments of the ND1 and CO1 mitochondrial genes was for the first time found in four liver fluke Fasciola hepatica samples from Ukraine, Belarus, Moscow region, and Mordovia. The ND1 and CO1 fragments were respectively 292 and 433 bp in size, with polymorphic sites accounting for 2.7 and 0.9% of the total sequence. Seven haplotypes were found in the four samples; two haplotypes (A and B) were most common (29.1 and 45.8%, respectively) in the pooled sample. The haplotype frequency distribution differed among the four populations. Haplotype B prevailed in the Mordovian and Moscow region samples. In addition, these samples had a higher number of unique haplotypes (A2, A3, B2). The results testify to genetic divergence of the four geographically distant populations of F. hepatica.     

A Case of Fasciola hepatica Infection Mimicking Cholangiocarcinoma and ITS-1 Sequencing of the Worm

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.     

Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran

Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica. In Iran, the distribution of these two species overlaps in most areas, including the northern human endemic province of Gilan where both fasciolids are simultaneously found in individual cattle and buffaloes. A phenotypic study of fasciolid adult flukes from naturally infected bovines from Gilan was carried out by means of an exhaustive morphometric analysis using traditional microscopic measurements and an allometric model. The Iranian fasciolids were compared to F. hepatica and F. gigantica standard populations, i.e. from geographical areas where both species do not co-exist (Bolivia and Burkina Faso, respectively). Although morphometric values somewhat overlapped, there were clear differences in allometric growth. The allometric function was adjusted to 25 pairs of variables. Results obtained revealed that Iranian F. hepatica-like specimens are larger than the F. hepatica standard and Iranian F. gigantica-like specimens are longer and narrower than the F. gigantica standard, but with smaller body area. Measurements which permit a specific differentiation in allopatric populations (distance between ventral sucker and posterior end of the body; ratio between body length and body width) overlap in the specimens from Gilan, thus proving the presence of intermediate forms. When compared to the standard populations, the different Iranian fasciolid morphs show greater differences in F. gigantica-like specimens than in F. hepatica-like specimens. This study shows that simple, traditional microscopic measurements may be sufficient for the morphometric characterisation of fasciolids, even in areas where intermediate forms are present.     

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.     

人sTNFR1基因原核表达及体外抗肝细胞凋亡的活性研究

采用RT-PCR,从Hela细胞的mRNA中扩增人sTNFR1基因,构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆,转化入大肠杆菌,测序证实其序列与基因数据库中sTNFR1基因一致.经异丙基-β-D半乳糖苷酶（IPTG）诱导表达,淀粉树脂亲和层析法纯化,得到融合蛋白sTNFR1-MBP.结果显示：经Western-blotting检测,sTNFR1-MBP具有免疫活性L流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用,这为今后的研究打下了基础.     

结核分枝杆菌重组38kD蛋白用于新疆南疆维吾尔族和湖南湘中汉族血清学诊断的研究

目的：对结核分枝杆菌38kD蛋白编码基因进行克隆表达及纯化,建立基于重组38kD蛋白的酶联免疫吸附法（EusA）检测结核病人血清标本,评价重组38kD蛋白用于结核病血清学诊断抗原的价值。并比较分析其在汉族和维吾尔族人群中的血清学诊断的差异。方法：用PCR方法扩增38kD蛋白的编码基因,构建重组质粒,转化到大肠杆菌BL21中,经IPTG诱导表达,得到纯化的38kD蛋白,建立以38kD蛋白为包被抗原的ELISA,并检测临床确诊的结核病人血清标本。结果：ELISA检测结核病患者血清标本的维吾尔族阳性率为34％（52／153）,汉族为52．4％（65／124）,两者对比有统计学差异（x2=9．538,P〈O．005）。在阴性对照中的维吾尔族特异度为96．4％（159／165）,汉族为98．8％（130／133）,结果无统计学意义（x2=0．111,P〉0．5）。结论：重组38kD蛋白用于血清学诊断的敏感度在维吾尔族和汉族中有差异,而其诊断特异度无差别。