Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

Identification of structural features of condensed tannins that affect protein aggregation

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r=-0.916; amw: r=-0.925; p<0.01; df=27), and gelatin (mDP: r=-0.961; amw: r=-0.981; p<0.01; df=12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r=0.677; p<0.05; df=9 and r=0.887; p<0.01; df=9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r=-0.941; p<0.01; df=5) and increased the apparent β-sheet content (r=0.916; p<0.05; df=5) of BSA

Anxiety and Depression in Adults with Autism Spectrum Disorder: A Systematic Review and Meta-analysis

Adults with autism spectrum disorder (ASD) are thought to be at disproportionate risk of developing mental health comorbidities, with anxiety and depression being considered most prominent amongst these. Yet, no systematic review has been carried out to date to examine rates of both anxiety and depression focusing specifically on adults with ASD. This systematic review and meta-analysis examined the rates of anxiety and depression in adults with ASD and the impact of factors such as assessment methods and presence of comorbid intellectual disability (ID) diagnosis on estimated prevalence rates. Electronic database searches for studies published between January 2000 and September 2017 identified a total of 35 studies, including 30 studies measuring anxiety (n = 26 070; mean age = 30.9, s.d. = 6.2 years) and 29 studies measuring depression (n = 26 117; mean age = 31.1, s.d. = 6.8 years). The pooled estimation of current and lifetime prevalence for adults with ASD were 27% and 42% for any anxiety disorder, and 23% and 37% for depressive disorder. Further analyses revealed that the use of questionnaire measures and the presence of ID may significantly influence estimates of prevalence. The current literature suffers from a high degree of heterogeneity in study method and an overreliance on clinical samples. These results highlight the importance of community-based studies and the identification and inclusion of well-characterized samples to reduce heterogeneity and bias in estimates of prevalence for comorbidity in adults with ASD and other populations with complex psychiatric presentations

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1 beta, IL-12 and IFN-gamma

International audienceAfter intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-$\alpha$, interleukin (IL)-1$\beta$, IL-12, and interferon (IFN)-$\gamma$ by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-$\alpha$ or IL-1$\beta$. Bovine PMN were shown to secrete TNF-$\alpha$, IL-1$\beta$, IL-12, IL-8 and IFN-$\gamma$ in response to LPS. Co-incubation of PMN with LPS and TNF-$\alpha$ increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-$\alpha$ had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines

Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8

Abstract Background Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity. Methods Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantum promastigotes and intracellular amastigotes, trypomastigotes of Trypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania. Results The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms of Leishmania. Ultrastructural studies with Leishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite’s death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM. Conclusions The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus against Leishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases

Antiparasitic activity of methanolic extract of Acacia oxyphylla (Leguminosae) against Raillietina echinobothrida

Acacia oxyphylla has been used traditionally by the natives of Mizoram against intestinal worm infections. In the present study, the crude methanolic extract of the plant was tested in vitro on the cestode parasite Raillietina echinobothrida to evaluate its potential anthelmintic efficacy. The test parasites were exposed to varying concentrations of the plant extract in phosphate buffered saline and they all slipped into a flaccid paralytic state at varying periods of time post incubation. Ultrastructural observations on the paralysed worms revealed wide scale destruction of the parasite tegument with intense vacuolization of the syncytium and swellings of the basal lamina accompanied by deformities in the cell organelles. To determine the exact mode of action of the botanicals on the parasite body surface, the tegumental enzymes viz. acid phosphatase (AcPase), alkaline phosphatase (AlkPase) and adenosine triphosphatase (ATPase) were investigated. A prominent decrease in the phosphatase activity, in comparison to the untreated control parasites was noticeable. In view of the alterations in the structural and functional integrity of the tegument, occurring in the treated parasites, it may be assumed that the changes transpire because of a compromise in the permeability of the tegument under the influence of the test plant-derived active chemical principles