Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors.
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ABSTRACT:
SUBMITTER: Yatsuda AP
PROVIDER: S-EPMC1418636 | biostudies-literature | 2006 Mar
REPOSITORIES: biostudies-literature
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Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors.
Yatsuda Ana P AP Bakker Nicole N Krijgsveld Jeroen J Knox David P DP Heck Albert J R AJ de Vries Erik E
Infection and immunity 20060301 3
Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-secretory products of Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors were employed to localize active CBLs in 2-D protein gels. Mass spectrometry provided the identification of AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered expressed sequence tags. ...[more]
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Project description:Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of sheep, is particularly adept at developing resistance to the anthelmintic drugs used in its treatment and control. The basis of anthelmintic resistance is poorly understood for many commonly used drugs with most research being focused on mechanisms involving drug targets or drug efflux. Altered or increased drug metabolism is a possible mechanism that has yet to receive much attention despite the clear role of xenobiotic metabolism in pesticide resistance in insects. The cytochrome P450s (CYPs) are a large family of drug-metabolising enzymes present in almost all living organisms, but for many years thought to be absent from parasitic nematodes. In this paper, we describe the CYP sequences encoded in the H. contortus genome and compare their expression in different parasite life-stages, sexes and tissues. We developed a novel real-time PCR approach based on partially assembled CYP sequences "tags" and confirmed findings in the subsequent draft genome with RNA-seq. Constitutive expression was highest in larval stages for the majority of CYPs, although higher expression was detected in the adult male or female for a small subset of genes. Many CYPs were expressed in the worm intestine. A number of H. contortus genes share high identity with Caenorhabditis elegans CYPs and the similarity in their expression profiles supports their classification as putative orthologues. Notably, H. contortus appears to lack the dramatic CYP subfamily expansions seen in C. elegans and other species, which are typical of CYPs with exogenous roles. However, a small group of H. contortus genes cluster with the C. elegans CYP34 and CYP35 subfamilies and may represent candidate xenobiotic metabolising genes in the parasite.
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Project description:The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.
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Project description:The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants, Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematode Caenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.
| S-EPMC387910 | biostudies-literature
Project description:BackgroundHaemonchus contortus, a blood-feeding parasite, is constantly surrounded by large quantities of heme released from the catabolism of host red blood cells. To cope with the toxicity of free heme, H. contortus needs to uptake and detoxify the heme, a process believed to be paramount for parasite survival.MethodsA heme-responsive gene Hc-hrg-2 was identified which is the homologue of Ce-hrg-2. The transcriptional levels in all developmental stages and heme-responsive ability of Hc-hrg-2 were analyzed by qRT-PCR. Immunofluorescence analysis and cell transfections were performed to analyze the expression pattern of Hc-HGR-2. Statistical analyses were performed with GraghPad Prism 6.0 using Student's t-test.ResultsTo investigate the heme homeostasis of H. contortus, we first identified a heme-responsive gene Hc-hrg-2, a homolog of Ce-hrg-2 that is involved in heme transport in the hypodermis of Caenorhabditis elegans. Using qRT-PCR, we showed that Hc-hrg-2 mRNA was expressed throughout all life-cycle stages of H. contortus with the highest level in the third-stage larvae (L3s). Notably, transcription of Hc-hrg-2 in the exsheathed L3s was significantly upregulated in the presence of high concentration of heme. We found that Hc-HRG-2 protein was mainly located in the hypodermal tissues of adult H. contortus in vivo and the endoplasmic reticulum in the transfected mammalian cells. Our in vitro assay demonstrated that Hc-HRG-2 is a heme-binding protein with glutathione S-transferase activity and heme had a significant effect on its enzymatic activity when a model substrate 1-chloro-2, 4-dinitrobenzene (CDNB) was used.ConclusionsHc-hrg-2 is a heme-responsive gene and engaged in heme homeostasis regulation in hypodermal tissues during the free-living stages of H. contortus.
| S-EPMC6988263 | biostudies-literature