Article

A cathepsin L protease essential for Caenorhabditis elegans embryogenesis is functionally conserved in parasitic nematodes

July 2002
Molecular and Biochemical Parasitology 122(1):21-33

July 2002
122(1):21-33

DOI:10.1016/S0166-6851(02)00066-X

Source
PubMed

Authors:

Collette Britton

University of Glasgow

Linda Bates Murray

Mississippi College

Proteolytic enzymes are involved in processes important to development and survival of many organisms. Parasite proteases are considered potential targets of parasite control yet, for most, their precise physiological functions are unknown. Validation of potential targets requires analysis of function. We have recently identified a cathepsin L (CPL) cysteine protease, Ce-CPL-1, which is essential for embryonic development of the free-living nematode Caenorhabditis elegans. We now show that CPL genes closely related to Ce-cpl-1 are expressed in the animal parasitic nematodes Haemonchus contortus, Dictyocaulus viviparus, Teladorsagia circumcincta, Ancylostoma caninum and Ascaris suum, as well as in plant parasitic nematodes. The similarities in gene structure and encoded amino acid sequence indicate that the parasite and C. elegans CPLs are homologous enzymes. We demonstrate functional compensation of the loss of C. elegans cpl-1 by transgenic expression of the H. contortus cpl-1 gene, rescuing the embryonic lethality. These genes may therefore be orthologues, sharing the same function in both species. Targeting of this enzyme has potential in inhibiting development and transmission of parasitic nematodes. In addition, the role of CPL is important to our understanding of nematode development.

Haemonchus contortus cathepsin L cysteine protease (cpl1) mRNA, complete cds

Nucleotide Sequence

July 2000

Collette Britton

Ancylostoma caninum cathepsin L-like protease (cpl-1) mRNA, partial cds

Nucleotide Sequence

November 2000

Collette Britton

Haemonchus contortus cathepsin L (cpl-1) gene, complete cds

Nucleotide Sequence

September 2001

Collette Britton

Haemonchus contortus cathepsin L cysteine protease (cpl-1) mRNA, complete cds

Nucleotide Sequence

August 2001

Collette Britton

Dictyocaulus viviparus cathepsin L 1 (cpl-1) mRNA, partial cds

Nucleotide Sequence

June 2001

Collette Britton

Dictyocaulus viviparus cathepsin L (cpl-1) gene, partial cds

Nucleotide Sequence

September 2001

Collette Britton

Ascaris suum cathepsin L (cpl-1) mRNA, partial cds

Nucleotide Sequence

December 2001

Collette Britton

Teladorsagia circumcincta cathepsin L 1 (cpl-1) gene, partial cds

Nucleotide Sequence

June 2001

Collette Britton

Peptidases in Parasitic Nematodes. A review

Chapter

Full-text available

Jan 2013

The nematodes are, after the insects, the group of organisms with the largest number of species identified. They include members of great medical, veterinary and agricultural significance, making this group one of the most important animal parasites. However there are many gaps in our knowledge of them. For example, there is still not a single nematode species for which we have detailed knowledge of feeding, digestion and nutritional requirements, showing that there are still many aspects to be learned about nutrition in nematodes [1]. Our understanding of the process of protein digestion, a very important function in the biology of any organism, is still poor since our knowledge is composed of fragmentary data for different groups of nematodes. It is believed that peptidases are essential during the development process and in the most critical moments of parasite-host interactions, and are thus directly involved with the growth and survival of the parasite. Their identification and characterization are important for basic understanding of the biology of the parasite, and their relevance to parasitic nematodes as virulence factors is clear. Consequently, peptidases are currently viewed as potential targets for vaccines, drugs and serodiagnosis. Despite this, in most cases, the precise physiological functions of peptidases in parasites are not known [2]. Peptidases comprise a large class of hydrolytic enzymes in parasitic nematodes, participating in nutrition through digestion of host proteins [3]. They also act in the moulting and resorption of the cuticle by processing and activating proenzymes or prohormones [4], degrading proteins that anchor the epidermis to the underlying cuticle (apolysis) [5], or by digesting the cuticle for resorption or facilitating its shedding (ecdysis) [6]. They are also active during embryonic development of the egg [7]. Peptidases are important in host-parasite relationships, being important virulence factors in some parasites [8]. The pathogenicity of several species of nematode has been significantly correlated with their peptidase activity. These include Strongyloides stercoralis [9], Anisakis simplex [10], Onchocerca volvulus [11], Trichinella spiralis [12], and Ancylostoma caninum [13]. All major types of peptidases have been described in nematodes. Aspartic peptidases have been described primarily in functions related to the digestion of nutrients. In invertebrates it is thought that, along with the cysteine peptidases, these have the same role as aspartic and serine peptidases in vertebrates [14]. In parasitic nematodes, the cysteine peptidases may be the class for which we have most information, since, owing to their great diversity, they cover virtually all functions in which peptidases are involved in parasitic nematodes. Cathepsins B and L are types of cysteine peptidases belonging to the papain family, and have been comprehensively studied in nematodes [15]. High variability has been found among the cathepsins B from different species of nematodes regarding optimum temperature and pH, and substrate affinity. It is thought that their main role is to digest nutrients and that the high interspecific variability observed is due to the nematode adapting to the ecological niche it occupies [16]. Cathepsins L also seem to be involved in the digestion of nutrients, as well as in processes of embryogenesis and moulting [2]. Many of these cathepsins L have counterparts in the free living nematode Caenorhabditis elegans, suggesting that they may be involved in conserved functions in different species of nematodes, but little is known about their precise functions [7]. The metallopeptidases are involved in the invasion of host tissues by the parasite, as they are able to degrade the extracellular matrix, and are also involved in the process of ecdysis and digestion of nutrients. The serine peptidases are also present in nematodes, and, along with the metallopeptidases, are believed to play the largest part in the invasion of host tissues by the parasites [10].

Toward integrative ‘omics of the barber’s pole worm and related parasitic nematodes

Article

Aug 2020
INFECT GENET EVOL

Advances in nucleic acid sequencing, mass spectrometry and computational biology have facilitated the identification, annotation and analysis of genes, transcripts, proteins and metabolites in model nematodes (Caenorhabditis elegans and Pristionchus pacificus) and socioeconomically important parasitic nematodes (Clades I, III, IV and V). Significant progress has been made in genomics and transcriptomics as well as in the proteomics and lipidomics of Haemonchus contortus (the barber's pole worm) – one of the most pathogenic representatives of the order Strongylida. Here, we review salient aspects of genomics, transcriptomics, proteomics, lipidomics, glycomics and functional genomics, and discuss the rise of integrative ‘omics of this economically important parasite. Although our knowledge of the molecular biology, genetics and biochemistry of H. contortus and related species has progressed significantly, much remains to be explored, particularly in areas such as drug resistance, unique/unknown genes, host-parasite interactions, parasitism and the pathogenesis of disease, by integrating the use of multiple ‘omics methods. This approach should lead to a better understanding of H. contortus and its relatives at a ‘systems biology’ level, and should assist in developing new interventions against these parasites.

Cloning and characterization of recombinant Hc-CPL-1 from gastrointestinal nematode Haemonchus contortus isolate from goat (Capra hircus) population in Penang, Malaysia

Article

Dec 2017
TROP BIOMED

Cathepsin L (CPL) cysteine protease is a proteolytic enzyme that involves in many biological processes in a wide range of organisms. In free-living nematode Caenorhabditis elegans, CPL plays important roles in embryogenesis and development processes. The CPL protein is also believed to have a role in degradation of blood meal in the gut of parasitic nematodes. Considering this enzyme might play the same functions in parasitic nematodes, CPL became a potential candidate for vaccination against Haemonchus contortus, a gastrointestinal nematode of small ruminants. H. contortus has been shown to have variations in term of morphology and genetic materials that correlated with different hosts and geographical areas. These variations could hinder the development of effective vaccines. Thus, the present study was conducted to clone and characterize recombinant Hc-CPL-1 from H. contortus isolated from a goat population in Penang, Malaysia. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify target complimentary DNA (cDNA) from total RNA and protein expression using Escherichia coli expression system was performed from constructed cDNA clone library. The identity of each protein band was confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis followed by De novo sequencing and database matching. The protein structure and its evolutionary relationship were also studied using several bioinformatics approaches. Basic local alignment search tool (BLAST) analysis of the strain retrieved from clone library showed 99% sequence similarity to the Haemonchus cathepsin L cysteine protease and a 47 kDA protein was successfully expressed. Bioinformatic analysis indicated that this protease has a close relationship with Dv-CPL-1, Sv-CPL-1 and Ce-CPL-1. These data might provide an insight on manipulating this enzyme for future novel vaccine development. © 2017, Malaysian Society for Parasitology. All rights reserved.

Developmental and functional characterization of cystatin and chitinase of Acanthocheilonema viteae

Thesis

May 2007

Smitha Pillai

Die Infektion mit Filarien (Nematoden) ruft massive Schädigungen beim Menschen hervor. Strategien zur Bekämpfung dieser Parasitose basieren auf einer Massenbehandlung mit Ivermectin und Derivaten. Allerdings ist die Behandlung der Patienten zeitaufwändig und teuer. In diesem Zusammenhang stellt Aufklärung molekularer Mechanismen, die die parasitische Lebensform dieser Würmer ermöglichen, einen neuen Ansatzpunkt für die Entwicklung von Therapeutika und Präventivmaßnahmen dar. Die Moleküle Cystatin und Chitinase wurden, auf Grund ihrer immunodulatorischen und katalytischen Eigenschaften, bei der Nager-Filarie Acanthocheilonema viteae als essentielle Proteine identifiziert. Ziel der vorliegenden Arbeit war daher die detaillierte, funktionale Charakterisierung dieser beiden Proteine. Um das räumliche Expressionsmuster und damit potentielle Funktionen des Sekretionsprotein Cystatins zu ermitteln, wurde der frei lebende Nematode Caenorhabditis elegans als heterologes Expressionssystem genutzt. Unter dem Einfluss des Cystatin-Promoters konnte GFP in pharyngealen und rektalen Zellen von C. elegans exprimiert werden. Möglicherweise ist das Cystatin damit bei A. viteae in den Häutungsprozess involviert, da bei C. elegans derartige Enzyme in den pharyngealen Zellen gespeichert werden. Des Weiteren wurde der Häutungsprozess der infektiösen L3 zum Stadium der L4 durch Ausschalten des Gens mittels RNAi um drei Tage verzögert. Allerdings war der Effekt transient und die Verzögerung des Häutungsprozesses beeinflusste weder die Viabilität noch die Infektiösität der Larven. Die Analyse der Regulation von Cystatin während der Entwicklung des Parasiten mittels der Real-Time PCR zeigte, dass das Gen im Stadium der Mikrofilarien, die der Immunantwort des Wirtes voll exponiert sind, maximal exprimiert wird. Für die Chitinase von A. viteae konnte eine essentielle Rolle im Häutungsprozess nachgewiesen werden. Das Ausschalten des Gens führte zu einer Hemmung der Häutung bei 90 % der L3 und damit zu ihrem Tod. Die maximale Expression im L3 Stadium des Parasiten ist ein weiterer Hinweis darauf, dass dieses Protein in den Häutungsprozess involviert ist. Mittels RNAi bei adulten Parasiten konnte die katalytische Rolle der Chitinase beim Abbau des Chitins im Ei bestätigt werden, da hier nur ungeschlüpfte Mikrofilarien ausgeschieden wurden. Die Ergebnisse dieser Arbeit liefern weitere Hinweise darauf, dass sowohl das Cystatin als auch die Chitinase von A. viteae auf Grund ihrer essentiellen endogenen Funtionen attraktive Zielmoleküle in der Bekämpfung von Filariosen darstellen.

Heterologous expression in Caenorhabditis elegans as an alternative approach to functional studies in Schistosoma mansoni

Article

Full-text available

May 2014

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p < 0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory respons.

Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

Article

Full-text available

Dec 2013
VET RES

With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection.

Cathepsin L-like cysteine proteases from Brugia malayi: cDNA cloning and comparison with Caenorhabditis elegans

Article

Full-text available

Dec 2004

Proteases are thought to be potentially useful targets for developing medicines to control filariasis caused by parasitic nematodes. To screen cysteine proteases essential for viability of nematodes, fifteen cathepsin B/L-like genes of Caenorhabditis elegans, as a model of the parasitic nematodes were interfered by RNAi. As a result, ∼100% embryonic lethality was observed only when Ce-cpl-1, encoding a cathepsin L-like protease, was knocked down. Subsequent attempts were made to identify the orthologs of Ce-cpl-1 in the parasitic nematode Brugia malayi by molecular cloning and sequencing of some EST clones. The sequences of five distinct open reading frames were identified, all of which are most homologous to Ce-CPL-1 in C. elegans. The consensus catalytic triad of cathepsin L-like proteases is conserved among four of the five predicted proteins. Phylogenetic analysis suggests that one of them, Bm-CPL-1, is closely related to the proteases from other filarial parasites. However, neither Bm-CPL-1 nor the other four predicted proteins belong to the same sub-branch of Ce-CPL-1 in the phylogenetic tree. In B. Malayi, the functions of Ce-CPL-1 are presumably shared by some of the predicted proteases including Bm-CPL-1, although the possibility cannot be ruled out that B. malayi has an unknown cysteine protease more resembling Ce-CPL-1.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis

Article

Full-text available

May 2024

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Proteomic Profiling and In Silico Characterization of the Secretome of Anisakis simplex Sensu Stricto L3 Larvae

Article

Full-text available

Feb 2022

Anisakis simplex sensu stricto (s.s.) L3 larvae are one of the major etiological factors of human anisakiasis, which is one of the most important foodborne parasitic diseases. Nevertheless, to date, Anisakis secretome proteins, with important functions in nematode pathogenicity and host-parasite interactions, have not been extensively explored. Therefore, the aim of this study was to identify and characterize the excretory-secretory (ES) proteins of A. simplex L3 larvae. ES proteins of A. simplex were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the identified proteins were then analyzed using bioinformatics tools. A total of 158 proteins were detected. Detailed bioinformatic characterization of ES proteins was performed, including Gene Ontology (GO) analysis, identification of enzymes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, protein family classification, secretory pathway prediction, and detection of essential proteins. Furthermore, of all detected ES proteins, 1 was identified as an allergen, which was Ani s 4, and 18 were potential allergens, most of which were homologs of nematode and arthropod allergens. Nine potential pathogenicity-related proteins were predicted, which were predominantly homologs of chaperones. In addition, predicted host-parasite interactions between the Anisakis ES proteins and both human and fish proteins were identified. In conclusion, this study represents the first global analysis of Anisakis ES proteins. The findings provide a better understanding of survival and invasion strategies of A. simplex L3 larvae.

Plant serine protease inhibitor (SPI): A potent player with bactericidal, fungicidal, nematicidal and antiviral properties

Article

Full-text available

Jan 2020

Scanning electron microscopy reveals deleterious effects of Moringa oleifera seed exuded proteins on root-knot nematode Meloidogyne incognita eggs

Article

Nov 2019
INT J BIOL MACROMOL

Plant seeds can exudate active molecules with inhibitory effects against several soil pathogens, including nematodes. This study aimed to characterize and evaluate the nematicidal properties against Meloidogyne incognita of exuded proteins from Moringa oleifera seeds. M. oleifera seeds were soaked in distilled water, and exudates were harvested and analyzed for the presence of defense proteins and anthelmintic activity. Enzymatic assays revealed the existence of PR-proteins such as β-1,3-glucanases (0.18 ± 0.003 nkatal mg-1 of protein), chitinases (0.22 ± 0.004 nkatal mg-1 of protein), proteases (261.30 ± 6.405 AU mg-1 of protein min-1), serine (190.30 ± 5.574 IA mg-1 of protein) and cysteine (231.70 ± 0.923 IA mg-1 of protein), protease inhibitors. The exuded proteins presented ovicidal activity and caused 100% mortality of second-stage juveniles (J2s). Scanning electron microscopy (SEM) revealed deleterious effects on M. incognita eggs, such as invaginations, cracks, scratched surface, and loss of internal content. These findings confirm the presence of anthelmintic proteins in M. oleifera seed exudate, possibly involved in plant defense during seed germination. Besides this, the exuded proteins exhibited strong biotechnological potential for use in the biocontrol of M. incognita infections, which are responsible for millions of dollars in staple crop losses every year.

Differential proteolytic activity of Anisakis simplex s.s. and Anisakis pegreffii, two species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis

Article

Full-text available

Jul 2019
ACTA TROP

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen–host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p≤0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecificdifferences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

Differential cleaving of specific substrates for cathepsin-like activity shows cysteine and serine protease activities and a differential profile between Anisakis simplex s.s. and Anisakis pegreffii, sibling species major etiologic agents of anisakiasis

Article

Full-text available

Nov 2019

Humans can contract anisakiasis by eating fish or squid containing live larvae of the third stage (L3) of the parasitic nematodes of the genus Anisakis, majorly from Anisakis simplex s.s. and Anisakis pegreffii, sibling species of the A. simplex s.l. complex. Most cases diagnosed molecularly are due to A. simplex s.s., although A. pegreffii has also been identified in human cases. Cathepsins are mostly lysosomal multifunctional cysteine proteases and can participate in the pathogenicity of parasites. Cathepsin B and L activities were investigated in the two sibling species of Anisakis mentioned. L3 and L4 of both species were collected during their in vitro development, and cathepsin activity was determined in the range of pH 4.0–8.5, using specific fluorogenic substrates. The activity detected with the substrate Z-FR-AMC (N-α-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl-coumarin) was identified as cathepsin L (optimum pH = 5.0, range 4.0–6.0, p < 0.001). Activity was highest in L3 freshly collected from fish, especially in A. simplex s.s., and decreased during development, which could be related to virulence, invasion of host tissues, and/or intracellular digestion. Cathepsin B-like activity was not identified with either of the substrates used (Z-RR-AMC [N-α-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methyl-coumarin] and Z-FR-AMC). With Z-RR-AMC, cleaving activity was detected almost exclusively in L4 of A. simplex s.s. (p < 0.05) with optimum pH = 8.0 (range 7.0–8.5). Assays with class-specific protease inhibitors showed that this activity was mainly due to serine proteases [up to 90% inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)], although metalloproteases (up to 40–45% inhibition with 1,10 phenanthroline) and slight cysteine protease activity (<15% inhibition with E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane]; putative cathepsin B-like) were also detected. These results show differential serine protease activity between sibling Anisakis species, regulated by larval development, at least in A. simplex s.s. The higher cathepsin L and serine protease activities detected in this species could be related to its greater pathogenicity, reported in experimental animals, compared to that of A. pegreffii.

Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus

Article

Full-text available

Jan 2019
INT J MOL SCI

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

Cysteine peptidases of Eudiplozoon nipponicum: A broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp

Article

Full-text available

Mar 2018

Background: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. Methods: Sequences of cathepsins L and B (EnCL and EnCB) were mined fromE. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. Results: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm’s digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. Conclusions: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

Cysteine Proteases of Parasitic Helminths

Chapter

Nov 2017

Cysteine protease and/or thiol peptidase/sulphydryl peptidase/cysteinyl peptidase invariably contain cysteine at their active site and hence collectively termed as cathepsins. This proteolytic enzyme, secreted and/or regurgitated/excreted by the in situ invasive forms of metazoan parasites of men and domestic livestock, often involve in the host tissue penetration, digestion of nutrients, and their availability to the parasite for its growth and development, evasion of host defense, and in of late documented their significant role in detection of in situ parasites at an early developmental stage(s) [Immunodiagnosis] beside an important entity to develop immunoprotection strategies against the livestock diseases, so much so, to contain and curb the colossal recurring losses, incidental to widely prevalent parasitic disease, to the fast developing livestock products based food industry in the developing world. Herein, the authors discuss the historical background, molecular structure, and classification of cathepsin proteases and update advancements in exploiting the enzyme for early diagnosis and planning immunoprotection strategies against metazoan parasitic diseases with special reference to tropical fasciolosis.

The cathepsin S cysteine proteinase of the burrowing nematode Radopholus similis is essential for the reproduction and invasion

Article

Full-text available

Dec 2016

Background: The nematode Radopholus similis is an important migratory endoparasite of plants. Cysteine proteinases such as cathepsin S (CPS) play key roles during embryonic development, invasion, and pathogenesis in nematodes and many other animal parasites. This study was designed to investigate the molecular characterization and functions of a cathepsin S protease in R. similis and to find new targets for its control. Results: Rs-CPS of R. similis, Hg-CPS of Heterodera glycines and Ha-CPS of H. avenae are closely genetically related and share the same branch of the phylogenetic tree. Rs-cps is a multi-copy gene that is expressed in the esophageal glands, ovaries, testes, vas deferens, and eggs of R. similis. Rs-cps mRNA transcripts are expressed at varying levels during all developmental stages of R. similis. Rs-cps expression was highest in females. The neurostimulant octopamine did not significantly enhance the ingestion of the dsRNA soaking solution by R. similis but instead had a detrimental effect on nematode activity. The dsRNA soaking solution diffused into the body of R. similis not only through the esophageal lumen but also through the amphids, excretory duct, vagina, anus and cloacal orifice. We confirmed that RNAi significantly suppressed the expression level of Rs-cps and reproductive capability and pathogenicity of R. similis. Conclusions: Our results demonstrate that Rs-cps plays important roles in the reproduction, parasitism and pathogenesis of R. similis and could be used as a new potential target for controlling plant parasitic nematodes.

Understanding Haemonchus contortus Better Through Genomics and Transcriptomics

Chapter

Apr 2016
ADV PARASIT

Parasitic roundworms (nematodes) cause substantial mortality and morbidity in animals globally. The barber's pole worm, Haemonchus contortus, is one of the most economically significant parasitic nematodes of small ruminants worldwide. Although this and related nematodes can be controlled relatively well using anthelmintics, resistance against most drugs in common use has become a major problem. Until recently, almost nothing was known about the molecular biology of H. contortus on a global scale. This chapter gives a brief background on H. contortus and haemonchosis, immune responses, vaccine research, chemotherapeutics and current problems associated with drug resistance. It also describes progress in transcriptomics before the availability of H. contortus genomes and the challenges associated with such work. It then reviews major progress on the two draft genomes and developmental transcriptomes of H. contortus, and summarizes their implications for the molecular biology of this worm in both the free-living and the parasitic stages of its life cycle. The chapter concludes by considering how genomics and transcriptomics can accelerate research on Haemonchus and related parasites, and can enable the development of new interventions against haemonchosis.

Immunity to Haemonchus contortus and Vaccine Development

Article

Dec 2016
ADV PARASIT

Sheep are capable of developing protective immunity to Haemonchus contortus through repeated exposure to this parasite, although this immune protection is the result of a complex interaction among age, gender, physiological status, pregnancy, lactation, nutrition and innate and adaptive immunity in the host animal. There are multiple effectors of the protective immune response, which differ depending on the developmental stage of the parasite being targeted, and our understanding of the effector mechanisms has developed considerably in the 2000s. The rational design of vaccines based on 'natural' or 'exposed' antigens depends on an understanding of this exposure-induced immunity. However, the most effective current vaccines rely on protection via the induction of high circulating antibody levels to 'hidden' gut antigens of H. contortus. The success of this latter strategy has resulted in the launch of a vaccine, which is based on extracts of the parasite's gut, to aid in the control of Haemonchus in Australia. The development of recombinant subunit vaccines based on the components of the successful native vaccine has not yet been achieved and most of the recent successes with recombinant subunit vaccines have focussed on antigens unrelated to the gut antigens. The future integration of an understanding of the immunobiology of this parasite with advances in antigen identification, expression (or synthesis) and presentation is likely to be pivotal to the further development of these recombinant subunit vaccines. Recent progress in each of the components underpinning this integrated approach is summarized in this review.

Isolation and characterization of antigens present in the protective ES-thiol fraction of Ostertagia ostertagi and their evaluation as vaccine candidate

Thesis

Jan 2008

Yves Meyvis

Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus

Article

Aug 2014
Exp Parasitol

Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata

Article

Jul 2014
FISH SHELLFISH IMMUN

Enzymology of the nematode cuticle: A potential drug target?

Article

Full-text available

Aug 2014

All nematodes possess an external structure known as the cuticle, which is crucial for their development and survival. This structure is composed primarily of collagen, which is secreted from the underlying hypodermal cells. Extensive studies using the free-living nematode Caenorhabditis elegans demonstrate that formation of the cuticle requires the activity of an extensive range of enzymes. Enzymes are required both pre-secretion, for synthesis of component proteins such as collagen, and post-secretion, for removal of the previous developmental stage cuticle, in a process known as moulting or exsheathment. The excretion/secretion products of numerous parasitic nematodes contain metallo-, serine and cysteine proteases, and these proteases are conserved across the nematode phylum and many are involved in the moulting/exsheathment process. This review highlights the enzymes required for cuticle formation, with a focus on the post-secretion moulting events. Where orthologues of the C. elegans enzymes have been identified in parasitic nematodes these may represent novel candidate targets for future drug/vaccine development.

Knocking-Down Meloidogyne incognita Proteases by Plant-Delivered dsRNA Has Negative Pleiotropic Effect on Nematode Vigor

Article

Full-text available

Dec 2013
PLOS ONE

The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

Article

Full-text available

Mar 2010

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance. Keywordproteomic analysis- Artemia sinica -copper sulfate-acute stress response

Screening of different classes of proteases in microfilarial and adult stages of Setaria cervi

Article

Full-text available

Jun 2009

Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory–secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host–parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.

Transcription profiling reveals stage- and function-dependent expression patterns in the filarial nematode Brugia malayi

Article

Full-text available

May 2012
BMC GENOMICS

Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite's development and survival in its mammalian and insect hosts. We used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched) while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched). We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes. Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of infection, development, reproduction, and survival in the host. Some of these may be useful targets for vaccines or new drug treatments for filariasis.

Cathepsin B- and L-like cysteine protease activities during the in vitro development of Hysterothylacium aduncum (Nematoda: Anisakidae), a worldwide fish parasite

Article

Mar 2010
Parasitol Int

Proteinases play an important role as virulence factors both in the life-cycle of parasites and in the pathogen–host relationship. Hysterothylacium aduncum is a worldwide fish parasite nematode which has been associated with non-invasive anisakidosis and allergic responses to fish consumption in humans. Cysteine proteinases have been associated with allergy to plant pollens, detergents and dust mites. In this study the presence of two types of cysteine proteinases (cathepsin B and cathepsin L) during in vitro development of H. aduncum is investigated. Specific fluorescent substrates were used to determine cathepsin activities. The activity detected with substrate Z-FR-AMC was identified as cathepsin L (optimum pH = 5.5; range 3.5–6.5). Cathepsin B activity was only identified with Z-RR-AMC (optimum pH = 7.0–7.5; range 5.0–8.0). The start of cultivation led to increased activity of both cathepsins (1.8-fold for cathepsin B and 6.3-fold for cathepsin L). These activities varied according to the developmental stage. Cathepsin B activity decreased after M4, returning to its initial level. Cathepsin L activity also decreased after M4, but still maintained a high level (4–6 times the initial level) in adult stages. Having considered these activity variations and the optimum pH values, we suggest that cathepsin L has a role in digestive processes while cathepsin B could be involved in cuticle renewal, among other possible functions.

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Article

Full-text available

Jul 2011
PLOS ONE

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.

Bioinformatic processing of sequencing data of Eudiplozoon nipponicum with a focus on the prediction and characterisation of functionally important molecules

Thesis

Full-text available

Apr 2023

Jiří Vorel

Class Monogenea represents the most species-rich and host-specific group of predominantly fish parasites within Neodermata. Despite their ecological and economic importance, they are primarily studied in terms of taxonomy and morphology and for the population and evolutionary characteristics. Comprehensive omics studies targeting the description of nuclear genomes, stage-specific transcriptomes, and proteomes (e.g., excretory-secretory or tissue-specific) are neglected with a few exceptions. Only these comprehensive studies lead to the creation of robust sequential databases, using which it is possible to understand the molecular nature and biochemistry of monogenean parasites and identify important protein molecules involved in the host-parasite interaction, such as searching for a host, inhibition of hemostasis, modulation of host immunity, suppression of blood clotting by hematophagous parasites, digestion of macromolecules, and absorption of nutrients. The presented dissertation thesis is focused on bioinformatic (in silico) processing of genomic and transcriptomic sequencing data, de novo genome, mitogenome and transcriptome assembly, functional annotation of predicted proteins, molecular functions, and biochemical pathways and further characterisation of selected functionally important protein molecules in the biology of experimental organism Eudiplozoon nipponicum (Monogenea, Diplozoidae), a haematophagous ectoparasite of common carp Cyprinus carpio. Furthermore, this work characterises the composition of adult worms’ excretory-secretory proteome, so-called secretome, and presents proteins interacting with the fish host. 50.81 Gbp of genomic sequencing data (Illumina and Oxford Nanopore reads) was generated, bioinformatically processed, and assembled into the genome draft of length 0.94 Gbp, consisting of 21,044 contigs (N50 = 87.07 kbp). The final assembly represents 57% of the estimated genome size (~1.64 Gbp), with abundantly widespread repetitive and low-complexity regions (~64% of assembled length). In total, 36,626 predicted genes encode 33,031 protein-coding transcripts. A circular mitochondrial genome of 17,038 bp was also assembled from the genomic sequencing data. Another study dealing with the de novo assembly of the E. nipponicum adult worms’ transcriptome (assembled from 324,941 Roche 454 single-end and 149,697,864 Illumina paired-end reads) produced 37,062 protein-coding transcripts, and for 19,644 of them (53.0%) were identified sequential homologues. Mass spectrometry analysis of excretory-secretory proteome identified 721 protein molecules secreted by adults to the outer environment, predominantly with immunomodulatory and anti-inflammatory functions and the ability to digest host macromolecules. Although the amount of omics data and the number of characterised molecules of monogenean parasites have recently increased, monogeneans are still neglected organisms from the point of view of functional genomics, even despite their evident ecological and economic importance. To date, E. nipponicum is the most studied monogenean representative on the level of molecular biology and currently represents an organism with the largest genome within parasitic helminths. Results presented in this thesis represent an important milestone with a significant contribution to monogenean research, and created databases (genomic, transcriptomic and secretomic) have a considerable potential to serve as a source of information for further research of these organisms.

An investigation into the mechanism of action of emodepside and other novel anthelmintic compounds

Thesis

Jan 2008

Kathryn Bull

p>Anthelmintic drugs are used as a control measure for parasitic nematode infections. However, heavy reliance on these compounds has led to the development of parasite resistance and a consequent need to develop new anthelmintics that function via novel biological pathways. In this project, seven potential anthelmintics were examined for activity in the free-living nematode C. elegans. Of the seven compounds, those belonging to the cyclooctadepsipeptide group were shown to inhibit C. elegans pharyngeal pumping. The most potent of the compounds tested was emodepside, which has previously been shown to act as a potent broad-spectrum anthelmintic. The major aim of this project was to define and characterise the mechanism of action of emodepside using the free-living nematode C. elegans. Emodepside potently inhibited the locomotion and pharyngeal pumping of adult and larval stage 4 (L4) C. elegans. The L4 pharynx demonstrated reduced emodepside sensitivity in the presence, but not absence, of the intact cuticle, suggesting that the L4 cuticle reduces drug access. Continuous exposure of C. elegans to emodepside from egg to adult resulted in a slowing of worm development, possibly due to the paralysis of locomotion and pharyngeal pumping, which are important for food location and ingestion. Emodepside inhibited C. elegans egg laying behaviour, but not egg production or egg hatching. These results suggest that emodepside functions at the neuromuscular junction of the pharyngeal, vulval and body wall muscles to produce paralysis of pharyngeal pumping, egg laying and locomotion. Analysis of specific C. elegans gene mutants for their sensitivity to the inhibition of pharyngeal 5-HT response by emodepside strongly suggested that the anthelmintic functions primarily via the Ca2+- activated K+ channel SLO-1, with minor contributory roles performed by the G protein-coupled receptor LAT-1, and the G proteins Go;, and G(V The C. elegans slo-1 (js379) null demonstrated a high level of resistance to emodepside, which was abolished upon rescue of slo-1 expression in neurons. Rescue of slo- 1 expression in the pharyngeal muscles did not restore emodepside sensitivity, suggesting that neuronal SLO-1 is activated by emodepside to achieve paralysis of pharyngeal pumping. A gain-of-function mutant for SLO-1 that increases the Ca2+ sensitivity of the channel was found to be hypersensitive to emodepside, suggesting that emodepside manipulates the Ca2+ sensitivity of SLO-1 to facilitate its activity. To establish how emodepside is affecting SLO-1, the role of this channel in the pharynx was investigated. The s/o-7 Qs379) null possessed a longer mean pump duration and a disrupted pattern of pumping. Rescue of neuronal SLO-1 restored the wild type phenotype, suggesting that neuronal SLO-1 contributes to the control of pharyngeal pumping via the regulation of neurotransmitter release. Therefore, it is possible that emodepside may function by increasing the Ca2+ sensitivity of neuronal SLO-1 at the pharyngeal neuromuscular junction, resulting in an inhibition of neurotransmitter release and muscle paralysis. However, whole cell patch clamp recordings from pharyngeal muscle cells showed that emodepside inhibits SLO-1 currents rather than activating them. It is possible that the different intracellular environments afforded by pharyngeal neurons and muscle may result in the SLO-1 channel being modified differently in these two locations. This could produce a neuronal channel that is stimulated by emodepside and a muscle channel inhibited by the drug. Interestingly, the lat-1 (okl465) mutant was resistant to the effect of emodepside on pharyngeal pumping but not locomotion, suggesting that components of the molecular pathways involved in emodepside activity at the pharyngeal and body wall muscle neuromuscular junctions may be subtly different. The cyclooctadepsipeptides verticilide, PF1022-222 and PF1022-888 were also shown to inhibit C. elegans pharyngeal pumping. The slo-1 (js379) and lat-1 (okl465) null mutants demonstrated reduced sensitivity to all three compounds, suggesting that SLO-1 and LAT-1 are involved in the mechanism of action of these compounds as well as emodepside. SLO-1 and LAT-1 are not known to be involved in the mechanism of action of any other currently available anthelmintic, highlighting the novel functioning of the compounds tested in this project, and their potential as anthelmintics that could break resistance.</p

Gene Expression of Protease Inhibitors in Tomato Plants with Invasion by Root-Knot Nematode Meloidogyne incognita and Modulation of Their Activity with Salicylic and Jasmonic Acids

Article

Full-text available

Mar 2021

Progress in development of vaccines against most important gastrointestinal helminth parasites of humans and animals

Article

Dec 2003

Among many vaccine approaches developed against gastrointestinal helminths in the recent years, parenteral vaccination and use of recombinant helminth antigens expressed in various expression systems have been a major focus. Significant progress has been made towards cloning of protective worm antigens. The recombinant proteins have several benefits over classic vaccine technologies, including increased safety, economy, stability and versatility. Moreover, the identification of site and developmental stage of the parasite in which the expression of genes encoding for potential vaccine antigen become possible using a variety of molecular techniques including hybridization in situ, expressed sequence tags analysis or cDNA microarray technology. Unfortunately, in the research reported so far little attention has been paid to oral vaccination, which may be of particular importance for the development of protective immunity against enteric parasites. The biggest obstacles to vaccine production at present time are: lack of an efficient expression system, which could guarantee proper post-translational modifications of recombinant helminth antigens as well as lack of efficient mucosal delivery systems. These obstacles will be possibly successfully overcome by use of transgenic plants both for the antigen expression and as adjuvants.

Proteases as Vaccines Against Gastrointestinal Nematode Parasites of Sheep and Cattle

Article

Aug 2012

D P Knox

Parasitic nematodes contain and secrete various proteases (proteolytic enzymes) that are known or proposed to facilitate the penetration of host tissue barriers, the digestion of host protein for nutrients, and the evasion of the host antiparasite immune responses, in addition to their functions in internal processes such as tissue catabolism and apoptosis. These key functions underscore the reasons why proteases have been targeted as vaccine components. Several excreted/secreted (ES) proteases are known to be targeted by host-protective humoral immune responses and have been evaluated as vaccine candidates against nematode parasites of livestock. In particular, ES cysteine proteases are associated with vaccination-induced protective immunity in cattle and sheep against the abomasal parasites Ostertagia ostertagi and Haemonchus contortus, respectively. Moreover, proteases found on the intestinal surface of the latter are among the most effective vaccine antigens identified to date for any helminth parasite. These proteases include a variety of endo- and exopeptidases required to digest hemoglobin in the blood meal - a process that involves a partly conserved cascade or network of proteases also found in hookworms, schistosomes, and Plasmodium spp. In addition to cysteine proteases, other intestinal proteases include aspartic and metalloproteases, aminopetidases, and dipeptidyl peptidases. In general, protection by protease vaccines is mediated by antibody inhibition of enzyme activity that blocks either parasite invasion of the host or digestion, leading to parasite expulsion and/or death. A major barrier to vaccine production is that recombinant protease vaccines lack the efficacy of their native counterparts for a variety of reasons, including incorrect protein folding and/or the lack of, or inappropriate, post-translational modifications such as glycosylation. These issues have been addressed by the use of eukaryotic expression systems such as insect cells and recent work indicates that the free-living nematode, Caenorhabditis elegans, can be also engineered to produce functional heterologous nematode parasite proteases.

RNA Interference: A Potential Discovery Tool for Therapeutic Targets of Parasitic Nematodes

Article

Aug 2012

Collette Britton

Although gene sequence data for parasitic nematodes have increased significantly in the last decade, there is currently no reliable method to test gene function and validate putative control targets. Gene silencing by RNA interference (RNAi) has proven successful in the free-living nematode Caenorhabditis elegans and the ease of this technique has allowed it to be adapted for high-throughput genome-wide screening of essential gene function. This success encouraged a number of RNAi studies on parasitic nematodes of animals and plants. Whereas RNAi in parasitic nematodes, as well as some free-living nematodes, is not as effective as it is in C. elegans, specific gene silencing has been observed for some target genes. Recent work with Haemonchus contortus suggests that the success of RNAi can be improved by targeting genes expressed in sites accessible to the environment such as the intestine, excretory/secretory system, or amphids. One of the genes robustly silenced in H. contortus encodes the aminopeptidase H11. RNAi of H11 in larvae prior to oral infection resulted in significant reductions in worm burden and egg output, similar to vaccination with native H11 protein. This provides proof-of-principle that RNAi can be developed as a tool to identify essential gene function. Using current RNAi techniques, screening for essential genes as potential control targets may be limited by transcript accessibility to introduced double-stranded RNA. Optimization of double-stranded RNA delivery methods is discussed as enhanced uptake may allow RNAi to be fully exploited as a tool for both functional genomics and target discovery in parasitic nematodes.

Helminth Cysteine Proteases

Article

Dec 2013

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 444 is Helminth Cysteine Proteases.

Screening and analysis of Hc-ubq and Hc-gst related to desiccation survival of infective Haemonchus contortus larvae

Article

Mar 2015

Infective Haemonchus contortus larvae (L3s) are able to protect themselves from desiccation. To explore the molecular mechanisms of desiccation survival, mRNA differential display RT-PCR was used to screen differentially expressed genes in L3s upon desiccation, followed by RNAi experiments to define gene functions. In this, 58 differentially expressed transcripts were obtained. Among these, the BF-U01A and CH-U02A fragments represent genes with the highest identity percentage in bioinformatic analysis. They were named Hc-ubq and Hc-gst based on their respective homologous ubiquitin in Caenorhabditis elegans and glutathione S-transferase in H. contortus. Quantitive RT-PCR results indicated that they were both up-regulated in desiccated L3s. Hc-ubq and Hc-gst RNAi in H. contortus showed reduced survival rate of L3s, with unchanged locomotion behavior. Homologous Ce-ubq-2 and Ce-gst-7 RNAi in C. elegans also displayed higher larval death rate. These results suggest that ubq and gst may play important roles in nematode desiccation tolerance. Our study analyzed desiccation resistance related genes in H. contortus L3s, and revealed significant research implications on the mechanisms behind nematode desiccation survival. Copyright © 2015 Elsevier B.V. All rights reserved.

Die Chemie und der Wurm: Caenorhabditis elegans als Plattform für das Zusammenführen von chemischer und biologischer Forschung

Article

May 2011
Angew Chem

Dieser Aufsatz diskutiert die Nützlichkeit des Wurms Caenorhabditis elegans als Modellorganismus für Chemiker, die an der Untersuchung lebender Systeme interessiert sind. C. elegans, ein 1 mm langer Rundwurm, ist ein beliebter Modellorganismus in nahezu allen Gebieten der modernen Biologie. Der Wurm hat zahlreiche Eigenschaften, die ihn für die Biologie attraktiv machen: Er ist klein (<1000 Zellen), transparent und genetisch leicht zu manipulieren. Trotz seiner Schlichtheit weist der Wurm komplexe Phänotypen auf, die mit seiner Mehrzelligkeit zusammenhängen: Er hat differenzierte Zelltypen und Organe, er altert und hat eine wohldefinierte Lebenserwartung, er kann lernen und besitzt ein Erinnerungsvermögen. Der Aufsatz will verdeutlichen, dass diese Mischung aus Einfachheit und Komplexität den Wurm zu einem besonders nützlichen Werkzeug macht, um die Beziehungen zwischen Phänomenen auf molekularer Ebene und der Ebene des Gesamtorganismus zu erforschen (Altern, Verhalten, Kognition, Anfälligkeit für Krankheiten). Es werden vornehmlich solche Forschungsarbeiten vorgestellt, die chemisch relevant sind. Außerdem werden Instrumente und Arbeitstechniken – biologischer, chemischer und physikalischer Natur – vorgestellt, die uns zur Erforschung des Wurms zur Verfügung stehen.

Nematode Hsp90: Highly conserved but functionally diverse

Article

Apr 2014
Parasitology

SUMMARY Nematodes are amongst the most successful and abundant organisms on the planet with approximately 30 000 species described, although the actual number of species is estimated to be one million or more. Despite sharing a relatively simple and invariant body plan, there is considerable diversity within the phylum. Nematodes have evolved to colonize most ecological niches, and can be free-living or can parasitize plants or animals to the detriment of the host organism. In this review we consider the role of heat shock protein 90 (Hsp90) in the nematode life cycle. We describe studies on Hsp90 in the free-living nematode Caenorhabditis elegans and comparative work on the parasitic species Brugia pahangi, and consider whether a dependence upon Hsp90 can be exploited for the control of parasitic species.

It's No Fluke: The Planarian as a Model for Understanding Schistosomes

Article

Full-text available

Jul 2013
PLOS PATHOG

Schistosomiasis is among the most prevalent human parasitic diseases, affecting more than 200 million people in the developing world [1]. Indeed, some estimates suggest the global disease-associated disability resulting from schistosomiasis may reach levels on par with global killers including malaria, tuberculosis, or HIV/AIDS [2], [3]. The causative agents of this disease are flatworms (Schistosoma or blood flukes) that feed on blood in the veins surrounding the host's intestine or bladder. These worms produce hundreds to thousands of eggs per day, many of which lodge in host tissues and cause diverse pathologies, including hepatic fibrosis, splenomegaly, and in some cases, perhaps cancer. Despite the grim statistics, only a single therapeutic agent (praziquantel) is currently used to treat schistosome infection. With the looming possibility that praziquantel-resistant Schistosoma strains could arise, an urgent need exists to identify novel therapeutic agents to combat these parasites.

A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling*1

Article

May 2004
MOL BIOCHEM PARASIT

David B Guiliano

Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.

Synthetic and Natural Protease Inhibitors Provide Insights into Parasite Development, Virulence and Pathogenesis

Article

Mar 2013
CURR MED CHEM

Protease function is essential to many biological systems and processes. In parasites, proteases are essential for host tissue degradation, immune evasion, and nutrition acquisition. Helminths (worms) depend on several classes of proteases for development, host tissue invasion and migration, and for degradation of host hemoglobin and serum proteins. The protozoa, which cause malaria, depend on both cysteine and aspartic proteases to initiate host hemoglobin digestion. Other types of proteases are involved in erythrocyte cell invasion and cell exit. Surface metalloproteases in kinetoplastids are implicated in the evasion of complement-mediated cell lysis and cell entry. Cysteine proteases in Entamoeba facilitate invasion of the host colon. Giardia utilizes a cysteine protease for both encystation and excystation. This review will summarize published data using protease inhibitors as tools to identify the function of parasite proteases in the development, virulence, and pathogenesis of parasites; as well as the role of endogenous parasite protease inhibitors in regulation.

ESTUDIO CINÉTICO DE LA DETECCIÓN DE LARVAS EN HECES DE TERNEROS INFESTADOS DE FORMA NATURAL CON D. VIVIPARUS

Article

presencia de larvas (análisis larvoscópico) en heces mediante el ensayo de Baerman, utilizando como material de estudio un grupo de 25 terneros sanos; los cuales fueron expuestos a una infestación natural con D. viviparus durante 10 semanas. En las primeras cuatro semanas la detección de larvas fue prácticamente negativa. La máxima expulsión de larvas se produce en la semana cinco y seis, por lo que el ensayo alcanza su mayor sensibilidad. Estos resultados nos permiten valorar que la detección de larvas está asociada al fenómeno de intermitencia en la expulsión de huevos debido a baja sensibilidad encontrada en este estudio. ABSTRACT. The dynamics of the presence of larva was studied (larvascopic analysis) in feces by means of the rehearsal of Baerman, using as study material a group of 25 healthy calves; which were exposed to a natural infestation with D. viviparus during 10 weeks. In the first four weeks the detection of larva was practically negative. The maximum expulsion of larva takes place in the week five and six, for what the rehearsal reaches its biggest sensibility. These results allow us to value that the detection of larva is associated to the intermittence phenomenon in the expulsion of eggs due to low sensibility found in this study.

C. elegans as a Resource for Studies on Plant Parasitic Nematodes

Chapter

Apr 2011

C. elegans provides a suitable model to study basic and conserved nematode biology. The short life-cycle, adult size, easy maintenance in large numbers and the tractability of C. elegans facilitate its use in translational biology. The C. elegans genome project has greatly assisted the mapping, sequencing and annotation of parasitic nematode genomes. Furthermore, the development of RNAi in C. elegans has not only aided functional analysis of conserved proteins in parasitic species but has also provided a platform for the development of RNAi in these target species. Application of RNAi in plant-parasitic nematodes has enhanced gene functional analysis as well as highlighted the potential for its development as a transgenic control. Keywords C. elegans -Model organism-RNAi-Genome projects-Homologues

The development of RNA interference (RNAi) in gastrointestinal nematodes

Article

Apr 2012
Parasitology

Despite the utility of RNAi for defining gene function in Caenorhabditis elegans and early successes reported in parasitic nematodes, RNAi has proven to be stubbornly inconsistent or ineffective in the animal parasitic nematodes examined to date. Here, we summarise some of our experiences with RNAi in parasitic nematodes affecting animals and discuss the available data in the context of our own unpublished work, taking account of mode of delivery, larval activation, site of gene transcription and the presence/absence of essential RNAi pathway genes as defined by comparisons to C. elegans. We discuss future directions briefly including the evaluation of nanoparticles as a means to enhance delivery of interfering RNA to the target worm tissue.

Molecular cloning and characterization of an intestinal cathepsin L protease from the root-knot nematode Meloidogyne incognita

Article

Sep 2003

A cathepsin L protease full-length cDNA (named Mi-cpl-1) was characterised from infective second-stage juveniles of the root-knot nematode Meloidogyne incognita by SL1-primed PCR and 3′ rapid amplification of cDNA ends (3′-RACE). A single open reading frame was identified, encoding a putative 383-amino acid protein predicted to contain a putative N-terminal short secretion signal peptide, which suggests that Mi-CPL-1 should function as an extracellular protein. The putative mature enzyme included several conserved amino acid residues/motives, among which the typical catalytic triad of the active site. In situ mRNA hybridization analyses showed that transcripts of Mi-cpl-1 accumulated specifically in the intestinal cells of specimens, which suggests that the protein could be involved in the digestive processes of the nematode. However, expression of Mi-cpl-1 was shown to occur exclusively in the developmental stages which are in close interaction with the root tissues (i.e. second-stage juveniles and females). This may indicate that some function of the cathepsin L cysteine protease in M. incognita is more directly related to the parasitic aspects of the plant-nematode relationship, e.g. pathogenicity and/or evasion of primary host plant defence systems.

cDNA cloning, mRNA expression and recombinant expression of a cathepsin L-like cysteine protease from Neobenedenia melleni (Monogenea: Capsalidae)

Article

Sep 2007
AQUACULTURE

The monogenean Neobenedenia melleni is a worldwide virulent pathogen to many species of sea-caged fishes. Its outbreaks have caused fearful economic losses in fish mariculture. Cysteine proteases play vital biological roles in various important physiological and pathological processes. In the present paper the full-length cDNA of cathepsin L-like cysteine protease was isolated from the N. melleni using rapid amplification of cDNA ends (RACE). It is 1070 bp long and contains an open reading frame (ORF) of 1008 bp. The prepro-cathepsin L consists of a signal peptide of 17 amino acids (aa), a pro-region peptide of 100 aa and a mature peptide of 218 aa. The consensus motifs flanking conserved catalytic active sites (C25, H159 and N175, papain numbering), which are present in all papain-like cysteine proteases, were identified in the sequence. The cathepsin L gene comprises three introns and four exons. mRNA expression of cathepsin L was determined using semi-quantitative RT-PCR analysis. The cathepsin L mRNA was expressed in oncomiracidia, larvae and adult, but not in newly shed eggs and encysted oncomiracidia. The coding region of cathepsin L cDNA for the mature cathepsin L protein was subcloned into an expression plasmid pET-22b(+) and fused with a 6-His-Tag. Moreover, we have successfully developed an expression system in Escherichia coli to overproduce the recombinant cathepsin L of N. melleni. The present study is the first report of the cloning and description of cDNA encoding a cysteine protease from a monogenean, and provides essential information for further studies at the protein level, such as the function, regulation mechanism and possibility for use as a target molecule for an anti-monogenean drug.

Silencing of essential genes by RNA interference in Haemonchus contortus

Article

Feb 2012
Parasitology

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

41.5-kDa Cathepsin L protease from Clonorchis sinensis: Expression, characterization, and serological reactivity of one excretory-secretory antigen

Article

Mar 2012
Parasitol Res

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies.

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Article

Sep 2011
Exp Parasitol

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.

Autocatalytic Processing of Recombinant Human Procathepsin L. CONTRIBUTION OF BOTH INTERMOLECULAR AND UNIMOLECULAR EVENTS IN THE PROCESSING OF PROCATHEPSIN L IN VITRO

Article

Full-text available

Feb 1998

Robert Ménard

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.

Basic Local Alignment Search Tool

Article

Full-text available

Oct 1990

Stephen F Altschul

Genome Sequence of the Nematode C. elegans: A Platform for Investigating BiologyThe C. elegans Sequencing ConsortiumScience19982825396201220189851916

Article

Full-text available

Dec 1998

Charles Steward

The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.

A molecular evolutionary framework for the phylum Nematoda

Article

Full-text available

Mar 1998

Nematodes are important: parasitic nematodes threaten the health of plants, animals and humans on a global scale; interstitial nematodes pervade sediment and soil ecosystems in overwhelming numbers; and Caenorhabditis elegans is a favourite experimental model system. A lack of clearly homologous characters and the absence of an informative fossil record have prevented us from deriving a consistent evolutionary framework for the phylum. Here we present a phylogenetic analysis, using 53 small subunit ribosomal DNA sequences from a wide range of nematodes. With this analysis, we can compare animal-parasitic, plant-parasitic and free-living taxa using a common measurement. Our results indicate that convergent morphological evolution may be extensive and that present higher-level classification of the Nematoda will need revision. We identify five major clades within the phylum, all of which include parasitic species. We suggest that animal parasitism arose independently at least four times, and plant parasitism three times. We clarify the relationship of C. elegans to major parasitic groups; this will allow more effective exploitation of our genetic and biological knowledge of this model species.

Basic Local Aligment Search Tool

Article

Full-text available

Oct 1990

A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.

Isolation and sequence of a cDNA for human pro-(cathesin L)

Article

Full-text available

Aug 1988

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is the precursor to an acid proteinase with enzymic specificity similar to that of human cathepsin L. By cross-hybridization with a mouse MEP sequence, cDNA clones of the human form of MEP in an SV40 expression vector were isolated. A 1.6 kb cDNA showed 70% deduced amino acid sequence identity with mouse MEP. The deduced amino acid sequence of the cloned human MEP was the same, except for two amino acids, as the N-terminal sequence of mature human cathepsin L, thereby establishing that human MEP is human pro-(cathepsin L). Use of this human pro-(cathepsin L) cDNA clone allowed the detection of a 1.6-1.8 kb pro-(cathepsin L) mRNA in human cells which was not detected with a mouse pro-(cathepsin L) probe.

Organization and Expression of Eucaryotic Split Genes Coding for Proteins

Article

Full-text available

Feb 1981

Alignment/Phylogeny of the Papain Superfamily of Cysteine Proteases

Article

Full-text available

Mar 1995

An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference. The iterative approach resulted in significant improvements in the alignment/phylogeny. There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups. There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups. The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence. The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution. In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes.

Cloning, genomic organization, and chromosomal localization of human cathepsin L

Article

Full-text available

Feb 1993

Cathepsin L is a lysosomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors, and tumor promoters. Many human tumors express high levels of cathepsin L, which is a broad spectrum protease with potent elastase and collagenase activities. Two published human cathepsin L cDNA sequences differ only in their 5'-untranslated regions. In this study, we demonstrate the concurrent expression of two distinct human cathepsin L mRNAs (hCATL-A and hCATL-B) in adenocarcinoma, hepatoma, and renal cancer cell lines. Cloning of the human cathepsin L gene by polymerase chain reaction amplification of genomic DNA and subsequent sequencing reveals that hCATL-A and hCATL-B mRNAs are encoded by a single gene. The 3' end of the first intron contains the 5' portion of hCATL-B and is contiguous to the second exon of the gene. These data suggest either the possibility of alternative splicing or the presence of a second promoter within the first intron of the hCATL gene. We mapped the hCATL gene to chromosome 9q21-22. Sequencing of both the mouse and human cathepsin L genes demonstrates almost complete conservation of exon and intron position, but significant divergence in intron structure, possibly reflecting differences in regulation of expression of the mouse and human cathepsin L genes.

Two Distinct Gene Subfamilies within the Family of Cysteine Protease Genes

Article

Full-text available

May 1993

A cDNA clone for a physiologically regulated Tetrahymena cysteine protease gene was sequenced. The nucleotide sequence predicts that the clone encodes a 336-amino acid protein composed of a 19-residue N-terminal signal sequence followed by a 107-residue propeptide and a 210-residue mature protein. Comparison of the deduced amino acid sequence of the protein with those of other cysteine proteases revealed a highly conserved interspersed amino acid motif in the propeptide region of the protein, the ERFNIN motif. The motif was present in all of the cysteine proteases in the data base with the exception of the cathepsin B-like proteins, which have shorter propeptides. Differences in the propeptides and in conserved amino acids of the mature proteins suggest that the ERFNIN proteases and the cathepsin B-like proteases constitute two distinct subfamilies within the cysteine proteases.

Host Movement and the Genetic Structure of Populations of Parasitic Nematodes

Article

Full-text available

Dec 1995

Mitochondrial DNA (mtDNA) sequence data were used to compare the population genetic structures of five species of parasitic nematodes from three different hosts: Ostertagia ostertagi and Haemonchus placei from cattle, H. contortus and Teladorsagia circumcincta from sheep, and Mazamastrongylus odocoilei from white-tailed deer. The parasites of sheep and cattle showed a pattern consistent with high gene flow among populations. The parasite of deer showed a pattern of substantial population subdivision and isolation by distance. It appears that host movement is an important determinant of population genetic structure in these nematodes. High gene flow in the parasites of livestock also indicates great opportunity for the spread of rare alleles that confer resistance to anthelmintic drugs. All species, including the parasite of deer, had unusually high within-population diversities (averages of 0.019-0.027 substitutions per site between pairs of individuals from the same population). Large effective population sizes (Ne), perhaps in combination with rapid mtDNA evolution, appear to be the most likely explanation for these high within-population diversities.

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

Article

Full-text available

Dec 1996

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

Article

Full-text available

Feb 1997

We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW server.

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Article

Full-text available

Dec 1998

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III

Article

Full-text available

Dec 2000

Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans

Article

Full-text available

Mar 2002

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.

Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences

Article

Dec 1991

We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae 1 Note: Nucleotide sequence data reported here are available in GenBank database under the accession number U53172. 1

Article

May 1998
MOL BIOCHEM PARASIT

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide susbstrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5′ end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

Plasma Membrane-Associated Cysteine Proteinases in Human and Animal Tumors

Article

Jan 1987
PATHOBIOLOGY

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.

Characterization of two cDNAs encoding cysteine proteinases from the soybean cyst nematode Heterodera glycines

Article

Jun 1997
Parasitology

P. E. URWIN

Identification of promoter elements of parasite nematode genes in transgenic Caenorhabditis elegans

Article

Nov 1999
MOL BIOCHEM PARASIT

Transformation of the free-living nematode Caenorhabditis elegans with promoter/reporter gene constructs is a very powerful technique to examine and dissect gene regulatory mechanisms. No such transformation system is available for parasitic nematode species. We have exploited C. elegans as a heterologous transformation system to examine activity and specificity of parasitic nematode gene promoters. Using three different parasite promoter/lac Z reporter constructs strict tissue-specific expression is observed. Upstream sequences of the Haemonchus contortus gut pepsinogen gene pep-1 and cysteine protease gene AC-2 direct expression exclusively in gut cells, while promoter sequence of the Ostertagia circumcincta cuticular collagen gene colost-1 directs hypodermal-specific expression. Mutation analysis indicates that AC-2 promoter function is dependent on a GATA-like motif close to the translation start site, similar to our findings with the C.eleganscpr-1 cysteine protease gene. While the spatial expression of these parasite promoters in C. elegans correlates with their expression in the parasite, the exact timing of expression does not. This suggests that regulatory mechanisms influencing the timing of expression may have evolved more rapidly than those controlling spatial expression of structural genes.

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae

Article

Jun 1998
MOL BIOCHEM PARASIT

Genome Sequence of the Nematode C. elegans: A Platform for Investigating Biology The C

Article

Jan 1998

Ability of activated complement component C3 to induce lysosomal enzyme release from macrophages

Article

Jun 1976

THE mechanism underlying chronic inflammation is of considerable medical importance both in temperate countries-where diseases such as rheumatoid arthritis are common-and in tropical countries-where various chronic inflammatory conditions cause much disease and disability. It is generally accepted that the macrophage is the central cell in chronic inflammation. We have drawn attention to the parallelism between the capacity of various agents to induce chronic inflammation in vivo and selective lysosomal enzyme secretion from cultures of macrophages1,2. The mechanism by which macrophage enzyme secretion is induced is unknown: we now report a mechanism mediated by cleavage products of the complement component C3.

Isolation of putative cysteine protease genes of Ostertagia ostertagi

Article

Dec 1992
MOL BIOCHEM PARASIT

Recombinant phage containing putative Ostertagia ostertagi cysteine protease genes have been isolated from a lambda EMBL-3:genomic DNA library using a Haemonchus contortus cathepsin B-like cysteine protease cDNA as hybridization probe. Restriction enzyme maps of the phages suggest that they identify at least 3 genes, 2 of which appear to be linked in tandem. The complete nucleotide sequence of one gene, CP-1, was determined. The CP-1 gene appears to be organized into 12 exons than span 4.5 kb of DNA. The number and sizes of the exons are essentially identical to those in the H. contortus AC-2 cysteine protease gene. Partial nucleotide sequences obtained for a second O. ostertagi gene, CP-3, revealed a similar organization for exons 8-12 in this gene. Like other cathepsin B-like cysteine proteases, CP-1 appears to be synthesized initially as a preproprotein that is proteolytically processed to its mature form. The amino acid identity between the presumptive CP-1 and CP-3 proteins is 66%, which is similar to the level of homology between the presumed mature protein regions of CP-1 and AC-2. Amino acid identity between CP-1 and AC-2 is greatest in the mature protein region and lowest in the signal sequence and propeptide regions. The CP-3 protein appears to be most closely related to the H. contortus AC-5 protein. CP-1 and CP-3 display significantly greater homology to H. contortus cysteine proteases than they do to human cathepsin B or the Sm31 cysteine protease of Schistosoma mansoni (about 40% identity with each).

Cloning and sequence comparisons of four distinct cysteine proteases expressed by Haemonchus contortus adult worms

Article

May 1992
MOL BIOCHEM PARASIT

Three new members of a developmentally regulated cysteine protease gene family of the parasitic nematode Haemonchus contortus have been isolated and characterized. One of the new genes, AC-3, was found to be linked in tandem to the previously characterized AC-2 gene. Nucleotide sequence analyses revealed that the first 90 amino acids of AC-3 are organized into four exons, similar to the situation in AC-2. A cDNA that appears to be a near full-length copy of the AC-3 gene was isolated using the polymerase chain reaction (PCR) technique to amplify cDNAs from adult worm poly(A)+ mRNAs. In addition to AC-3, a distinct cysteine protease cDNA, AC-4, was amplified by the same oligonucleotide primers. cDNAs encoding a fifth cysteine protease, AC-5, were isolated from an adult worm cDNA expression library using specific rabbit antisera and by PCR. Comparison of the predicted amino acid sequences of AC-3, AC-4 and AC-5 reveal that they share 64-77% identity with one another and with the previously reported AC-1 and AC-2 sequences. The amino acids surrounding the active site cysteine are highly conserved, as are the positions of other cysteine residues in the mature protein sequences. The H. contortus proteases are more similar to one another than they are to human cathepsin B (38-44% amino acid identity) or to the Sm31 cysteine protease of Schistosoma mansoni (36-40% identity). Our studies indicate that H. contortus adult worms express mRNAs for several distinct cysteine proteases. The significant primary sequence differences between the proteases suggest that they differ in their substrate specificities and precise physiological functions.

Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms

Article

Jul 1990
MOL BIOCHEM PARASIT

We have cloned cDNAs encoding a 35-kilodalton cysteine protease that is a major component of protective extracts isolated from blood-feeding Haemonchus contortus adult worms. Near full-length cDNAs for the protease were isolated by immunoscreening an adult worm cDNA expression library with a rabbit antiserum prepared against the protein eluted from preparative SDS gels and by rescreening the library with oligonucleotide probes. The protein predicted from the nucleotide sequence of the cDNAs and of a genomic DNA clone comprises 342 amino acids and contains an N-terminal signal sequence, 16 cysteine residues and four potential N-linked glycosylation sites. The enzyme appears to be glycosylated in vivo. The H. contortus protease, called AC-1, displays an overall 42% sequence identity with the human lysosomal thiol protease cathepsin B. The similarities between cathepsin B and AC-1 are localized primarily to regions of cathepsin B that comprise the mature, active form of the enzyme. A stretch of six amino acids that includes the active site cysteine of cathepsin B is conserved, and is present in the same relative location in AC-1, suggesting that this region comprises the active site of the H. contortus enzyme.

Parasite proteases

Article

Feb 1989

James H Mckerrow

Plasma membrane-Associated cysteine proteinase in human and animal tumours

Article

Feb 1987

In vivo and in vitro evidence for the involvement of cysteine-proteinase in bone resorption

Article

Jan 1985

The excretion of cathepsin B, a lysosomal cysteine proteinase, by parathyroid hormone-stimulated embryonic mouse calvaria in culture, correlates closely with the extent of bone resorption evaluated by the loss of hydroxyproline and calcium and by the extension of resorption lacunae. E-64, a specific inhibitor of cysteine proteinases, inhibits reversibly the resorption of cultured bones without affecting the hormone-induced secretion of lysosomal hydrolases. Given in vivo to rats, the proteinase inhibitors, E-64 and leupeptin, both induce a concomitant fall in the serum calcium level and in the urinary excretion of hydroxyproline. These results provide evidence that cysteine proteinases, possibly lysosomal cathepsins, are necessary for bone resorption.

Characterization and localization of cathepsin B proteinases expressed by adult Ancylostoma caninum hookworms

Article

Jun 1995
MOL BIOCHEM PARASIT

The hookworm Ancylostoma caninum induces human eosinophilic enteritis (EE), probably via allergic responses to its secretions. Cysteine and metallo-proteinases may be the components of these secretions that elicit hypersensitivity reactions. In order to characterize genes encoding cysteine proteinases (CP) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with a cloned fragment of a hookworm CP gene. This fragment was obtained using consensus oligonucleotide, CP-gene-specific primers in the polymerase chain reaction. cDNAs encoding two CPs were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen CP of 343 amino acids (aa), predicted to be processed in vivo into a mature CP of 255 aa. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and to human cathepsin B (60%). The second gene, AcCP-2, encoded a mature CP of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. Rabbit antisera raised against recombinant AcCP-1 reacted with esophageal, amphidial and excretory glands in formalin-fixed, paraffin embedded sections of both male and female adult hookworms, and with an antigen of approx. 40 kDa in Western blot analysis of excretory/secretory products from adult hookworms. Together, these immuno-hybridization results strongly suggest that the CP encoded by the AcCP-1 gene is secreted by hookworms. These are the first reported CP genes from hookworms. Proteinases encoded by these genes may be responsible for the CP activity that we have shown previously to be secreted by adult A. caninum.

Developmentally Regulated Secretion of Cathepsin L-like Cysteine Proteases by Haemonchus contortus

Article

Sep 1995

Cysteine protease activity was present in media collected after 24 hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen, and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with M(r) values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4, but not the L3 larval stage. The active and developmentally regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition, immune evasion, or both.

Molecular Cloning and Sequencing of cDNA That Encodes Cysteine Proteinase in the Eggs of the Silkmoth, Bombyx mori

Article

Jan 1995

We have isolated and sequenced a 1,486-base-pair near full-length cDNA coding for Bombyx egg cysteine proteinase. The cDNA encodes 344 amino acid residues containing a typical signal peptide sequence (16 residues), pro-peptide (104 residues), and the sequence for mature enzyme (224 residues). Sequence alignments show that the egg cysteine proteinase is similar to lobster cysteine proteinase (61% identity), barley cysteine proteinase, Aleurain (52%), rice cysteine proteinase, Oryzain (54%), and rat cathepsin L (59%). The amino-terminal sequencing of the egg cysteine proteinase indicates that the enzyme purified as an inactive form from eggs is a pro-enzyme. Pro-egg cysteine proteinase was detected in other silkmoth tissues such as ovary, fat body, hemocyte, and hemolymph by immunoblotting.

Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles

Article

Dec 1993
MOL BIOCHEM PARASIT

Cathepsin L-like activity was demonstrated in the excretory/secretory (E/S) products of Fasciola hepatica newly excysted juveniles (NEJ), 3-week-old, 5-week-old and mature flukes using the fluorogenic substituted 7-amino-4-methylcoumarin substrates Z-phe-arg-AMC, Z-arg-arg-AMC and Z-arg-AMC. Gelatin-substrate polyacrylamide gel analysis revealed that the E/S from each of these stages contained multiple proteolytic enzymes; however, the pattern of proteinases obtained for NEJ E/S differed markedly from that of all other stages examined. The four NEJ proteinases identified were inhibited by leupeptin and Z-phe-ala-diazomethyl ketone indicating that each had cathepsin L-like activity. The E/S products of all four developmental stages contain an enzyme capable of cleaving immunoglobulin at the hinge region, the activity of which is also inhibited by Z-phe-ala-diazomethyl ketone. Using in vitro cell attachment assays we show that the cathepsin L-like proteinase purified from the E/S products of adult F. hepatica can prevent the antibody-mediated attachment of eosinophil to NEJ. These experiments indicate that this proteinase has an important biological function in immune evasion.

Temporal reiteration of a precise gene expression pattern during nematode development

Article

Aug 1996

The nematode Caenorhabditis elegans is contained within a multifunctional exoskeleton, the cuticle, that contains a large number of distinct collagens. As the nematode proceeds from the egg through four larval stages to the adult, transition between larval stages is marked by synthesis of a new cuticle and subsequent moulting of the old one. This is a cyclically repeated developmental event, frequently described as the moulting cycle. We have examined the temporal expression of a group of six genes encoding distinct cuticular collagens. As expected, mRNA abundance for each of the six genes tested is found to oscillate, peaking once during each larval stage. Unexpectedly, the periods of abundance for each gene do not coincide, different genes being expressed at different times relative to one another within the moulting cycle. We detect a programme of temporally distinct waves of collagen gene expression, the precise pattern of which is repeated during each of the four larval stages. This multiphasic pattern of oscillating cuticular collagen gene expression indicates an unexpected complexity of temporal control during the nematode moulting cycle and has implications for collagen trimerization and cuticle synthesis.

Structural analysis of the catalytic site of AcCP-1, a cysteine proteinase secreted by the hookworm Ancylostoma caninum

Article

Dec 1996
Biochim Biophys Acta

Previously, we have reported that hookworms secrete cysteine proteinase activity that is capable of cleaving the cathepsin L-specific substrate Z-Phe-Arg-AMC. We have also reported the gene sequences of novel cathepsin B-like proteinases from hookworms, but have been unable to locate cathepsin L-like genes that could account for the presence of the cathepsin L activity in these parasites. Here we present an homology model for the secreted hookworm cysteine proteinase AcCP-1 based upon the crystal structure co-ordinates of human cathepsin B. The model predicts that substrate binding and specificity differs between AcCP-1 and cathepsin B, and demonstrates that AcCP-1 would preferentially cleave Phe-Arg over Arg-Arg. This thereby provides an explanation for our previous observations that the hookworm proteinase, while structurally cathepsin B-like, displays a cathepsin L-like substrate specificity.

Characterisation of two cDNAs encoding cysteine proteases from the soybean cyst nematode Heterodera glycines

Article

Jul 1997

Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long propeptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-I delta D86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Article

Mar 1998

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.

Double-Stranded RNA Induces mRNA Degradation in Trypanosoma brucei

Article

Jan 1999

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the alpha-tubulin mRNA 5' untranslated region (5' UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic alpha-tubulin 5' UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of alpha-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to alpha-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

Use of dsRNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway

Article

Jan 1999
CELL

We investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. In contrast, single-stranded RNA weakly interfered with gene activity. The method was used to determine the reception mechanism of the morphogen Wingless. Interference of the frizzled and Drosophila frizzled 2 genes together produced defects in embryonic patterning that mimic loss of wingless function. Interference of either gene alone had no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. Our results demonstrate that dsRNA interference can be used to analyze many aspects of gene function.

Proteinases and Associated Genes of Parasitic Helminths

Article

Feb 1999
ADV PARASIT

Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus.

The lysosomal cystein proteases

Article

Feb 1999
ANNU REV BIOPH BIOM

Mary E. McGrath

A significant number of exciting papain-like cysteine protease structures have been determined by crystallographic methods over the last several years. This trove of data allows for an analysis of the structural features that empower these molecules as they efficiently carry out their specialized tasks. Although the structure of the paradigm for the family, papain, has been known for twenty years, recent efforts have reaped several structures of specialized mammalian enzymes. This review first covers the commonalities of architecture and purpose of the papain-like cysteine proteases. From that broad platform, each of the lysosomal enzymes for which there is an X-ray structure (or structures) is then examined to gain an understanding of what structural features are used to customize specificity and activity. Structure-based design of inhibitors to control pathological cysteine protease activity will also be addressed.

The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans

Article

Nov 1999
CELL

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus

Article

Nov 1999

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

Predicting Subcellular Localization of Proteins Based on their N-terminal Amino Acid Sequence

Article

Aug 2000

A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

Comparative molecular modelling identifies a common putative IgE epitope on cysteine proteases of diverse sources

Article

Oct 2000

Previous approaches for studying common allergenic epitopes have mainly focused on sequence comparisons, which unfortunately yield little or no information on the shape of the epitope which is the most important determinant of cross-reactivity. The aim of this study was to investigate the structural basis for cross-reactivity between a previously identified immunodominant epitope of the house dust mite allergen Der p 1 (Leu147-Gln160) and the corresponding epitopes on other allergens that are either taxonomically closely related (i.e. cysteine proteases of other mite species) or representing evolutionary conserved structures (i.e. plant, human and parasite cysteine proteases). We carried out comparative molecular modelling on a range of cysteine proteases, including those of other mite species (Der f 1 and Eur m 1), human (cathepsins B, K, L, S and O), plants (papain, chymopapain and actinidin) and parasites (cruzain, cathepsin L-like Leishmania protease, Entamoeba ACP1 protease and Schistosoma Q26534, Q11003 and cathepsin L proteases). Our study shows that all the cysteine proteases investigated here display an epitope corresponding to that previously identified on Der p 1, but with varying shapes and degree of accessibility. It appears that the core of the epitope on these homologous cysteine proteases consists of a centrally located conserved Tyr residue flanked on either sides by accessible amino acids. Therefore, these cysteine proteases seem to use similar accessible structures, which may form the basis for the rational design of generic epitope-directed treatment strategies for controlling allergic diseases.

Functional genomic analysis of C. elegans chromosome I by systematic RNA interference

Article

Dec 2000

Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

200 000 Nematode expressed sequence tags on the Net

Article

Sep 2001

PAUP: phyogenetic analysis using parsimony. Champagne, IL: Illinois Natural History Survey

Jan 1991

Dl Swofford

Swofford DL. PAUP: phyogenetic analysis using parsimony. Champagne, IL: Illinois Natural History Survey, 1991.

The nematode Caenorhabditis elegans . Cold Spring Harbor

Jan 1988
587-606

J Sulston
J Hodgkin
Methods

Sulston J, Hodgkin J. Methods. In: Wood WB, editor. The nematode Caenorhabditis elegans. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1988:587 Á/606.

A cathepsin L protease essential for Caenorhabditis elegans embryogenesis is functionally conserved in parasitic nematodes

Abstract

No full-text available

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