Cysteine proteases of parasitic organisms

Microorganisms

Cysteine proteases, also known as thiol proteases, are a class of nucleophilic proteolytic enzymes containing cysteine residues in the enzymatic domain. These proteases generally play a pivotal role in many biological reactions, such as catabolic functions and protein processing, in all living organisms. They specifically take part in many important biological processes, especially in the absorption of nutrients, invasion, virulence, and immune evasion of parasitic organisms from unicellular protozoa to multicellular helminths. They can also be used as parasite diagnostic antigens and targets for gene modification and chemotherapy, as well as vaccine candidates, due to their species and even life-cycle stage specificity. This article highlights current knowledge on parasitic cysteine protease types, biological functions, and their applications in immunodiagnosis and chemotherapy.

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.

Clinical and Vaccine Immunology, 2000

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.

Journal of Biological Chemistry, 2008

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy. . 2 The abbreviations used are: Z-Phe-Ala-CHN 2 , benzyloxycarbonylphenylalanylalanyl diazomethane; RNAi, RNA interference; FR, flanking region; D-PBS, Dulbecco's phosphate-buffered saline; Z-FR-AMC, benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin; DTT, dithiothreitol; PS-SCL, positional scanning combinatorial synthetic combinatorial library; ACC, 7amino-4-carbamoylmethylcoumarin; Tf-R, transferrin receptor; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

Biological Chemistry, 2000

Parasites have evolved different virulence strategies to manipulate host physiological functions. The parasitoid wasp Cotesia congregata induces developmental arrest and immune suppression of its Lepidopteran host Manduca sexta. In this interaction, a symbiotic virus (C. congregata Bracovirus, CcBV) associated with the wasp is essential for parasitism success. The virus is injected into the host with wasp eggs and virus genes are expressed in host tissues. Among potential CcBV virulence genes, cystatins, which are tight binding inhibitors of C1A cysteine proteases, are suspected to play an important role in the interaction owing to their high level of expression. So far, however, potential in vivo targets in M. sexta are unknown. Here, we characterized for the first time four M. sexta C1A cysteine proteases corresponding to cathepsin L and cathepsin B and two different '26-29 kDa' cysteine proteases (MsCath1 and MsCath2). Our analyses revealed that MsCath1 and MsCath2 are transcriptionally downregulated in the course of parasitism. Moreover, viral Cystatin1 and MsCath1 co-localize in the plasma following parasitism, strongly suggesting that they interact. We also show that parasitism induces a general increase of cysteine protease activity which is later controlled. The potential involvement of cysteine proteases in defense against parasitoids is discussed.

Molecular and Biochemical Parasitology, 1990

Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.

Eukaryotic Cell, 2003

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

International Journal of Molecular Sciences

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens’ virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CP...

International journal for parasitology, 2014

Lysosomal soluble proteins are targeted to endosomes and lysosomes by specific receptors resident in the endoplasmic reticulum and/or the Golgi apparatus. The enteric protozoan parasite Entamoeba histolytica has a novel class of lysosomal targeting receptors, named the cysteine protease binding protein family (CPBF). Among 11 CPBFs (CPBF1-11), ligands for three members, CPBF1, CPBF6 and CPBF8, were previously shown to be cysteine proteases, α- and γ- amylases, and β-hexosaminidase and lysozymes, respectively. To further understand the heterogeneity of the ligands of CPBFs, we attempted to isolate and identify the ligands for other members of CPBFs, namely CPBF2, 3, 4, 5, 7, 9, 10 and 11, by immunoprecipitation and mass spectrometric analysis. We found that CPBF2 and CPBF10 bound to α-amylases while CPBF7 bound to β-hexosaminidases. It is intriguing that cysteine protease are exclusively recognised by CPBF1, whereas three α-amylases and β-hexosaminidases are redundantly recognised by...

Trends in Parasitology, 2003