Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

Reed, Sharon L.; Bouvier, Jacques; Pollack, Andrew; Engel, Juan C.; Brown, M A; Hirata, Ken; Que, Xuchu; Eakin, Ann E.; Hagblom, Per; Gillin, Frances D.

doi:10.1172/jci116359

Cited by 80 publications

(48 citation statements)

References 34 publications

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“…It could therefore be worth studying variation in genes in D. fragilis which are potentially involved in pathogenicity. Similar studies have been performed on Entamoeba histolytica and resulted in the identification of polymorphic gene products that are correlated with virulence in amebiasis (17,19) and gene products that could be used to determine the geographic origin of isolates and routes of transmission (12,27,28). Similar studies on D. fragilis should begin with identifying such protein coding genes, as to date only the coding region of the ssu rRNA gene has been published (20).…”

Section: Discussionmentioning

confidence: 85%

Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Peek¹,

Reedeker

Gool

2004

J Clin Microbiol

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Dientamoeba fragilis is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect D. fragilis DNA directly from human stool samples. The genetic diversity of D. fragilis from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that D. fragilis sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that D. fragilis shows remarkably little variation in its small-subunit rRNA gene.Dientamoeba fragilis is a protozoan parasite found in the mucosal crypts of the large intestines of humans. Originally D. fragilis was considered an ameba, but based on ultrastructural characteristics (2), antibody data (8), and phylogenetic data originating from 16S-like rRNA gene sequences, it has been established that it is a trichomonad (20), with no identified cyst stage. Most recent literature accepts that D. fragilis is an important enteric pathogen (7,10,18), with an estimated incidence of symptomatic infection of between 4 and 91% (11,21,25,26). Symptoms include abdominal pain, bloating, and diarrhea.Because of the lack of a cyst stage, diagnosis can be performed only on freshly passed stool or by the use of fixatives and permanent stains. In addition, day-to-day shedding is highly variable, which imposes the need for multiple sampling (24). These features have likely led to an underestimation of the prevalence of D. fragilis, which is reported to vary between 0.2% and more than 19% depending upon the population studied (3,9,15,16,26).Despite the relatively high prevalence of D. fragilis and its apparent role in patients presenting with gastrointestinal complaints, surprisingly little is known about its pathogenicity, route of transmission, epidemiology, and genetics. Because only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with nonpathogenic and pathogenic variants with similar morphology but different pathogenicities. This has been suggested for other lumendwelling protozoa such as Giardia duodenalis (13) and demonstrated for Entamoeba histolytica/Entamoeba dispar (4, 6). In a first report on variability in D. fragilis, the 16S-like ribosomal subunit DNA sequence of cultured D. fragilis from a small number of patients was analyzed by restriction fragment length polymorphism (RFLP) (14). Two of the 11 isolates gave a different restriction fragment pattern, indicating that there was genetic diversity in these D. fragilis cultures.The cumbersome and time-consuming techniques to maintain D. fragilis in culture in combination with the rapid disappearance of trophozoites from fresh feces have probably precluded more extensive and detailed studies of the genetic variability in D. fragilis. In addition, culture is not suitable for molecular ana...

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Section: Discussionmentioning

confidence: 85%

Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Peek¹,

Reedeker

Gool

2004

J Clin Microbiol

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“…Following three freeze-thaw cycles and a 15-s sonication, the proteinase activity was measured in the soluble fraction. Substrate specificity was tested for the liberation of the fluorescent leaving group, AMC, from the synthetic peptide substrates to determine the preferred cleavage of the P1 and P2 sites (21). The initial rate of substrate hydrolysis (nmol/min/mg protein) is based on the rate of increase of fluorescence using a Labsystems Fluoroskan Spectrofluorometer.…”

Section: Methodsmentioning

confidence: 99%

The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

Que¹,

Ngô²,

Lawton³

et al. 2002

Journal of Biological Chemistry

100

130

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Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative 1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.The protozoan, Toxoplasma gondii, is an obligate intracellular parasite that can invade and replicate within any nucleated cell of vertebrate hosts, including humans (1-3). Invasion by T. gondii tachyzoites is mediated by the sequential regulated release of specialized secretory organelles of the parasite including the micronemes, rhoptries, and dense granules (4). In early observations, the penetration of host cells by tachyzoites was enhanced by the addition of partially purified lysosomal enzymes (5). In addition, proteinases have been implicated in host cell invasion in other members of the Apicomplexa such as Plasmodium (6) and Eimeria tenella (7). We now show that a cathepsin B, toxopain-1, is strongly implicated in T. gondii invasion and that infection can be interrupted with specific cathepsin B inhibitors.Another unusual feature of Toxoplasma is the absence of a morphologically identifiable lysosomal system. In higher eukaryotic cells, acidic cathepsins in lysosomes are important in protein processing and breakdown. The mammalian precursor of cathepsin B is targeted to the lysosomal compartment by mannose 6-P for proteolytic activation (8). Thus, the apparent lack of a lysosomal system in Toxoplasma raises a number of questions regarding cellular proteinase functions within the parasite. The rhoptries are club-shaped organelles located at the apical end of the parasites with no known counterpart outside of the phylum Apicomplexa (9). Although nine rhoptry proteins (ROP1-9) 1 have been identified to date (10), a definitive function has only been determined for ROP2, which mediates binding of host mitochondria to the parasitophorous membrane (11). How rho...

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“…Studies in a human intestinal xenograft model of disease indicated that E. histolytica trophozoites that were transfected with an antisense plasmid to the ehcp5 gene failed to induce intestinal epithelial cell production of the inflammatory cytokines IL-1␤ and IL-8 and caused significantly less intestinal inflammation and tissue damage (33). We asked whether amebic proteinases could also act as an IL-18 activator in vitro.Cysteine proteinases are the major extracellular enzymes responsible for in vitro cytopathology and degradation of the extracellular matrix during the first steps of bowel invasion (16,(23)(24). To date, seven genes encoding cysteine proteinases have been identified in E. histolytica (4,11,24).…”

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confidence: 99%

“…To date, seven genes encoding cysteine proteinases have been identified in E. histolytica (4,11,24). One particular cysteine proteinase (designated EhCP5) is located on the surface of E. histolytica trophozoites and is not expressed in the closely related but noninvasive species, Entamoeba dispar (15).…”

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confidence: 99%

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A Surface Amebic Cysteine Proteinase Inactivates Interleukin-18

Que¹,

Kim

Sajid

et al. 2003

Infect Immun

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Amebiasis is a major cause of morbidity and mortality worldwide. Invasion by Entamoeba histolytica trophozoites causes secretion of proinflammatory cytokines from host epithelial cells, leading to a local acute inflammatory response, followed by lysis of colonic cells. Extracellular cysteine proteinases from amebic trophozoites are key virulence factors and have a number of important interactions with host defenses, including cleavage of immunoglobulin G (IgG), IgA, and complement components C3 and C5. Amebic lysates have also been shown to activate the precursor to interleukin 1-beta (proIL-1␤), mimicking the action of caspase-1. IL-18 is also a central cytokine, which induces gamma interferon (IFN-␥) and activates macrophages, one of the main host defenses against invading trophozoites. Because proIL-18 is also activated by caspase-1, we evaluated whether amebic proteinases had a similar effect. Instead, we found that recombinant proIL-18 was cleaved into smaller fragments by the complex of surface-associated and released amebic proteinases. To evaluate the function of an individual proteinase from the complex pool, we expressed an active surface proteinase, EhCP5, which is functional only in E. histolytica. Recombinant EhCP5 expressed in Pichia pastoris had kinetic properties similar to those of the native enzyme with respect to substrate specificity and sensitivity to proteinase inhibitors. In contrast to the activation of proIL-1␤ by amebic lysates, the purified proteinase cleaved proIL-18 and mature IL-18 to biologically inactive fragments. These studies suggest that the acute host response and amebic invasion result from a complex interplay of parasite virulence factors and host defenses. E. histolytica may block the host inflammatory response by a novel mechanism, inactivation of IL-18.Entamoeba histolytica is an enteric protozoan parasite that causes amebic dysentery and liver abscesses. Trophozoites invade the bowel by attaching to the epithelium through a galactose-inhibitable lectin (21), degrading the extracellular matrix by the action of neutral cysteine proteinases (16), lysing epithelial cells via an amebapore (20), and penetrating into the mucosa. During this process, multiple potent chemoattractant and proinflammatory cytokines are released by host epithelial cells (10), initiating an acute inflammatory response, which is seen in animal models (6) and human intestinal xenografts (29). Multiple factors enter in the control of the acute inflammatory response. Interleukin 8 (IL-8) and growth-related oncogene alpha released from epithelial cells act as chemoattractants and activators of neutrophils (2), while neutral cysteine proteinases from the amebae degrade the anaphylatoxins C3a and C5a (25).IL-18, which is also expressed in intestinal epithelial cells (8), is a coinducer of the Th1 response. The resulting stimulation of gamma interferon (IFN-␥) then activates macrophages, the major cell capable of killing E. histolytica trophozoites (27). Unlike most other cytokines, IL-18 and IL-1␤ lack a si...

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Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

Cited by 80 publications

References 34 publications

Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

A Surface Amebic Cysteine Proteinase Inactivates Interleukin-18

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