Cloning and expression of chitinases of Entamoebae

Vega, Humberto de la; Specht, Charles A.; Semino, Carlos E.; Robbins, Phillips W.; Eichinger, Daniel; Caplivski, D; Ghosh, Sudip Kumar; Samuelson, John

doi:10.1016/s0166-6851(96)02817-4

Cited by 68 publications

(72 citation statements)

References 38 publications

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“…However, more than one-third of the isolates tested gave the same restriction fragment pattern (8). The chitinase gene also encodes tandem repeats of a degenerate 7-amino-acid sequence (10), and a report on the use of chitinase repeat polymorphisms to distinguish isolates of E. histolytica has been published recently (14). However, there still exists a need for additional reliable polymorphic E. histolytica DNA markers.…”

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confidence: 99%

Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

Zaki

Clark

2001

J Clin Microbiol

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An important gap in our understanding of the epidemiology of amebiasis is what determines the outcome ofEntamoeba histolytica infections. To investigate the possible existence of invasive and noninvasive strains as one factor, the ability to differentiate individual isolates of E. histolytica is necessary. Two new loci containing internal repeats, locus 1-2 and locus 5-6, have been isolated. Each contains a single repeat block with two types of related direct repeats arranged in tandem. Southern blot analysis suggests that both loci are multicopy and may themselves be arranged in tandem arrays. Three other previously reported, internally repetitive loci containing at least two repeat blocks each with one or more related repeat units were also investigated. PCR was used to study polymorphism at each of these loci, which was detected to various degrees in each case. Variation was seen in the total number of bands obtained per isolate and their sizes. Nucleotide sequence comparison of loci 1-2 and 5-6 in five axenic isolates revealed differences in the number of repeat units, which correlated with the observed PCR product size variation, and in repeat sequence. Use of multiple loci collectively allowed differentiation of a majority of the 13 isolates studied, and we believe that these loci have the potential to be used as polymorphic molecular markers for investigating the epidemiology of E. histolytica and the potential existence of genetically distinct invasive and noninvasive strains.

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confidence: 99%

Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

Zaki

Clark

2001

J Clin Microbiol

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“…7F). Organelles containing Hsp60 were also distinct from spherical secretory vesicles where the amebic chitinase, which has a secretory signal sequence at its amino terminus that obeys the -3, -1 rule, goes when expressed in transfected parasites under an actin promoter (data not shown) (9,12,53). Consistent with their cytosolic localization, amebic ADH1 and ferredoxin lack either signal sequences or organelle-tar- FIG.…”

Section: Resultsmentioning

confidence: 97%

Hsp60 Is Targeted to a Cryptic Mitochondrion-Derived Organelle (“Crypton”) in the Microaerophilic Protozoan Parasite Entamoeba histolytica

Mai¹,

Ghosh²,

Frisardi³

et al. 1999

Molecular and Cellular Biology

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173

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Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42°C. 5 and 3 rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-aminoacid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacterialike enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.

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“…Alternatively, one can study cysts of E. invadens, an important pathogen of reptiles in zoos, which readily form cysts in axenic cultures (49). All of the abundant proteins in E. invadens cyst walls are present in cyst walls of E. histolytica, and the enzymes that make, degrade, and deacetylate chitin are the same (6,24,34,(50)(51)(52)(53)(54)(55).…”

Section: Third Strategy: Studies Of Cyst and Oocyst Walls Of Nonhumanmentioning

confidence: 99%

“…As a starting point, we describe a four-lectin model of the cyst wall of Entamoeba, which we have studied for the longest time and in the most depth ( Fig. 1 and 5A) (6,8,24,34,(50)(51)(52)(53)(54)(55). Three abundant cyst wall proteins of Entamoeba, each of which was identified by mass spectrometry with excellent coverage, are chitin-binding lectins that cross-link chitin fibrils (Jacob), hydrolyze chitin (chitinase), or self-aggregate (Jessie lectins) and form the daub that holds walls together and makes them impermeable ( Fig.…”

Section: Fifth Strategy: Make Models For the Structure Of Cyst And Oomentioning

confidence: 99%

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

et al. 2013

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bCysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, ␤-1,3-linked glucose for Toxoplasma, and ␤-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the ␤-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (␤-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins. Acid-fast lipids, Fibrils of beta-glucan, Dityrosine glows.-Anonymous, Haiku of the oocyst wall I n this minireview, we discuss strategies that have worked well to discover structural components of protist walls that allow them to travel by the fecal-oral route from one person to the next (1-5). Proteins and sugars are the major components of cyst walls of Entamoeba and Giardia, which cause amebic dysentery and diarrhea, respectively (6-8). In addition to proteins and carbohydrates, lipids are important structural components of oocyst walls of Cryptosporidium and Toxoplasma, which cause diarrhea and disseminated infections, respectively (9-11).To characterize the structural components of walls of so many different organisms, we have had to focus on what is likely to matter most. Many of these wall-oriented strategies are based upon a set of assumptions that may not be obvious to investigators working on stages of the parasites that are motile and dividing (12). Therefore, we review here the strategies and assumptions most useful to determine the protein, carbohydrate, and lipid compositions of cyst and oocyst walls. The compositions of these cyst and oocyst walls are then used to make simple, if incomplete, models for their structure and assembly.

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Cloning and expression of chitinases of Entamoebae

Cited by 68 publications

References 38 publications

Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

Isolation and Characterization of Polymorphic DNA from Entamoeba histolytica

Hsp60 Is Targeted to a Cryptic Mitochondrion-Derived Organelle (“Crypton”) in the Microaerophilic Protozoan Parasite Entamoeba histolytica

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

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