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A parasite cysteine protease is key to host protein degradation and iron acquisition.

O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, Zhou M, Craik CS, Caffrey CR, McKerrow JH - J. Biol. Chem. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158-2550, USA.

ABSTRACT
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.

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Related in: MedlinePlus

Growth rate (replication) of 90-13 versus tbcatb+/–T. brucei. One hundred thousand parasites were cultured at a density of 1 × 104 cells/ml and counted using a hemocytometer after 24, 48, and 72 h (n = 3). Diamonds, strain 90-13 (wild type); open squares, tbcatb+/–.
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fig1: Growth rate (replication) of 90-13 versus tbcatb+/–T. brucei. One hundred thousand parasites were cultured at a density of 1 × 104 cells/ml and counted using a hemocytometer after 24, 48, and 72 h (n = 3). Diamonds, strain 90-13 (wild type); open squares, tbcatb+/–.

Mentions: Single-allele Deletion of T. brucei Cathepsin B Gene Leads to Decreased Parasite Replication—Silencing of tbcatB by RNAi produced a marked phenotype (4). Parasites exhibited endosomal or lysosomal swelling, decreased growth rate, arrest in cytokinesis, and eventual death. However, this dramatic phenotype was associated with only a modest decrease in tbcatB mRNA, protein, and protease activity (4). Therefore, to validate and extend the RNAi results, a tbcatB heterozygous knock-out clone (tbcatb+/–) was produced by homologous recombination using a tbcatb targeting vector. The rate of replication of the tbcatb+/– clones was ∼40% less than the control 90-13 strain after 72 h (Fig. 1). tbcatb–/– clones could not be isolated due to presumed lethality.


A parasite cysteine protease is key to host protein degradation and iron acquisition.

O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, Zhou M, Craik CS, Caffrey CR, McKerrow JH - J. Biol. Chem. (2008)

Growth rate (replication) of 90-13 versus tbcatb+/–T. brucei. One hundred thousand parasites were cultured at a density of 1 × 104 cells/ml and counted using a hemocytometer after 24, 48, and 72 h (n = 3). Diamonds, strain 90-13 (wild type); open squares, tbcatb+/–.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570886&req=5

fig1: Growth rate (replication) of 90-13 versus tbcatb+/–T. brucei. One hundred thousand parasites were cultured at a density of 1 × 104 cells/ml and counted using a hemocytometer after 24, 48, and 72 h (n = 3). Diamonds, strain 90-13 (wild type); open squares, tbcatb+/–.
Mentions: Single-allele Deletion of T. brucei Cathepsin B Gene Leads to Decreased Parasite Replication—Silencing of tbcatB by RNAi produced a marked phenotype (4). Parasites exhibited endosomal or lysosomal swelling, decreased growth rate, arrest in cytokinesis, and eventual death. However, this dramatic phenotype was associated with only a modest decrease in tbcatB mRNA, protein, and protease activity (4). Therefore, to validate and extend the RNAi results, a tbcatB heterozygous knock-out clone (tbcatb+/–) was produced by homologous recombination using a tbcatb targeting vector. The rate of replication of the tbcatb+/– clones was ∼40% less than the control 90-13 strain after 72 h (Fig. 1). tbcatb–/– clones could not be isolated due to presumed lethality.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158-2550, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus