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90-kilodalton heat shock protein, Hsp90, as a target for genotyping Cryptosporidium spp. known to infect humans.

ABSTRACT: Small-subunit (SSU) rRNA-based methods have been commonly used in the differentiation of Cryptosporidium species or genotypes. In order to develop a new tool for confirming the genotypes of Cryptosporidium species, parts of the 90-kDa heat shock protein (Hsp90) genes of seven Cryptosporidium species and genotypes known to infect humans (C. hominis, C. parvum, C. meleagridis, C. canis, C. muris, C. suis, and the cervine genotype), together with one from cattle (C. andersoni), were sequenced and analyzed. With the exception of C. felis from cats and C. baileyi from birds, the Hsp90 genes of all tested Cryptosporidium species were amplified. Phylogenetic analysis of the hsp90 sequences from all these species is congruent with previous studies in which the SSU rRNA, 70-kDa heat shock protein, oocyst wall protein, and actin genes were analyzed and showed that gastric and intestinal parasites segregate into two distinct clades. In this study, the secondary products of hsp90 produced after PCR-restriction fragment length digestion with StyI and HphI or with BbsI showed that parasites within the intestinal or gastric clade could be differentiated from each other. These data confirm the utility of the Hsp90 gene as a sensitive, specific, and robust molecular tool for differentiating species and/or genotypes of Cryptosporidium in clinical specimens.

SUBMITTER: Feng Y

PROVIDER: S-EPMC2669192 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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90-kilodalton heat shock protein, Hsp90, as a target for genotyping Cryptosporidium spp. known to infect humans.

Feng Yaoyu Y Dearen Theresa T Cama Vitaliano V Xiao Lihua L

Eukaryotic cell 20090123 4

Small-subunit (SSU) rRNA-based methods have been commonly used in the differentiation of Cryptosporidium species or genotypes. In order to develop a new tool for confirming the genotypes of Cryptosporidium species, parts of the 90-kDa heat shock protein (Hsp90) genes of seven Cryptosporidium species and genotypes known to infect humans (C. hominis, C. parvum, C. meleagridis, C. canis, C. muris, C. suis, and the cervine genotype), together with one from cattle (C. andersoni), were sequenced and a ...[more]

PMID: 19168758

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Project description:BackgroundRecently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host-pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated.ResultsWe demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish.ConclusionThis study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Dataset Information

90-kilodalton heat shock protein, Hsp90, as a target for genotyping Cryptosporidium spp. known to infect humans.

Publications

90-kilodalton heat shock protein, Hsp90, as a target for genotyping Cryptosporidium spp. known to infect humans.

Similar Datasets

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