Modulation of gene expression of three Cryptosporidium parvum ATP-binding cassette transporters in response to drug treatment.
We studied three ATP-binding cassette (ABC) transporters (cgd1_1350, cgd7_4510, and cgd7_4520) of Cryptosporidium parvum that were identified to share a high level of homology with nucleotide binding domains of other parasitic ABC transporters and therefore could be potential candidates of efflux of drugs and/or contribute to the intrinsic resistance to chemotherapy observed of this parasite. Partial characterization and expression analysis of three C. parvum ABC transporters was determined by standard semi-quantitative real-time polymerase chain reaction. Expression of mRNA of the three ABC transporters was detected at different time points in infected cell cultures over the 48-h period, corresponding to the development of the sexual and asexual stages of the parasite. To determine if these three transporters were modulated in response to drug treatment, infected HCT 8 cells were then incubated for 48 h with different concentrations of paromomycin and a single concentration of cyclosporin A. Our results indicated that treatment by paromomycin resulted in approximately five to eightfold upregulation of cgd1_1350 transcript and about threefold of upregulation of Cgd7_4510 transcript levels. Cyclosporin A had a similar upregulating effect on cgd1_1350 and cgd7_4510 RNA levels: fivefold and 2.6-fold, respectively. On the contrary, drug treatment had little effect on cgd7_4520 transcript levels. Therefore, two of these transporters may be of value as tools for the study and the rational drug design of specific inhibitors to counteract C. parvum's intrinsic drug resistance.
Benitez
Alvaro J
AJ
Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA.
McNair
Nina
N
Mead
Jan
J
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002460:Cell Line; D016785:Cryptosporidium parvum / Q000187:drug effects* / Q000472:pathogenicity; D016572:Cyclosporine / Q000494:pharmacology*; D004847:Epithelial Cells / Q000469:parasitology*; D005786:Gene Expression Regulation; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000469:parasitology*; D008969:Molecular Sequence Data; D010303:Paromomycin / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018528:ATP-Binding Cassette Transporters; D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; D012333:RNA, Messenger; D010303:Paromomycin; D016572:Cyclosporine
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