Characterisation of a novel transporter from Cryptosporidium parvum.
P-ATPases are transmembrane proteins that hydrolyse ATP to drive cations or other substances across biomembranes. In this study we present the characterisation of a novel P-ATPase from the apicomplexan parasite Cryptosporidium parvum (CpATPase3), an opportunistic pathogen in autoimmune deficiency syndrome patients, for which no treatment is available. The single copy gene encodes 1488 amino acids, predicting a protein of 169.7 kDa. Primary sequence analysis, as well as an extensive phylogenetic reconstruction, indicated CpATPase3 belongs to a novel class of eukaryotic-specific P-ATPases (Type V) with undefined substrate preferences. Transcription and translation of the gene were confirmed by reverse-transcriptase polymerase chain reaction, and Western blot analysis of sporozoite protein extracts. Immunofluorescent microscopy of C. parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase-CpATPase3 (GST-ATP3) fusion protein showed that the parasite transporter was located within the apical complex associated with the parasite host-invasion machinery. Overall, these data demonstrate the diversity of C. parvum transporters, and raise the potential of Type V P-ATPases as apicomplexan-specific drug targets.
LaGier
Michael J
MJ
Wadsworth Center, New York State Department of Health and SUNY Albany School of Public Health, Department of Biomedical Sciences, P.O. Box 22002, 12201-2002, USA.
Keithly
Janet S
JS
Zhu
Guan
G
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis / Q000096:biosynthesis; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*
D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases