Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii.
Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.
Kawase
Osamu
O
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
Nishikawa
Yoshifumi
Y
Bannai
Hiroshi
H
Zhang
Houshuang
H
Zhang
Guohong
G
Jin
Shigeki
S
Lee
Eung-Goo
EG
Xuan
Xuenan
X
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002413:Cations, Divalent / Q000378:metabolism; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D015800:Protozoan Proteins / Q000032:analysis* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014122:Toxoplasma / Q000737:chemistry / Q000378:metabolism*; D014709:Vero Cells
D002413:Cations, Divalent; D010446:Peptide Fragments; D020543:Proteome; D015800:Protozoan Proteins; D002118:Calcium