bio sphere
Delving deep into the parasite proteome Digitonin helps researchers uncover new proteins in Leishmania.
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© 2006 AMERICAN CHEMICAL SOCIETY
(a)
(b)
CDC/WHO
CDC/MARTIN D. HICKLIN
nsect bites are often irritating—literally—but in the U.S. they generally aren’t a major health concern. But people in 88 countries throughout the world, from Central and South America to the Middle East and parts of Asia, Africa, and Southern Europe, have more to worry about. A bite from a sand fly could carry the Leishmania parasite, a protozoan that can infect vertebrates such as humans, dogs, and rodents with leishmaniasis. In less severe cutaneous cases of the disease, a large craterlike boil appears at the bite site, usually on the face. In more severe cases, the infection spreads to internal organs, such as the liver or spleen. This form can be fatal. To gain a better understanding of Leishmania and its life cycle, Marc Ouellette and colleagues at the Centre Hospitalier de l’Université Laval (Canada) have turned to proteomics. This approach is ideal because gene expression does not seem to occur at the level of transcription in this organism, says Ouellette. “The regulation is either at the level of stability of the RNA or at the level of translation—at the level of proteins,” he explains. Investigators have analyzed wholecell lysates in previous studies of Leishmania and other parasite proteomes, but only a small fraction of the thousands of predicted proteins have been observed. “One of the big problems in proteomics is that when you look at total proteins, you always get the same abundant proteins,” says Ouellette. “So we decided to develop a number of different fractionation protocols.” One of these protocols, which features digitonin extraction, was reported in the July issue of the Journal of Proteome Research (2006, 5, 1741–1750). Ouellette and colleagues extracted subcellular fractions of Leishmania infantum with digitonin, a detergent that pokes holes in membranes by binding to and then precipitating sterols. Differ-
(a) The sand fly Phlebotomus, which can carry Leishmania. (b) A skin sample shows signs of leishmaniasis.
ent cellular membranes have varying concentrations of sterols, so the researchers could focus on proteins in certain subcellular locations. The idea was the brainchild of Aude Foucher, a postdoc in Ouellette’s laboratory. When reviewing the literature, she read that digitonin often is used to make mitochondrial preparations. She figured that different concentrations of digitonin would allow the researchers to fractionate and localize proteins in L. infantum. Her hunch paid off. Digitonin extraction greatly increased the amount of total protein obtained and the number of protein spots that were visible on 2DE gels of L. infantum samples. The researchers used five concentrations of the detergent, resulting in five fractions. Fractions 1 and 2 produced spot patterns that were almost identical, so fraction 1 was not used in subsequent analyses. Fraction 2 contained cytosolic proteins. Fractions 3 and 4 contained proteins that are
known to reside in various organellar compartments, such as mitochondria and nuclei. Fraction 5 contained insoluble material such as cytoskeletal and cytoskeleton-associated proteins, which are highly abundant in whole-cell preparations and can swamp out the signals from rare proteins. The researchers verified the subcellular localizations of some of the proteins by fluorescent staining. Astonishingly, >70% of the proteins identified in this work had not been observed in previous proteomics studies on Leishmania species. Some of these proteins are homologous to proteins in other organisms, so the researchers have a good idea how these proteins function in L. infantum. Other proteins, however, are hypothetical; they are predicted to exist on the basis of the parasite’s genome sequence but have no known homologues. Ouellette says that this study validates the existence of these hypothetical proteins and provides information on their subcellular locations. Finally, the researchers used the method to compare the proteomes of two life-cycle stages of L. infantum. Sand flies carry the promastigote stage of the parasite, which infects animals. In an animal, the promastigote develops into an amastigote, which causes infection. In the study, several proteins were found to be differentially regulated in the various fractions of promastigotes and amastigotes. According to Ouellette, one potential limitation of the method is that it relies on sterol concentrations, which could vary among organisms and could be affected by the growth media. He says, however, that the researchers have obtained highly reproducible results in tests so far. Currently, the researchers are applying the fractionation technique to several projects, including the study of membrane-bound transport proteins. a —Katie Cottingham
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