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RESEARCH PROFILES
Delving deep into the parasite proteome
issue of JPR (pp 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. Different 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,
as mitochondria and nuclei. Fraction 5 contained insoluble material such as Insect bites are often irritating, but cytoskeletal and cytoskeleton-associin the U.S. they generally aren’t a maated proteins, which are highly abunjor health concern. For example, we dant in L. infantum whole-cell prepamight scratch a mosquito bite for a few rations; these proteins can swamp out days, but before we know it, the small the signals from rare proteins. The reswelling disappears. But people in 88 searchers also verified the subcellular countries throughout the world, from localizations of some of the proteins by Central and South America to the Midfluorescent staining. dle East and parts of Asia, Africa, and Astonishingly, >70% of the proteins Southern Europe, have more to worry identified in this work had not been obabout. A bite from a sand fly could carserved in previous proteomics studry the Leishmania parasite, ies on Leishmania species. a protozoan that can inSome of these proteins are (a) (b) fect vertebrates such as huhomologous to proteins in mans, dogs, and rodents other organisms, so the rewith a disease known as searchers have a good idea leishmaniasis. In less sehow these proteins function vere cutaneous cases of the in L. infantum. Other prodisease, a large craterlike teins, however, are hypoboil appears at the bite site, thetical; they are predicted usually on the face. In more to exist on the basis of the severe cases, the infection parasite’s genome sequence Glowing parasites. L. infantum cells were transfected with fluorescently spreads to internal organs, but have no known homolabeled proteins that were isolated in (a) fraction 2 and (b) fraction 3. The such as the liver or spleen. logues. Ouellette says that protein from fraction 2 is cytosolic, whereas the protein from fraction 3 This form of leishmaniasis this study validates the exisappears to localize to the mitochondria. can be fatal. tence of these hypothetical To gain a better underproteins and provides inforstanding of the Leishmania organism she read that digitonin often is used to mation on their subcellular locations. and its life cycle, Marc Ouellette and prepare mitochondrial preparations. Finally, the researchers used the colleagues at the Centre Hospitalier de She figured that different concentramethod to compare the proteomes of l’Université Laval (Canada) have turned tions of digitonin would allow the retwo life-cycle stages of L. infantum. to proteomics. This approach is ideal searchers to fractionate subcellular Sand flies carry the promastigote stage organelles and localize proteins in L. because gene expression does not seem of the parasite, which infects animals. to occur at the level of transcription in infantum. Ouellette says he was skepIn an animal, the promastigote develthis organism, says Ouellette. “The regtical at first. Despite Ouellette’s initial ops into an amastigote, which causes ulation is either at the level of stabilhesitation, Foucher tested her hunchinfection. In the study, several proteins ity of the RNA or at the level of transes—and they paid off. were found to be differentially regulatlation—at the level of proteins,” he Digitonin extraction greatly ined in the various fractions of promastiexplains. creased the amount of total protein obgotes and amastigotes. Investigators have analyzed wholetained and the number of protein spots According to Ouellette, one potential cell lysates in previous studies of Leishthat were visible on 2DE gels of L. inlimitation of the method is that it relies mania and other parasite proteomes, fantum samples. The researchers used on sterol concentrations, which could but only a small fraction of the thoufive concentrations of the detergent, revary between organisms and could be sands of predicted proteins have been sulting in five fractions. Fractions 1 and affected by the growth media. He says, observed. “One of the big problems in 2 produced spot patterns that were alhowever, that the researchers have obproteomics is that when you look at tomost identical, so fraction 1 was not tained highly reproducible results untal proteins, you always get the same used in subsequent analyses. Proteins der all of the conditions they have testabundant proteins,” says Ouellette. “So in the other four fractions were idened so far. Currently, the researchers are tified. Fraction 2 contained cytosolic we decided to develop a number of difapplying the fractionation technique to ferent fractionation protocols.” proteins. Fractions 3 and 4 contained several projects, including the study of One of these protocols features digiproteins that are known to reside in membrane-bound transport proteins. tonin extraction and is reported in this various organellar compartments, such —Katie Cottingham
1524 Journal of Proteome Research • Vol. 5, No. 7, 2006
© 2006 American Chemical Society
