The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.
"Interestingly, antibodies raised against L. mexicana gp63 recognised H. megaseliae proteins (Fig. 3). In previous works of our research group and others, the production of a similar cell-surface metalloproteinase of 60–66 kDa in some monoxenous trypanosomatids has been shown, including Crithidia fasciculata (Etges, 1992), C. deanei, Crithidia desouzai and Crithidia oncopelti (d'A ´ vila-Levy et al., 2001), C. guilhermei (Nogueira de Melo et al., 2002), "
[Show abstract][Hide abstract] ABSTRACT: The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.
International Journal for Parasitology 05/2006; 36(4):415-22. DOI:10.1016/j.ijpara.2005.12.006 · 3.87 Impact Factor
"This and additional functions may be further suggested by the finding of similar ectoproteases in other trypanosomatids (e.g.   ). In contrast, gp63 is thought to play little or no role for L. major in the vectors, since knockout mutants were found to develop and survive as well as the wild type in three Old World Phlebotomus species  . "
[Show abstract][Hide abstract] ABSTRACT: The zinc protease (gp63) of promastigotes was found to play a role in the sand fly part of the Leishmania life cycle. Lutzomyia longipalpis females were fed with promastigotes (10(6) per ml) of a Leishmania amazonensis clone whose gp63 was up- and down-regulated by directional cloning into P6.5 for sense- and anti-sense transcription. Early development was found to differ significantly between the sense- and anti-sense transfectants 2 days post-feeding. The sense transfectants overexpressing gp63 were found similar to those with the vector alone: both developed in the gut at high rates of approximately 90-100% and at a high density with moderate to heavy parasite loads in >70% of the infected females. In contrast, the anti-sense transfectants with gp63 down-regulated developed at a lower rate (approximately 70%) and, significantly, at a very low density, with moderate to heavy parasite loads only in approximately 30% of the infected females. On day 9 post-feeding, all three groups of transfectants developed at a similar rate of approximately 50% with comparable parasite loads. Thus, gp63 plays a role at the early stage of L. amazonensis establishment in L. longipalpis.
Microbes and Infection 07/2004; 6(7):646-9. DOI:10.1016/j.micinf.2004.03.003 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.
Canadian Journal of Microbiology 11/2003; 49(10):625-32. DOI:10.1139/w03-081 · 1.22 Impact Factor
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