Migration through the Extracellular Matrix by the Parasitic ProtozoanLeishmaniaIs Enhanced by Surface Metalloprotease gp63

Abstract

Leishmania species engineered to express high levels of the surface metalloprotease gp63 have enhanced capacity of migration through extracellular matrix in vitro. This correlates with gp63 degradation of extracellular matrix components, such as collagen type IV and fibronectin, and suggests an important role for gp63 in the pathogenesis of leishmaniasis.Leishmania species are digenetic protozoa that alternately parasitize their sand fly vectors and mammalian macrophages. Parasites are deposited in the mammalian skin by infected sand flies and thereafter must interact with and overcome a variety of obstacles, including extracellular matrix (ECM) and basement membrane (BM) proteins, to establish infection within macrophage phagolysosomes (6). The 63-kDa glycoprotein gp63 is a zinc-dependent metalloprotease found on the surface of the parasite that facilitates complement inactivation in serum (3), interaction with the host macrophage (2, 10, 11) and intraphagolysosomal survival (8, 11). Structural and biochemical similarities exist between gp63 and members of the matrix metalloproteases (4, 14). The latter are important for enhancing the migration of some tumor cells through the ECM and BM, aiding in their metastasis (15,16).To test the hypothesis that gp63 facilitates parasite migration through the ECM, we employed a commercial invasion system in which parasites are placed in a cell culture insert and assessed for their ability to pass through the insert's ECM (Matrigel)-impregnated 8-m-diameter pores (Becton Dickinson, Franklin Lakes, N.J.) (16). An attenuated, gp63-deficient variant of Leishmania amazonensis (12) was transfected with the following plasmid constructs for use in this study: (i) pX, an episomal expression vector; (ii) pX-gp63, which expresses wildtype gp63; and (iii) pX-E265D, which expresses an equivalent level of a proteolytically inactive form of gp63 (12). Stationaryphase promastigotes of each transfectant line were washed and suspended at a concentration of 10 8 cells ml Ϫ1 in Hanks buffered saline solution (Invitrogen, Carlsbad, Calif.). One milliliter of each parasite solution was applied to two cell culture inserts, one embedded with Matrigel and the other without Matrigel (control insert). At increasing times postinoculation, the numbers of parasites present in the lower wells were determined and the percentage of migration was calculated by dividing the number of parasites migrating through the inserts containing Matrigel by the number migrating through the control inserts. No differences were seen in the migrations of different transfectants across control inserts, with complete equilibration of parasite density achieved within 20 min.Migration was assessed over a 48-h period (Fig. 1A). Approximately 40% of the pX-gp63 promastigotes had migrated into the lower chamber at 12 h after inoculation, while only 7% of pX transfectants had migrated by this time. At 24 and 48 h after inoculation, the differences were even more pronounced, with 80 and nearly 100% of the pX-gp63 transfectants, res...

Fibronectin Binding and Proteolytic Degradation byLeishmaniaand Effects on Macrophage Activation

“…Thrice-washed cells were fixed in 0.5% glutaraldehyde (Glu) in phosphate-buffered saline (PBS) for 30 min on ice and then washed and resuspended at 10 8 per ml. It has been previously shown that the Glu fixation of cells does not significantly diminish the surface proteolytic activity of leishmanolysin (16)(17)(18). Purified bovine FN and laminin (LM) were purchased from a commercial source (Sigma-Aldrich), divided into aliquots, and stored at Ϫ20°C prior to use.…”

“…The surface biotinylation and preparation of detergent lysates of L. amazonensis were done as described previously (18,19). Briefly, the biotinylation of parasite surface proteins (using 10 7 washed, live cells) or 10 to 50 mg of FN or LM was done by incubation in PBS containing 1 mg/ml of EZ-Link N-hydroxysulfosuccinimide-long chain-biotin reagent (Pierce) on ice for 30 min.…”

“…To test the capacity of parasites to digest components of FN, 10 mg each of FN was incubated at 37°C with 10 7 stationary-phase, Glu-fixed promastigotes or amastigotes for increasing lengths of time (from 5 min to 24 h). The Glu fixation of parasites has been shown to not affect gp63 proteolytic activity (17)(18)(19). Supernatants of the reaction mixtures were analyzed by SDS-PAGE and Coomassie blue staining of gels.…”

“…and collagen type IV (18). FN is a large, multifunctional protein, the sequence of which is well conserved across species, that has multiple domains for interaction with ECM components such as heparin, collagen, and fibrin (12).…”

Leishmania, the phagosome, and host responses: The journey of a parasite

“…This major surface antigen exerts several functions before and after internalisation by phagocytic cells. It has been shown to degrade the extracellular matrix at a sub-cutaneous level, which is believed to facilitate the parasite movement under the skin following inoculation of promastigotes into the dermis [11]. GP63 is also able to inhibit complement activity by binding C3b and to increase the conversion of C3b into C3bi, an inactive form of C3, through direct cleavage, and thereby reducing fixation of the terminal C5-C9 membrane attack complex [12].…”