Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

Abstract

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t 1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by M␤CD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t 1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions.The digenetic protozoa of Leishmania spp. shuttle between an extracellular promastigote form in the sand fly vector and an intracellular amastigote form in mammalian hosts, including humans. In the sand fly, the avirulent procyclic promastigotes develop to the virulent metacyclic organisms, a process termed metacyclogenesis that can be mimicked by in vitro cultivation of logarithmic-to stationary-phase promastigotes (36). Leishmania causes 1.5 to 2 million new cases of human leishmaniasis annually, with manifestations ranging from selfhealing cutaneous sores to life-threatening visceral leishmaniasis (8, 9). Among a few well-characterized Leishmania virulence factors is the major surface protease (MSP), also called GP63. MSP plays several important roles during Leishmania spp. infection of mammals, including (i) enhancing promastigote phagocytosis by macrophages, (ii) facilitating promastigote evasion of complement-mediated lysis, and (iii) promoting amastigote survival in the phagolysosomes of macrophages (see reference 45 for a review). There is also evidence suggesting that in the sand fly MSP plays a role in the early-stage development of promastigotes (16) and contributes to promastigote adhesion in the guts and salivary glands (see reference 37 for a review).MSP is encoded by a family of highly conserved genes organized in a tandem array. MSP genes (MSPs) and homologues have been found in all Leishmania spp. studied to date, as well as in other trypanosomatids, including the monoxenous insect parasite Crithidia and the extracellular mammalian parasite Trypanosoma brucei...

Major Surface Protease of Trypanosomatids: One Size Fits All?

“…The release of internal, but not surface-oriented, MSPs from promastigotes of L. chagasi incubated with the extracellular matrix increased at normal mammalian body temperature (37°C) but not at room temperature. Conversely, the release of surface-localized MSPs increased at room temperature but was inhibited at 37°C (106). Release of MSP at 37°C could facilitate close contact between promastigotes and host cells such as Mø and fibroblasts and thus could be favorable for parasite entry and/or long-term survival.…”

“…These experiments revealed that one-fourth of the nascent MSPs remained internally for up to 6 days following biosynthesis, strongly supporting the existence of a stable internal subpopulation of MSPs. It appeared that only the mature 63-kDa form, not the 59-kDa protein, accounts for this subpopulation of internal MSPs (106,109). We have not formally ruled out two less likely possibilities: (i) that some surface MSP isoforms are inaccessible to surface biotinylation due to the presence of densely packed LPG, a situation reminiscent of the failure of immunogold to label some MSPs on the cell surfaces of L. major promastigotes (86), and (ii) that MSPs are constantly internalized with biotin removal.…”

“…1) that considers the three subpopulations of MSP in Leishmania spp. including surface-localized, intracellular, and released MSPs (106).…”

Structures of Glycolipids Found in Trypanosomatids: Contribution to Parasite Functions

“…release proteolytically active gp63 into the extracellular medium presumably facilitating the propagation of the parasite [131,132].…”

Immunology of Leishmaniasis and Future Prospective of Vaccines

“…contains multiple MSPs and can be found on the promastigote surface as well as in the parasite cytoplasm. Surface MSP is involved in parasite development within sandfly and the cytoplasmic MSP which is in preformed form is ready to use by the mammalian host (Yao, Donelson et al 2007). This action is analogous to various effectors that are used by type III secretion system in bacteria which behaves like syringe and needle to inject various factors into cells (Winnen, Schlumberger et al 2008).…”