Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

Abstract

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (M␤CD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. M␤CD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, M␤CD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of M␤CD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that M␤CD promotes the release of proteins into the extracellular medium, including both MSP and MSPlike protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. Leishmania protozoa shuttle between a mammalian host as intracellular amastigotes and a sand fly vector as flagellated promastigotes. The sand fly acquires amastigote-laden cells during a blood meal. In the sand fly midgut, procyclic promastigotes derived from transformation of amastigotes undergo multiplication and development via intermediate stages, eventually yielding metacyclic promastigotes (1). The infectious metacyclic promastigote is inoculated into a pool of blood in the dermis of a mammalian host formed during a sand fly bite. Parasites are phagocytized by host macrophages, where they transform to amastigotes and multiply in parasitophorous vacuoles. Amastigotes spread to new macrophages at local or disseminated sites, perpetuating the infection and ultimately resulting in asymptomatic infections or symptomatic leishmaniasis (2). Various forms of leishmaniasis are endemic in 88 countries on four continents, leading to approximately two million new cases and 59,000 deaths annually (3).Both host and parasite factors contribute to the success of infection. On one hand, mammalian host environmental risk factors and genetic background influence the clinical manifestations of infection (4). On the other hand, parasite virulence determinants required for disease development include, but are not limited to, the major surface protease (MSP) (also called GP63 or leishmanolysin) and lipophosphoglycan (LPG) (5-8). These two molecules play both overl...

Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form

“…A Brazilian strain of Leishmania infantum (previously called L. chagasi ) (MHOM/BR/00/1669) was serially passaged in golden hamsters to maintain parasite virulence. Amastigotes were isolated from infected hamster spleens and allowed to transform to promastigotes in HOMEM (Hemoflagellate modified Minimal Essential Medium) with 10 % fetal calf serum (FCS) and hemin made from commercially available ingredients as previously described [ 8 ]. Serum-free medium (SFM) was prepared similar to HOMEM but lacking FCS and with additional biopterin, adenosine and bovine serum albumin.…”

“…Metacyclic promastigotes could not be recovered from similar cultures of avirulent promastigotes (data not shown). In some experiments, metacyclic promastigotes were incubated in 25 mM MβCD (Sigma, St. Louis, MO) freshly prepared in RPMI 1640 (GIBCO) at a cell density of 2 × 10 8 cells/ml at room temperature for 1 h to deplete cellular sterols, and then washed twice by centrifugation in Hanks’ balanced salt solution (GIBCO) as described [ 8 , 15 ].…”

“…Our prior studies using the lipid-raft disrupting agent methyl-β-cyclodextrin (MβCD) suggested that lipid rafts play a pivotal role in the virulence of Leishmania spp. [ 8 ]. The anti-fungal agent amphotericin B is widely used in patients with leishmaniasis, especially in regions where parasites are resistant to standard therapy with antimony compounds [ 9 , 10 ], or in patients co-infected with Leishmania sp.…”

Deception and Manipulation: The Arms of Leishmania, a Successful Parasite

“…LPG avoids the ultimate step of the complement cascade through prevention of the attachment of the C5b-C9-complex to the parasite surface ( 83 , 84 ). On the other hand, GP63 inactivates C3b preventing the formation of the C5 convertase complex ( 85 , 86 ). Albeit, Dominguez et al showed that under physiological conditions 85–100% of L. donovani, L. infantum, L. major , and L. amazonensis promastigotes are killed by complement after 2.5 min in human blood ( 87 ).…”

Innate immunity againstLeishmaniainfections

“…L . major parasites lacking these glycocalyx receptors are highly susceptible to complement killing (Joshi et al ., , Spath et al ., 2003b, Yao et al ., ). Genetic deletion of GP63 in L .…”