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Eukaryotic Cell, October 2007, p. 1905-1912, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00073-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes{triangledown}

Chaoqun Yao,1,2* John E. Donelson,3,4 and Mary E. Wilson1,2,4,5,6

Departments of Internal Medicine,1 Biochemistry,3 Microbiology,5 Epidemiology,6 Program in Molecular Biology, University of Iowa, Iowa City, Iowa 52242,4 VA Medical Center, Iowa City, Iowa 522462

Received 9 March 2007/ Accepted 20 July 2007


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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 t1/2 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 MßCD, 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 t1/2 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.


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INTRODUCTION
 
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 self-healing 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 (11, 13, 45). The number of MSPs in individual trypanosomatids ranges from seven in L. major, to dozens in L. braziliensis, to hundreds in T. cruzi (12, 28, 39, 41). At least 18 MSPs are present in L. chagasi, the causative protozoan of visceral leishmaniasis in Latin America (33, 35). During in vitro promastigote growth of virulent strain L. chagasi from the logarithmic to the stationary phase, MSP protein abundance increases 14-fold. Concomitantly, the number of MSP isoforms observed on two-dimensional gel electrophoresis (2-DE) increases from 4 to 11 (46-48). In the present study we use "MSP" or "MSPs" when referring to properties of all MSP isoforms and "MSP isoforms" when referring to specific MSP isoforms.

In addition to detecting surface MSP, we and other groups have independently found that MSP is released into the extracellular medium from Leishmania spp. and other trypanosomatids (7, 10, 18, 26, 46). Moreover, a subpopulation of internal MSPs has been detected and appears to be stable for several days (42, 47). Collectively, data generated from several laboratories, including our own, have demonstrated the existence of three subpopulations, i.e., surface-localized MSP, internal MSP, and released MSP.

We hypothesize that these three MSP subpopulations are separately trafficked through the cell to interact with the environment, and that internal MSP serves as a pool ready for rapid release after inoculation of metacyclic promastigotes into mammalian skin. We previously showed that the half-life (t1/2) of surface-localized 63-kDa MSP in virulent L. chagasi increases 75% during promastigote growth from the logarithmic to the stationary phase (47). In the present study, we demonstrate that this growth-associated regulation of surface-localized MSP t1/2 diminished in the attenuated L5 L. chagasi strain. Furthermore, we report that the membrane lipid disruption reagent methyl-ß-cyclodextrin (MßCD) enhanced the release of surface-localized MSP into the extracellular medium, whereas the internal MSP was released only after environmental exposure to an in vitro extracellular matrix modeling basement membrane, but only at the elevated temperature characteristic of a mammalian host. These data suggest that the different MSP pools are regulated independently and play distinct functions during the life cycle of Leishmania spp. A model illustrating the potential relevance of these findings during the parasite life cycle is presented.


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MATERIALS AND METHODS
 
Parasites. A Brazilian strain of L. chagasi (MHOM/BR/00/1669) was continuously passaged, by intracardiac injection of amastigotes, in golden hamsters to maintain its virulence. Amastigotes were isolated from the spleens of infected hamsters and transformed to promastigotes at 26°C, in hemoflagellate-modified minimal essential medium with 10% heat-inactivated fetal calf serum (HOMEM; reagents from Gibco, Rockville, MO). Virulent promastigotes were passaged weekly in HOMEM and used within five passages. The attenuated L5 strain of L. chagasi has been continuously cultured in vitro in HOMEM for over 9 years (43). Strain L5 differs from the virulent strain in several respects, including (i) less-abundant MSP and the expression of only MSPL genes (6, 43, 48), (ii) a shorter and simpler lipophosphoglycan (27), and (iii) reduced virulence for rodent models (43). In some experiments, virulent promastigotes were spread on semisolid M199 agar plates to obtain clonal cells (19). A total of 124 clones were established in two independent experiments. Promastigote cultures were started at a cell density of 106 cell/ml at day 0 of cultivation. Logarithmic- and stationary-phase promastigotes were collected between days 2 and 4 and days 6 and 9, respectively, with phases defined according to cell density and morphology as previously described (49).

Chemicals and antibodies. Sulfo-NHS-biotin, streptavidin agarose beads, and growth factor reduced Matrigel matrix were purchased from Pierce (Rockford, IL), Sigma (St. Louise, MO), and BD Biosciences (Bedford, MA), respectively. MßCD, protein G-agarose beads and Promix containing [35S]methionine-cysteine were bought from Sigma, CalBioChem (San Diego, CA), and Amersham Pharmacia Biotech (Piscataway, NJ), respectively. Polyclonal rabbit and sheep antisera to MSP were raised against purified L. chagasi MSP as previously described (43). Monoclonal antibody to {alpha}-tubulin (AB-1) was purchased from Oncogene (San Diego, CA). Peroxidase conjugated anti-rabbit, anti-sheep, and anti-mouse antisera were purchased from CalBioChem, Kirkegaard & Perry Laboratories (Gaitherburg, MA), and Bio-Rad Laboratories (Richmond, CA), respectively.

Metabolic labeling and surface biotinylation. These procedures were conducted by using previously published protocols (47). Briefly, the promastigotes were pulsed in Hanks balanced salt solution (HBSS; Gibco) with Promix for 0.5 h, followed by surface biotinylation for an additional 0.5 h in sulfo-NHS-biotin. Samples were taken between 0 and 72 h of "chase" in serum-free, bovine serum albumin-free medium. Both cells and cell-free spent medium were collected. Newly synthesized MSP, localized either on the cell surface or intracellularly, was isolated by streptavidin-affinity purification and immunoprecipitated from the streptavidin-cleared fraction, respectively, and detected by autoradiography as previously described (47, 48). The efficiency of pull-down via biotin-streptavidin was routinely monitored by peroxidase-conjugated ExtrAvidin (Sigma) and ECL Western blotting detection reagents (Amersham). In contrast to the streptavidin pull-down fractions, the streptavidin-cleared fractions exhibited no detectable signals.

MßCD treatment of promastigotes. Promastigotes were washed twice by centrifugation in HBSS and incubated for 48 h at 2 x 107 cells/ml in freshly prepared MßCD in RPMI 1640 (Gibco) ranging in concentration from 0 to 15 mM. All conditions were done in triplicate. To monitor cell viability, MßCD-treated or control (0 mM MßCD) promastigotes were metabolically labeled in HBSS with Promix for 0.5 h, and triplicate samples were assayed by a liquid scintillation analyzer for incorporation of radioisotope after total proteins were precipitated with trichloroacetic acid as described previously (3). The relative 35S-labeled amino acid incorporation in the presence of MßCD was compared to that in control (untreated) promastigotes. A ratio of 1.0 indicated that MßCD had no effect on promastigote viability. To investigate whether membrane lipid chelation reagent MßCD affects the release of surface-localized MSP, stationary-phase promastigotes were incubated in either 0 or 15 mM MßCD for 3 h after surface biotinylation. Spent medium was collected and concentrated as previously described (48). Both biotinylated proteins and the internal MSP were analyzed.

MSP release into Matrigel matrix. Stationary-phase promastigotes in the first passage after being converted from amastigotes isolated from hamsters were surface biotinylated. Triplicate samples of cells were suspended to a density of 2 x 108 cells/ml, in ice-cold HBSS (100 µl) plus Matrigel matrix (200 µl). Cultures were then incubated at either room temperature or 37°C for 1 to 3 h. Matrigel matrix solidified under these conditions. The mixtures were transferred to 4°C overnight to liquefy the matrix, and promastigote cells were separated from the liquefied Matrigel matrix by centrifugation. Biotinylated proteins and the nonbiotinylated MSP were collected from both the whole cellular lysate and the liquefied Matrigel matrix.

Electrophoresis and protein detection. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blotting were conducted as described previously (46). Autoradiogram was achieved by exposing X-ray MR films (Kodak, Rochester, NY). Samples analyzed by 2-DE were separated in the first dimension by isoelectric focusing (IEF) in Immobiline Drystrips pH 4-7 (Amersham) and in the second dimension according to size in SDS-7.5% polyacrylamide gels (48). In the case of cellular lysates of individual clones, the samples were separated in Immobiline Dryplates pH 4-7 (Amersham), after which proteins were transferred to a nitrocellulose membrane and MSP was detected by Western blotting.


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RESULTS
 
MSP expression in clonal L. chagasi lines. Virulent stationary-phase L. chagasi promastigotes contain at least 11 isoforms of MSP according to 2-DE immunoblots. However, there have been no documented differences in the function or localization of these MSP isoforms. At least some of the different MSP isoforms are derived from different MSPs (47, 48). We approached the question of differential function by carefully characterizing MSP isoforms. Our prior work documents MSP isoforms in uncloned L. chagasi populations (47, 48); Fig. 1 shows 2-DE MSP profiles in clonal L. chagasi cell lines. Clonal lines were derived from a stationary-phase promastigote population on semisolid M199 agar plates. Proteins were separated by IEF and transferred to nitrocellulose membranes, and MSPs were detected by immunoblotting with polyvalent antiserum against MSP. An MSP profile similar to that of the entire population was observed for all 124 clones examined; representative examples shown in Fig. 1. Eight bands between isoelectric points (pI) 5.8 and 6.7 were detected, along with an additional three bands between pI 4.8 and 5.2. Each of these bands formed a spot in the second dimension, as shown in immunoblots of 24 representative clones (Fig. 1). As we anticipated but thought it important to investigate, we did not detect clonal variation in MSP expression during growth in vitro.


Figure 1
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  		FIG. 1. Homogeneity of MSP expression in clonal lines of L. chagasi. Single clones were selected from M199 plates prepared from virulent L. chagasi promastigote populations. After expanding, total cell lysates of stationary phase promastigote clones were subjected to IEF (A) or 2-DE (B) and transferred to nitrocellulose membranes. MSP proteins were detected by immunoblotting. (A) Immunoblot of total cellular proteins separated by IEF in a lysate of stationary-phase cells from the uncloned parental population (lane 1) or from four representatives from a total of 124 clones (lanes 2 to 5). (B) Representative of the 2-DE MSP profiles conducted on two dozen individual clones. Eleven previously described MSP isoforms from uncloned promastigotes are labeled for reference (47, 48).

Surface-localized MSP isoforms are differently regulated in attenuated and virulent strains. The MSP proteins of L. chagasi promastigotes are found in three cellular locations, i.e., internal, surface, and released into the environment (46, 47). The cellular distribution of MSP proteins changes during in vitro growth. We previously showed that an increase in total promastigote MSP content during "in vitro metacyclogenesis" is associated with a decrease in the rate of MSP release into the environment. To study the population of MSP released we contrasted MSP synthesis in, and loss from, virulent promastigotes compared to a nonvirulent attenuated line of parasites (L5) that does not undergo changes in total MSP content during metacyclogenesis (Fig. 2). For the purposes of comparison, we contrasted the 63-kDa MSP, an isoform that is expressed in both promastigote lines.


Figure 2
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  		FIG. 2. Surface-localized MSPs of attenuated versus virulent L. chagasi are differentially regulated. (A) Example of a pulse-chase experiment demonstrating the rates of surface or internal MSP loss from virulent L. chagasi promastigotes. Cultured promastigotes on day 3 (L) or day 7 (S) of in vitro growth were metabolically labeled with [35S]methionine-cysteine and surface biotinylated, followed by a chase without radioactivity for up to 72 h. Biotinylated surface proteins were separated from the nonbiotinylated internal proteins using streptavidin beads, and internal MSP was immunoprecipitated from bead-cleared fractions. Autoradiograms are shown. The 66-kDa isoform seen at 0 h in the nonbiotinylated samples is most likely MSP containing its propeptide (23). The t1/2 of the 63-kDa surface biotinylated MSP was determined by a linear regression of f(x) = a + bx, using densitometric analysis of the bands. Graphic results shown are the average ± the standard deviation of densitometric measurements of four independent experiments. *, P < 0.01 as determined by paired Student t test. (B) The rates of MSP synthesis in attenuated L5 versus virulent L. chagasi were measured during logarithmic (L)- or stationary (S)-phase growth were determined by metabolically labeling promastigote pro- teins with [35S]methionine-cysteine and immunoprecipitation with polyclonal antisera to MSP or P36. The ratio of MSP to P36 was standardized to virulent stationary-phase promastigotes, which was at one arbitrary unit. The data shown are representative of two independent experiments. (C) Release of surface-localized MSP into the extracellular medium. Attenuated L5 or virulent L. chagasi promastigotes during logarithmic (L)- or stationary (S)-phase growth were surface biotinylated and resuspended in fresh medium. Extracellular medium was collected 48 h later. MSP was detected in the streptavidin-bead pull-down fraction by Western blotting. Shown is the relative MSP abundance standardized to stationary-phase virulent promastigotes. The results for one of two independent experiments are shown.

First, the rate of MSP synthesis in both virulent and attenuated lines, in both logarithmic- and stationary-growth phases, was almost identical (Fig. 2B). L5 or virulent L. chagasi promastigotes were metabolically labeled with [35S]methionine, MSPs were immunoprecipitated, and newly synthesized MSPs were detected by autoradiogram. Cytosolic P36, which is constitutively expressed (22, 47), was used as a control. The ratio of MSP to P36 remained constant in the logarithmic- and stationary-phase promastigotes of both L5 and virulent strains.

Second, the t1/2 of cellular surface MSP was longer in stationary virulent promastigotes than in logarithmic virulent promastigotes, coinciding with its increased abundance in stationary virulent promastigotes. In contrast, surface MSP was lost at a uniform rate in the growth phases of attenuated L5 parasites (Fig. 2A). Both virulent and attenuated promastigotes were surface labeled by biotinylation during the logarithmic or stationary phase of growth and chased over the next 72 h. Immunoblotting was used to confirm that the indicated bands were indeed MSPs (not shown). The surface MSP of virulent L. chagasi promastigotes had a shorter t1/2 (52 h) when labeled in logarithmic growth phase than MSP proteins labeled in stationary phase (90 h; Fig. 2A). Whether this is due to the predominant MSP isoforms synthesized in the different growth stages or to other factors inherent in the growth phase of parasites cannot be determined (46, 47). In contrast, the t1/2 of surface-localized 63-kDa MSP in the attenuated L5 strain of L. chagasi promastigotes remained unchanged during growth from the logarithmic (51 h) to the stationary (52 h) phase. Indeed, the MSP t1/2 was almost identical to that of logarithmic-growth-phase virulent strain promastigotes (52 h) (Fig. 2A). In contrast to surface MSP, the internal MSP of both virulent and attenuated L5 strains remained stable throughout promastigote growth (Fig. 2A) (47).

Third, the mechanism differentiating the t1/2 of surface MSP in virulent as opposed to attenuated L5 promastigotes was a difference in the rate of MSP shedding into the medium. Surface MSP was labeled by biotinylation in both L5 and virulent strain parasites. Parasites were then incubated in fresh medium, and surface biotinylated MSPs were detected by Western blotting of the streptavidin bead-purified fraction of the spent media after 48 h of incubation. A minimum of fourfold more MSPs was found in the spent media of both the logarithmic and the stationary phases of L5 strain and the logarithmic phase of the virulent strain than the stationary phase of the virulent strain (Fig. 2C). Collectively, these data indicate the increase in surface-localized MSP in the stationary-phase virulent promastigotes is associated with a decrease in the rate of shedding into the environment compared to logarithmic-phase virulent promastigotes. There is no similar growth phase-dependent regulation of MSP in the L5 attenuated strain of L. chagasi.

MßCD enhances the release of the surface-localized MSP isoforms. The unique retention of surface MSP by virulent stationary-phase promastigotes could be due to its association with surface lipid-containing membrane domains. MßCD depletes lipid rafts from the plasma membranes of a variety mammalian cells by chelating and transiently removing membrane cholesterol (14, 17, 21, 24, 31, 40). Based on the hypothesis that differential association of MSP with membrane lipids could account for its release by logarithmic promastigotes and retention by stationary promastigotes, we reasoned that membrane lipid disruption with MßCD could enhance MSP release from the Leishmania membrane. In replicate experiments, virulent L. chagasi promastigotes were treated with 0, 5, 10, or 15 mM MßCD for 48 h. A dose-dependent augmented release of MSP into the extracellular medium was observed. Specifically, control cells (0 mM MßCD) released ca. 35% of MSP, whereas the cells in 15 mM MßCD released ~80% of MSP into the extracellular medium (Fig. 3A and B).


Figure 3
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  		FIG. 3. MßCD enhances a dose-dependent release of surface-localized MSP. (A and B) Virulent promastigotes on different days of growth were treated with the indicated concentration of MßCD (0 to 15 mM) for 48 h. Filtered supernatants and cells were subjected to SDS-polyacrylamide gel electrophoresis. MSP was detected by immunoblotting. The results for one of three independent experiments, each of which was conducted in triplicate, are shown. (A) Extracellular (E) and cellular (C) MSP content in parasites incubated with the indicated MßCD concentration is shown. (B) The abundance of extracellular MSP in the medium of cells shown in panel A was quantified by densitometric analysis. Values represent the percentage ([extracellular/total MSP] x 100) for each concentration. Statistical analysis findings for 0 versus 10 or 15 mM MßCD are indicated as follows: *, P < 0.05; and **, P < 0.01. (C) MßCD effect on total incorporation of [35S]methionine-cysteine into newly synthesized proteins. MßCD-treated cells were metabolically labeled, and newly synthesized total protein was monitored as described in Materials and Methods. Values represent the total incorporation of radioisotope into the newly synthesized proteins of treated parasites relative to the controls (0 mM MßCD). One of two independent experiments, with three replicate conditions, is shown. (D) MßCD treatment enhances release of surface-localized MSP into the extracellular medium. Stationary-phase virulent L. chagasi promastigotes were surface biotinylated in HBSS and incubated for 3 h at room temperature in the absence (0 mM) or presence (15 mM) of MßCD as described in Materials and Methods. Biotinylated (S) and nonbiotinylated (I) MSPs were isolated from filtered spent medium with streptavidin beads and immunoprecipitation, respectively. MSP was detected by immunoblotting. To monitor for cell lysis, the cytosolic protein P36 was measured in the cleared fraction after biotin-streptavidin affinity and MSP immunoprecipitation by immunoblotting. Total cell lysates (cell) were included. The results for one of three independent replicate experiments are shown.

To eliminate the possibility that the enhanced MSP release was due to a toxic effect of MßCD on promastigotes, the rate of promastigote protein synthesis was measured in the absence or presence of MßCD under the experimental conditions. Comparable levels of 35S-radioisotopes were incorporated into newly synthesized proteins of untreated or MßCD-treated cells (Fig. 3C), indicating that MßCD treatment of promastigotes under these conditions is not detrimental to the cells. Furthermore, the growth curves of untreated or MßCD-treated cells in HOMEM were similar (data not shown). Thus, perturbing the plasma membrane lipid-containing domains of stationary-phase promastigotes accelerates MSP release into the extracellular medium, although it does not appear to harm the promastigotes in culture.

To test the hypothesis that disruption of membrane lipid-containing domains with MßCD only promotes release of surface-localized MSP, stationary-phase promastigotes were treated with 15 mM MßCD for 3 h after surface biotinylation. Control cells were treated identically but received no MßCD. Spent medium was collected, from which surface-biotinylated proteins were isolated by streptavidin affinity purification. Internal MSP was purified by immunoprecipitation from the streptavidin-cleared fraction of the spent medium. Immunoblots were used to assay for the presence of MSP. As shown in Fig. 3D, nonbiotinylated, internal MSP was not detectable in extracellular medium. In contrast, biotinylated, surface MSP was 34.6% ± 12.8% (n = 3) more abundant in the spent media of the MßCD-treated cells than in controls. Furthermore, no cytoskeletal ß-tubulin and cytosolic P36 markers were detected by immunoblotting in the clear fraction of the same spent media after biotin-streptavidin affinity purification and MSP immunoprecipitation (Fig. 3D and data not shown), which eliminates the possibility that MSP release is due to cell lysis. These data indicate that MßCD enhances the release of only surface-localized MSP, a result consistent with the possibility that MSP stabilization in the surface membrane requires an association with cholesterol/ergosterol-containing lipid domains.

Release of internal MSP isoforms is stimulated by the Matrigel matrix, specifically at 37°C. Although surface MSP can be artificially released by disrupting membrane lipid domains, the natural evolution of stationary promastigotes in the sand fly is to a cellular state that retains surface MSP. Metacyclic promastigotes are inoculated by sand flies into mammalian tissues, whereupon they initially encounter an elevated temperature and components of extracellular mammalian environment. We investigated whether MSP would be released under conditions that mimic the in vivo setting. First, we tested whether the highest mammalian body temperature encountered by the parasite, i.e., 37°C, would stimulate internal MSP release. Stationary-phase promastigotes were metabolically labeled, surface biotinylated, and subsequently incubated at 37°C for 24 h to test for release of surface versus internal MSP. Similarly treated control promastigotes were incubated at room temperature. Surface and internal MSPs were immunoprecipitated from the streptavidin bead-enriched or -cleared cellular lysates and detected by autoradiography. Under these conditions, there was no detectable change in internal versus surface MSP in the promastigotes after 24 h at a higher temperature (data not shown). These data suggest that a temperature increase to 37°C is by itself insufficient to stimulate internal MSP release.

We then incubated stationary-phase promastigotes in the Matrigel matrix at 37°C to test whether this combination would stimulate the release of internal MSP. Matrigel matrix is a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, which has been used to study the metastasis of cancer cells (29, 34). One prominent feature of this matrix is that it is a liquid at 4°C but it gels at room temperature and above, forming a reconstituted basement membrane. Consequently, when promastigotes are incubated in the matrix at 37°C, this setting experimentally mimics the site of sand fly inoculation into a mammalian host.

Stationary-phase promastigotes were surface biotinylated prior to incubation in either Matrigel matrix or HBSS. Promastigotes incubated in HBSS released surface MSP but little or no internal MSP into the extracellular medium at room temperature. Neither MSP form, either surface or internal, was substantially released at 37°C (Fig. 4A and B). In contrast, incubation of promastigotes in the Matrigel matrix for 3 h at 37°C stimulated release of mostly internal MSP (Fig. 4A and B). This effect was enhanced by a longer (3 versus 1 h) incubation time. Strikingly, the effect of Matrigel on release of internal MSP was significantly lower at room temperature, whereas more surface MSP was released under these conditions (Fig. 4A and B). Furthermore, the level of total internal MSP was significantly higher in parasites incubated in Matrigel compared to HBSS, although there was no change in internal MSP when parasites were incubated at room temperature versus 37°C (Fig. 4C). Hence, it is very unlikely that the specific release of internal MSP stimulated by a combination of Matrigel matrix and 37°C was due to leakiness of intracellular content from damaged promastigotes, even though we cannot formally eliminate this possibility at this time. Overall, these results lead us to conclude that surface MSP is released at room temperature and that this release is inhibited at 37°C, whereas internal MSP is released in response to the presence of Matrigel matrix, specifically at 37°C (Fig. 4).


Figure 4
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  		FIG. 4. Release of internal MSP. (A) Stationary-phase promastigotes in the first passage after isolation from hamsters were incubated in HBSS (control, 3 h) or the Matrigel matrix for 1 or 3 h after surface biotinylation at either 37°C or room temperature. After transfer to 4°C, parasites were removed by centrifugation. Streptavidin beads were used to isolate biotinylated surface MSP from both the whole-cell lysate and the extracellular medium. The internal MSP was purified from streptavidin-bead cleared fractions by immunoprecipitation and was detected by Western blotting. Each condition was performed in triplicate. (B and C) Quantitation of released and internal MSP. The band intensity of the extracellular (B) and internal (C) fractions for each experiment in panel A was determined by densitometric analysis, and the averages and standard deviations of the triplicate samples were determined. The results from one of three experiments are presented.


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DISCUSSION
 
MSPs are among the most abundant cellular proteins in promastigotes of all Leishmania spp. studied to date. Indeed, in L. mexicana, MSPs account for 1 and 0.1% of total proteins in promastigotes and amastigotes, respectively (1). Promastigote cell-associated MSP is predominantly attached to the cell surface by glycosylphosphatidylinositol anchors (4, 5). However, our laboratory and others have observed that as much as one-third of the cell-associated MSP is located intracellularly, as determined by a combination of surface biotinylation, immunoelectron microscopy, and cytofluorometry (42, 47). Furthermore, the internal MSP in L. chagasi is so stable that no reduction in abundance is detected for up to 6 days using pulse-chase analysis (47). We hypothesized that the surface-localized and internal MSPs are regulated separately via different mechanisms. Furthermore, the role and origin of the MSP released by promastigotes into extracellular medium has as yet been uncharacterized. In the present study we showed by using MßCD that the decreased release of surface MSP by the virulent stationary promastigotes is associated with the content of membrane lipids, since MßCD-mediated removal of cholesterol/ergosterol specifically enhanced the release of surface-localized MSP into extracellular medium. This likely reflects changes in the promastigote membrane during metacyclogenesis, in that a lipid-rich membrane retaining MSP in metacyclic parasites could promote retention of high surface levels of this virulence factor. In contrast, exposure to conditions mimicking mammalian tissue with Matrigel at 37°C stimulated the release of internal, but not surface, MSP. These data demonstrate for the first time that the surface-localized and internal MSPs are trafficked out of the promastigote cell in response to different external stimuli.

Phenotypic variation has been found in isoforms of a 235-kDa rhpoptry protein between clones of Plasmodium yoelii parasites. This protein is encoded by a multigene family of ~50 genes and may be involved in the selection of red blood cells for invasion by merozoites (2, 15, 20, 30, 38). Because there are at least 11 MSP isoforms in stationary-phase virulent L. chagasi promastigotes (47, 48), we hypothesized that similar variation between L. chagasi parasites could yield clonal isolates that express one or a few MSPs. However, we were not able to document clonal variation in MSP expression by cells expanded from individual clones by using 2-DE immunoblotting. This does not prove that all parasite clones express all MSP isoforms or that individual parasite clones cannot express only one or a few MSP isoforms in vivo. Nonetheless, according to our ability to detect we tentatively conclude that at least some L. chagasi parasites are able to express the majority of MSP isoforms when derived from a single cloned cell.

The three MSP classes of mRNAs (MSPL, MSPS, and MSPC) in L. chagasi are posttranscriptionally regulated. In the case of MSPL mRNA this regulation is known to occur specifically at the level of mRNA stability (6, 32, 44). Regarding MSP regulation at the protein level, we showed herein that the measurable rate of MSP synthesis was very similar throughout promastigote growth in vitro from logarithmic to stationary phase, a finding consistent with our earlier report (47). Therefore, the growth-associated 14-fold increase in the abundance of cell-associated MSP must be posttranslationally regulated. An increase in protein stability, associated with decreased MSP shedding, accounts for a fivefold increase (47). We show here that the internal pool of MSP is extremely stable throughout growth of both attenuated L5 and virulent parasite strains. Consequently, internal MSP appears not to be affected by the growth-associated regulation of MSP stability. We also demonstrate that the t1/2 of surface-localized 63-kDa MSP in the attenuated strain is similar to that of the lower MSP-expressing, logarithmic-phase promastigotes of the virulent strain, regardless of the growth phase (Fig. 2). One plausible explanation for this difference between attenuated and virulent strains during growth is the different rates of MSP shedding. We documented that the rate of MSP shedding by stationary-phase virulent promastigotes is slower than that of logarithmic-phase virulent promastigotes and that MSP shedding by L5 attenuated promastigotes is more rapid than virulent L. chagasi in all growth phases (Fig. 2C).

The biochemical mechanisms by which Leishmania spp. promastigotes regulate MSP release are not well understood. Released MSPs have electrophoretic mobilities similar to those of their cell-associated counterparts (46). At least some surface-localized L. amazonensis MSP is released through autoproteolytic activity, as shown by site-specific mutation and inhibition by a zinc chelator (26). We previously determined that released MSP does not bind to a antibody against the cross-reactive determinant, suggesting it is not released by a phosphatidylinositol-specific phospholipase C (46) similar to the released MSP of L. amazonensis. Released L. amazonensis MSP does not contain ethanolamine, suggesting it lacks a glycosylphosphatidylinositol membrane anchor (26). The data generated here by using lipid chelation suggests that the decreased release of MSP from stationary virulent promastigotes is due to remodeling of the surface membrane such that MSP is retained in association with lipids. We cannot rule out the additional possibility that there may also be recycling and degradation of MSP as a means of decreasing cellular levels of MSP, but this has yet to be tested.

In addition to the above evidence that MSP release by virulent promastigotes requires a specific membrane lipid composition, we approached the mechanisms by which Leishmania spp. promastigotes release MSP using a model of in vivo conditions. The Matrigel matrix contains laminin, collagen IV, entacin, heparin sulfate proteoglycan, growth factors, collagenases, and other undefined components. We demonstrated here that a combination of this matrix and mammalian body temperature is sufficient to stimulate internal MSP release. We suggest that the mechanism of MSP release in mammalian tissue differs from release in promastigote axenic culture. Whether this reflects differences between MSP trafficking in the sand fly versus the mammalian hosts is not clear.

The goal of the present study was to address how the three MSP subpopulations (surface, internal, and released) are regulated during metacyclogenesis and in response to the mammalian host environment. A model for MSP regulation in the different promastigote environments is illustrated in Fig. 5. In this model MSP is abundantly released by the dividing, procyclic promastigotes in the sand fly gut, as simulated by the logarithmic growth of L. chagasi in culture. This released MSP might be related to the nutrient requirements of Leishmania in the insect gut environment, where residual mammalian blood from the sand fly meal is a main source of nutrients. Indeed, it has been shown that downregulation of MSP in L. amazonensis reduces the parasites' early development in sand flies (16). As procyclic promastigotes develop to metacyclic promastigotes, the rate of released surface-localized MSP decreases and the abundance of surface-localized MSP increases (47). Our data suggest that this increase is due to association of metacyclic MSP with lipid-containing membrane domains. Internal MSP is not released during metacyclogenesis. However, after inoculation into mammalian subcutaneous tissue by a sand fly vector, metacyclic promastigotes encounter a temperature increase, host extracellular matrix, and innate immune mechanisms such as complement, antimicrobial peptides and phagocytotic cells. In response to these stimuli, promastigotes could release internal MSP into mammalian tissue. It is thus logical to consider the possibility that the surface-localized MSP plays a role in the promastigotes' evasion of complement-mediated killing and their phagocytosis and/or internalization by macrophages and other cells. Internal MSP, on the other hand, may play a role in the degradation of extracellular matrix components such as collagen IV and fibronectin, as suggested in a prior report on an L. amazonensis (25). As such, it could facilitate promastigote migration toward cells such as macrophages, dendritic cells, and fibroblasts that are favorable for parasite entry and long-term survival. Thus, it is likely that the many isoforms of MSP protease facilitate parasite survival through different mechanisms in the diverse host and vector environments encountered by the parasite.


Figure 5
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  		FIG. 5. Model of MSP release and possible functions during metacyclogenesis in the sand fly vector and inoculation into the mammalian hosts. Solid and open circles represent surface-localized and internal MSP, respectively. Arrows show MSP being released into the extracellular environment. The width of the arrow is proportional to the amount of released MSP. In the sand fly panel, the upper diagram depicts MSP release from procyclic (logarithmic-growth-phase) promastigotes, and the lower diagram depicts release from their metacyclic (stationary-phase) counterparts. In the mammalian host, a metacyclic promastigote is depicted. M{phi}, macrophage.


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ACKNOWLEDGMENTS
 
C.Y. was supported by a Veterans' Affairs Merit Review Entry grant and a Veterans' Affairs Merit Review grant. Other support for this study included NIH R01 grants AI32135 and AI059451 (M.E.W. and J.E.D.); NIH R01 grants AI045540, AI048822, and AI067874 (M.E.W.); and Merit Review and Gulf War RFA grants from the Department of Veterans' Affairs (M.E.W.).


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FOOTNOTES
 
* Corresponding author. Mailing address: 471 EMRB, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-6807. Fax: (319) 353-4565. E-mail: chaoqun-yao{at}uiowa.edu Back

{triangledown} Published ahead of print on 10 August 2007. Back


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Eukaryotic Cell, October 2007, p. 1905-1912, Vol. 6, No. 10
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