leishmania accepted - ec.asm.org · 58 dimensional gel electrophoresis (2-de) increases from four...

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Internal and surface-localized MSP of Leishmania and their differential release from 1

promastigotes 2

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

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Departments of Internal Medicine1, Biochemistry3, Microbiology5, and Epidemiology6, Program 7

in Molecular Biology4, University of Iowa, Iowa City, IA 52242; VA Medical Center2, Iowa 8

City, IA 52246 9

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*Corresponding author: Chaoqun Yao, 471 EMRB, Department of Internal Medicine, University 12

of Iowa, Iowa City, IA 52242. Tel: (319)335-6807; Fax: (319)353-4565; E-mail: chaoqun-13

[email protected] 14

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Running Title: Major surface protease of Leishmania 17

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00073-07 EC Accepts, published online ahead of print on 10 August 2007

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Abstract 18

MSP (major surface protease), also called GP63, is a virulence factor of Leishmania spp. 19

protozoa. There are three pools of MSP, located either internally within the parasite, anchored to 20

the surface membrane, or released into the extracellular environment. The regulation and 21

biological functions of these MSP pools are unknown. Herein we investigated the trafficking 22

and extrusion of surface versus internal MSPs. Virulent L. chagasi undergo a growth-associated 23

lengthening in the T½ of surface-localized MSP, but this did not occur in the attenuated L5 24

strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by 25

MβCD, which chelates membrane cholesterol/ergosterol. Furthermore, incubation of 26

promastigotes at 37oC with Matrigel matrix, a soluble basement membrane extract of EHS 27

tumor cells, stimulated release of internal MSP but not surface-located MSP. Taken together, 28

these data indicate that MSP subpopulations in distinct cellular locations are released from the 29

parasite under different environmental conditions. We hypothesize that the internal MSP with its 30

lengthy T½ does not serve as a pool for promastigote surface MSP in the sand fly vector, but that 31

it instead functions as an MSP pool ready for a quick release upon inoculation of metacyclic 32

promastigotes into mammals. We present a model in which these different MSP pools are 33

released under distinct life cycle-specific conditions. 34

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The digenetic protozoa of Leishmania spp. shuttle between an extracellular promastigote 35

form in the sand fly vector and an intracellular amastigote form in mammalian hosts, including 36

humans. In the sand fly, the avirulent procyclic promastigotes develop to the virulent metacyclic 37

organisms, a process termed metacyclogenesis that can be mimicked by in vitro cultivation of 38

logarithmic to stationary phase promastigotes (36). Leishmania causes 1.5 to 2 million new 39

cases of human leishmaniasis annually, with manifestations ranging from self-healing cutaneous 40

sores to life-threatening visceral leishmaniasis (8, 9). Among a handful of well-characterized 41

Leishmania virulence factors is the major surface protease (MSP), also called GP63. MSP plays 42

several important roles during Leishmania spp. infection of mammals, including: (i) enhancing 43

promastigote phagocytosis by macrophages; (ii) facilitating promastigote evasion of 44

complement-mediated lysis; and (iii) promoting amastigote survival in the phagolysosomes of 45

macrophages [see (45) for a review]. There is also evidence suggesting that in the sand fly MSP 46

plays a role in the early-stage development of promastigotes (16), and contributes to 47

promastigote adhesion in the guts and salivary glands [see (37) for a review]. 48

MSP is encoded by a family of highly conserved genes organized in a tandem array. 49

MSP genes (MSPs) and homologues have been found in all Leishmania spp. studied to date, as 50

well as in other trypanosomatids, including the monoxenous insect parasite Crithidia and the 51

extracellular mammalian parasite Trypanosoma brucei (11, 13, 45). The number of MSPs in 52

individual trypanosomatids ranges from seven in L. major, to dozens in L. braziliensis, to 53

hundreds in T. cruzi (12, 28, 39, 41). At least 18 MSPs are present in L. chagasi, the causative 54

protozoan of visceral leishmaniasis in Latin America (33, 35). During in vitro promastigote 55

growth of virulent strain L. chagasi from logarithmic to stationary phase, MSP protein 56

abundance increases 14 fold. Concomitantly, the number of MSP isoforms observed on two-57

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dimensional gel electrophoresis (2-DE) increases from four to eleven (46-48). In this publication 58

we will use “MSP” when referring to properties of all MSP isoforms, and “MSPs” or “MSP 59

isoforms” when referring to the different MSP isoforms. 60

In addition to surface MSP, we and other groups have independently found that MSP is 61

released into the extracellular medium from Leishmania spp. and other trypanosomatids (7, 10, 62

18, 26, 46). In addition, a subpopulation of internal MSPs has been detected and appears to be 63

stable for several days (42, 47). Collectively, data generated from several laboratories, including 64

our own, demonstrate the existence of three subpopulations, i.e., surface-localized MSP, internal 65

MSP and released MSP. 66

We hypothesize that these three MSP subpopulations are separately trafficked through 67

the cell to interact with the environment, and that internal MSP serves as a pool ready for rapid 68

release after inoculation of metacyclic promastigote into mammalian skin. We previously 69

showed that the half life (T½) of surface-localized 63-kDa MSP in virulent L. chagasi increases 70

75% during promastigote growth from logarithmic to stationary phase (47). In the current report, 71

we demonstrate that this growth-associated regulation of surface-localized MSP T½ diminished 72

in the attenuated L5 L. chagasi strain. Furthermore, we report that the membrane lipid disruption 73

reagent methyl-β-cyclodextrin (MβCD) enhanced the release of surface-localized MSP into the 74

extracellular medium, whereas the internal MSP was released only after environmental exposure 75

to an in vitro extracellular matrix modeling basement membrane, but only at the elevated 76

temperature characteristic of a mammalian host. These data suggest that the different MSP pools 77

are regulated independently and play distinct functions during the life cycle of Leishmania spp. 78

A model illustrating the potential relevance of these findings during the parasite life cycle is 79

presented. 80

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Materials and Methods 81

Parasites. A Brazilian strain of L. chagasi (MHOM/BR/00/1669) was continuously passaged, by 82

intracardiac injection of amastigotes, in golden hamsters to maintain its virulence. Amastigotes 83

were isolated from the spleens of infected hamsters and transformed to promastigotes at 26oC, in 84

hemoflagellate-modified minimal essential medium with 10% heat-inactivated fetal calf serum 85

(HOMEM; reagents from GIBCO, Rockville, MO). Virulent promastigotes were passaged 86

weekly in HOMEM, and used within five passages. The attenuated L5 strain of L. chagasi has 87

been continuously cultured in vitro in HOMEM for over nine years (43). Strain L5 differs from 88

the virulent strain in several respects including (i) less abundant MSP and the expression of only 89

MSPL genes (6, 43, 48), (ii) a shorter and simpler lipophosphoglycan (27), and (iii) reduced 90

virulence for rodent models (43). In some experiments, virulent promastigotes were spread on 91

semi-solid M199-agar plates to obtain clonal cells (19). A total of 124 clones were established in 92

two independent experiments. Promastigote cultures were started at a cell density of 1 × 106 93

cell/ml at day 0 of cultivation. Logarithmic and stationary phase promastigotes were collected 94

between days 2-4 and 6-9, respectively, with phases defined according to cell density and 95

morphology as previously described (49). 96

Chemicals and antibodies. Sulfo-NHS-Biotin, streptavidin agarose beads, and growth factor 97

reduced Matrigel™ matrix were purchased from Pierce (Rockford, IL), Sigma (St. Louise, MO), 98

and BD Biosciences (Bedford, MA), respectively. MβCD, protein G-agarose beads and Promix 99

containing [35S]-methionine and -cysteine were bought from Sigma, CalBioChem (San Diego, 100

CA) and Amersham Pharmacia Biotech (Piscataway, NJ), respectively. Polyclonal rabbit and 101

sheep antisera to MSP were raised against purified L. chagasi MSP as previously described (43). 102

Monoclonal antibody to α-tubulin (AB-1) was purchased from Oncogene (San Diego, CA). 103

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Peroxidase conjugated anti-rabbit, anti-sheep, and anti-mouse antisera were purchased from 104

CalBioChem, Kirkegaard & Perry Laboratories (Gaitherburg, MA) and Bio-Rad Laboratories 105

(Richmond, CA), respectively. 106

Metabolic labeling and surface biotinylation. These procedures were conducted using 107

previously published protocols (47). Briefly, the promastigotes were pulsed in Hank’s balanced 108

salt solution (HBSS, GIBCO) with Promix for 0.5 h, followed by surface biotinylation for an 109

additional 0.5 h in Sulfo-NHS-Biotin. Samples were taken between 0 and 72 h of “chase” in 110

serum-free, bovine serum albumin-free medium. Both cells and cell-free spent medium were 111

collected. Newly synthesized MSP, localized either on the cell surface or intracellularly, was 112

isolated by streptavidin-affinity purification and immunoprecipitated from the streptavidin-113

cleared fraction, respectively, and detected by autoradiography as previously described (47, 48). 114

The efficiency of pull-down via biotin/streptavidin was routinely monitored by peroxidase 115

conjugated ExtrAvidin (Sigma) and ECL™ western blotting detection reagents (Amersham). In 116

contrast to the streptavidin pull-down fractions, the streptavidin-cleared fractions exhibited no 117

detectable signals. 118

MβCD treatment of promastigotes. Promastigotes were washed twice by centrifugation in 119

HBSS, and incubated for 48 h at 2 × 107 cells/ml in freshly prepared MβCD in RPMI 1640 120

(GIBCO) ranging in concentration from 0 to 15 mM. All conditions were done in triplicate. To 121

monitor cell viability, MβCD-treated or control (0 mM MβCD) promastigotes were 122

metabolically labeled in HBSS with Promix for 0.5 h, and triplicate samples were assayed by a 123

liquid scintillation analyzer for incorporation of radioisotope after total proteins were 124

precipitated with trichloroacetic acid as described (3). The relative [35S]-amino acid 125

incorporation in the presence of MβCD was compared to that in control (untreated) 126

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promastigotes. A ratio of 1.0 indicated that MβCD had no effect on promastigote viability. To 127

investigate whether membrane lipid chelation reagent MβCD affects the release of surface-128

localized MSP, stationary phase promastigotes were incubated in either 0 or 15 mM MβCD for 3 129

h after surface biotinylation. Spent medium was collected and concentrated as previously 130

described (48). Both biotinylated proteins and the internal MSP were analyzed. 131

MSP release into Matrigel™ matrix. Stationary phase promastigotes in the first passage after 132

being converted from amastigotes isolated from hamsters were surface biotinylated. Triplicate 133

samples of cells were suspended to a density of 2 × 108 cells/ml, in ice-cold HBSS (100 µl) plus 134

Matrigel™ matrix (200 µl). Cultures were then incubated at either room temperature (RT) or 135

37oC for 1 to 3 h. Matrigel™ matrix solidified under these conditions. The mixtures were 136

transferred to 4oC overnight to liquefy the matrix, and promastigote cells were separated from 137

the liquefied Matrigel™ matrix by centrifugation. Biotinylated proteins and the non-biotinylated 138

MSP were collected from both the whole cellular lysate and the liquefied Matrigel™ matrix. 139

Electrophoresis and protein detection. SDS-polyacrylamide gel electrophoresis and western 140

blotting were conducted as described (46). Autoradiogram was achieved by exposing X-ray MR 141

films (Kodak, Rochester, NY). Samples analyzed by 2-DE were separated in the first dimension 142

by isoelectric focusing (IEF) in Immobiline™ Drystrips pH 4-7 (Amersham), and in the second 143

dimension according to size, in 7.5% SDS-polyacrylamide gels (48). In the case of cellular 144

lysates of individual clones, the samples were separated in Immobiline™ Dryplates pH 4-7 145

(Amersham), after which proteins were transferred to a nitrocellulose membrane and MSP was 146

detected by western blotting. 147

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Results 148

MSP expression in clonal L. chagasi lines. Virulent stationary phase L. chagasi promastigotes 149

contain at least eleven isoforms of MSP according to 2-DE immunoblots. However, there have 150

been no documented differences in the function or localization of these MSP isoforms. At least 151

some of the different MSP isoforms are derived from different MSPs (47, 48). We approached 152

the question of differential function by carefully characterizing MSP isoforms. Our prior work 153

documents MSP isoforms in uncloned L. chagasi populations (47, 48); Fig. 1 shows 2-DE MSP 154

profiles in clonal L. chagasi cell lines. Clonal lines were derived from a stationary phase 155

promastigote population on semi-solid M199-agar plates. Proteins were separated by IEF and 156

transferred to nitrocellulose membranes, and MSPs were detected by immunoblotting with 157

polyvalent antiserum against MSP. An MSP profile similar to that of the entire population was 158

observed for all 124 clones examined; representative examples shown in Fig. 1. Eight bands 159

between isoelectric points (pI) 5.8 and 6.7 were detected, along with an additional three bands 160

between pI 4.8 and 5.2. Each of these bands formed a spot in the second dimension, as shown in 161

immunoblots of 24 representative clones (Fig. 1). As we anticipated but thought it important to 162

investigate, we did not detect clonal variation in MSP expression during growth in vitro. 163

Surface-localized MSP isoforms are differently regulated in attenuated and virulent 164

strains. The MSP proteins of L. chagasi promastigotes are found in three cellular locations, i.e. 165

internal, surface, and released into the environment (46, 47). The cellular distribution of MSP 166

proteins changes during in vitro growth. We previously showed that an increase in total 167

promastigote MSP content during “in vitro metacyclogenesis” is associated with a decrease in 168

the rate of MSP release into the environment. To study the population of MSP released we 169

contrasted MSP synthesis in, and loss from, virulent promastigotes compared to a non-virulent 170

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attenuated line of parasites (L5) that does not undergo changes in total MSP content during 171

metacyclogenesis (Fig. 2). For the purposes of comparison we contrasted the 63-kDa MSP, an 172

isoform that is expressed in both promastigote lines. 173

First, the rate of MSP synthesis in both virulent and attenuated lines, in both logarithmic 174

and stationary growth phases, was almost identical (Fig. 2B). L5 or virulent L. chagasi 175

promastigotes were metabolically labeled with [35S]-methionine, MSPs were 176

immunoprecipitated, and newly synthesized MSPs were detected by autoradiogram. Cytosolic 177

P36, which is constitutively expressed (22, 47), was used as a control. The ratio of MSP to P36 178

remained constant in logarithmic and stationary phase promastigotes of both the L5 and virulent 179

strains. 180

Secondly, the T½ of cellular surface MSP was longer in stationary virulent promastigotes 181

than in logarithmic virulent promastigotes, coinciding with its increased abundance in stationary 182

virulent promastigotes. In contrast, surface MSP was lost at a uniform rate in the growth phases 183

of attenuated L5 parasites (Fig. 2A). Both virulent and attenuated promastigotes were surface 184

labeled by biotinylation during logarithmic or stationary phase of growth, and chased over the 185

next 72 h. Immunoblotting was used to confirm that the indicated bands were indeed MSPs (not 186

shown). The surface MSP of virulent L. chagasi promastigotes had a shorter T1/2 (52 h) when 187

labeled in logarithmic growth phase than MSP proteins labeled in stationary phase (90 h; Fig. 188

2A). Whether this is due to the predominant MSP isoforms synthesized in the different growth 189

stages, or to other factors inherent in the growth phase of parasites cannot be determined (46, 190

47). In contrast, the T½ of surface-localized 63-kDa MSP in the attenuated L5 strain of L. 191

chagasi promastigotes remained unchanged during growth from logarithmic (51 h) to stationary 192

phase (52 h). Indeed, the MSP T½ was almost identical to that of logarithmic growth-phase 193

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virulent strain promastigotes (52 h) (Fig. 2A). In contrast to surface MSP, the internal MSP of 194

both virulent and attenuated L5 strains remained stable throughout promastigote growth [Fig. 2A 195

and (47)]. 196

Thirdly, the mechanism differentiating the T½ of surface MSP in virulent as opposed to 197

attenuated L5 promastigotes was a difference in the rate of MSP shedding into the medium. 198

Surface MSP was labeled by biotinylation in both L5 and virulent strain parasites. Parasites 199

were then incubated in fresh medium, and surface biotinylated MSPs were detected by western 200

blotting of the streptavidin bead-purified fraction of the spent media after 48 h incubation. A 201

minimum of four fold more MSPs was found in the spent media of both logarithmic and 202

stationary phase of L5 strain and the logarithmic phase of the virulent strain than the stationary 203

phase of the virulent strain (Fig. 2C). Collectively, these data indicate the increase in surface-204

localized MSP in the stationary phase virulent promastigotes is associated with a decrease in the 205

rate of shedding into the environment, compared to logarithmic phase virulent promastigotes. 206

There is not similar growth phase-dependent regulation of MSP in the L5 attenuated strain of L. 207

chagasi. 208

MβCD enhances release of the surface-localized MSP isoforms. The unique retention of 209

surface MSP by virulent stationary phase promastigotes could be due to its association with 210

surface lipid-containing membrane domains. MβCD depletes lipid rafts from the plasma 211

membranes of a variety mammalian cells by chelating and transiently removing membrane 212

cholesterol (14, 17, 21, 24, 31, 40). Based on the hypothesis that differential association of MSP 213

with membrane lipids could account for its release by logarithmic promastigotes and retention by 214

stationary promastigotes, we reasoned that membrane lipid disruption with MβCD could enhance 215

MSP release from the Leishmania membrane. In replicate experiments, virulent L. chagasi 216

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promastigotes were treated with 0, 5, 10 or 15 mM MβCD for 48 h. A dose-dependent 217

augmented release of MSP into the extracellular medium was observed. Specifically, control 218

cells (0 mM MβCD) released about 35% of MSP, whereas the cells in 15 mM MβCD released 219

~80% of MSP into the extracellular medium (Fig. 3A and B). 220

To eliminate the possibility that the enhanced MSP release was due to a toxic effect of 221

MβCD on promastigotes, the rate of promastigote protein synthesis was measured in the absence 222

or presence of MβCD under the experimental conditions. Comparable levels of [35S]-223

radioisotopes were incorporated into newly synthesized proteins of untreated or MβCD-treated 224

cells (Fig. 3C), indicating that MβCD treatment of promastigotes under these conditions is not 225

detrimental to the cells. Furthermore, the growth curves of untreated or MβCD-treated cells in 226

HOMEM were similar (data not shown). Thus, perturbing the plasma membrane lipid-227

containing domains of stationary phase promastigotes accelerates MSP release into the 228

extracellular medium, although it does not appear to harm the promastigotes in culture. 229

To test the hypothesis that disruption of membrane lipid-containing domains with MβCD 230

only promotes release of surface-localized MSP, stationary phase promastigotes were treated 231

with 15 mM MβCD for 3 h after surface biotinylation. Control cells were treated identically but 232

received no MβCD. Spent medium was collected, from which surface-biotinylated proteins were 233

isolated by streptavidin affinity purification. Internal MSP was purified by immunoprecipitation 234

from the streptavidin-cleared fraction of the spent medium. Immunoblots were used to assay for 235

the presence of MSP. As showed in Fig. 3D, non-biotinylated, internal MSP was undetectable in 236

extracellular medium. In contrast, biotinylated, surface MSP was 34.6±12.8% (n=3) more 237

abundant in the spent media of the MβCD-treated cells than controls. Furthermore, no 238

cytoskeletal β-tubulin and cytosolic P36 markers were detected by immunoblotting in the clear 239

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fraction of the same spent media post biotin-streptavidin affinity purification and MSP 240

immunoprecipitation (Fig. 3D, and data not shown), which eliminates the possibility that MSP 241

release is due to cell lysis. These data indicate that MβCD enhances release of only surface-242

localized MSP, a result consistent with the possibility that MSP stabilization in the surface 243

membrane requires an association with cholesterol/ergosterol-containing lipid domains. 244

Release of internal MSP isoforms is stimulated by the Matrigel™ matrix, specifically at 245

37oC. Although surface MSP can be artificially released by disrupting membrane lipid domains, 246

the natural evolution of stationary promastigotes in the sand fly is to a cellular state that retains 247

surface MSP. Metacyclic promastigotes are inoculated by sand flies into mammalian tissues, 248

whereupon they initially encounter an elevated temperature and components of extracellular 249

mammalian environment. We investigated whether MSP would be released under conditions 250

that mimic the in vivo setting. First we tested whether the highest mammalian body temperature 251

encountered by the parasite, i.e., 37oC, would stimulate internal MSP release. Stationary phase 252

promastigotes were metabolically labeled, surface biotinylated, and subsequently incubated at 253

37oC for 24 h to test for release of surface versus internal MSP. Similarly treated control 254

promastigotes were incubated at RT. Surface and internal MSP were immunoprecipitated from 255

the streptavidin bead-enriched or -cleared cellular lysates and detected by autoradiography. 256

Under these conditions, there was no detectable change in internal versus surface MSP in the 257

promastigotes after 24 h at higher temperature (data not shown). These data suggest that 258

temperature increase to 37oC is by itself insufficient to stimulate internal MSP release. 259

We then incubated stationary phase promastigotes in the Matrigel™ matrix at 37oC to test 260

whether this combination would stimulate the release of internal MSP. Matrigel matrix is a 261

soluble basement membrane extract of EHS tumor cells, which has been used to study the 262

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metastasis of cancer cells (29, 34). One prominent feature of this matrix is that it is a liquid at 263

4oC but it gels at RT and above, forming a reconstituted basement membrane. Consequently, 264

when promastigotes are incubated in the matrix at 37oC, this setting experimentally mimics the 265

site of sand fly inoculation into a mammalian host. 266

Stationary phase promastigotes were surface biotinylated prior to incubation in either 267

Matrigel matrix or HBSS. Promastigotes incubated in HBSS released surface MSP but little or 268

no internal MSP into the extracellular medium at RT. Neither MSP form, either surface or 269

internal, was substantially released at 37oC (Fig. 4A and B). In contrast, incubation of 270

promastigotes in the Matrigel matrix for 3 h at 37oC stimulated release of mostly internal MSP 271

(Fig. 4A and B). This effect was enhanced by a longer (3 vs. 1 h) incubation time. Strikingly, 272

the effect of Matrigel on release of internal MSP was significantly lower at RT, whereas more 273

surface MSP was released under these conditions (Fig. 4A and B). Furthermore, the level of 274

total internal MSP was significantly higher in parasites incubated in Matrigel™ compared to 275

HBSS, although there was no change in internal MSP when parasites were incubated at RT 276

versus 37oC (Fig. 4C). Hence, it is very unlikely that the specific release of internal MSP 277

stimulated by a combination of Matrigel™ matrix and 37oC was due to leakiness of intracellular 278

content from damaged promastigotes, even though we can not formally eliminate this possibility 279

at this time. Overall, these results lead us to conclude that surface MSP is released at RT and 280

that this release is inhibited at 37oC, whereas internal MSP is released in response to the presence 281

of Matrigel matrix, specifically at 37oC (Fig. 4). 282

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Discussion 283

MSPs are among the most abundant cellular proteins in promastigotes of all Leishmania 284

spp. studied to date. Indeed, in L. mexicana, MSPs account for 1% and 0.1% of total proteins in 285

promastigotes and amastigotes, respectively (1). Promastigote cell-associated MSP is 286

predominantly attached to the cell surface by glycosylphosphatidylinositol anchors (4, 5). 287

However, our laboratory and others have observed that as much as one-third of the cell-288

associated MSP is located intracellularly, as determined by a combination of surface 289

biotinylation, immunoelectron microscopy and cytofluorometry (42, 47). Furthermore, the 290

internal MSP in L. chagasi is so stable that no reduction in abundance is detected for up to six 291

days using pulse-chase analysis (47). We hypothesized that the surface-localized and internal 292

MSPs are regulated separately via different mechanisms. Furthermore, the role and origin of the 293

MSP released by promastigotes into extracellular medium has as yet been uncharacterized. In 294

the current study we showed using MβCD that the decreased release of surface MSP by the 295

virulent stationary promastigotes is associated with the content of membrane lipids, since 296

MβCD-mediated removal of cholesterol/ergosterol specifically enhanced the release of surface-297

localized MSP into extracellular medium. This likely reflects changes in the promastigote 298

membrane during metacyclogenesis, in that a lipid-rich membrane retaining MSP in metacyclic 299

parasites could promote retention of high surface levels of this virulence factor. In contrast, 300

exposure to conditions mimicking mammalian tissue with Matrigel at 37oC stimulated the 301

release of internal, but not surface, MSP. These data demonstrate for the first time that the 302

surface-localized and internal MSPs are trafficked out of the promastigote cell in response to 303

different external stimuli. 304

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Phenotypic variation has been found in isoforms of a 235-kDa rhpoptry protein between 305

clones of Plasmodium yoelli parasites. This protein is encoded by a multigene family of ~50 306

genes and may be involved in the selection of red blood cells for invasion by merozoites (2, 15, 307

20, 30, 38). Because there are at least 11 MSP isoforms in stationary phase virulent L. chagasi 308

promastigotes (47, 48), we hypothesized that similar variation between L. chagasi parasites 309

could yield clonal isolates that express one or a few MSPs. However, we were not able to 310

document clonal variation in MSP expression by cells expanded from individual clones, using 2-311

DE immunoblotting. This does not prove that all parasite clones express all MSP isoforms, or 312

that individual parasite clones cannot express only one or a few MSP isoforms in vivo. 313

Nonetheless, according to our ability to detect we tentatively conclude that at least some L. 314

chagasi parasites are able to express the majority of MSP isoforms when derived from a single 315

cloned cell. 316

The three MSP classes of mRNAs (MSPL, MSPS and MSPC) in L. chagasi are post-317

transcriptionally regulated. In the case of MSPL mRNA this regulation is known to occur 318

specifically at the level of mRNA stability (6, 32, 44). Regarding MSP regulation at the protein 319

level, we showed herein that the measurable rate of MSP synthesis was very similar throughout 320

promastigote growth in vitro from logarithmic to stationary phase, consistent with our earlier 321

report (47). Therefore, the growth-associated 14-fold increase in the abundance of cell-322

associated MSP must be post-translationally regulated. An increase in protein stability, 323

associated with decreased MSP shedding, accounts for a 5-fold increase (47). We show in the 324

current study that internal pool of MSP is extremely stable throughout growth of both attenuated 325

L5 and virulent parasite strains. Consequently, internal MSP appears not to be affected by the 326

growth-associated regulation of MSP stability. We also demonstrate that the T½ of surface-327

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localized 63-kDa MSP in the attenuated strain is similar to that of the lower MSP-expressing, 328

logarithmic phase promastigotes of the virulent strain, regardless of the growth phase (Fig. 2). 329

One plausible explanation for this difference between attenuated and virulent strains during 330

growth is the different rates of MSP shedding. We documented that the rate of MSP shedding by 331

stationary phase virulent promastigotes is slower than that of logarithmic phase virulent 332

promastigotes, and that MSP shedding by L5 attenuated promastigotes is more rapid than 333

virulent L. chagasi in all growth phases (see Fig. 2C). 334

The biochemical mechanisms by which Leishmania spp. promastigotes regulate MSP 335

release are not well understood. Released MSPs have similar electrophoretic mobility to their 336

cell-associated counterparts (46). At least some surface-localized L. amazonensis MSP is 337

released through autoproteolytic activity as showed by site-specific mutation and inhibition by a 338

zinc chelator (26). We previously determined that released MSP does not bind to a antibody 339

against the cross-reactive determinant, suggesting it is not released by a PI-PLC (46) similar to 340

the released MSP of L. amazonensis. Released L. amazonensis MSP does not contain 341

ethanolamine suggesting it lacks a GPI membrane anchor (26). Data generated in this report 342

using lipid chelation suggests that the decreased release of MSP from stationary virulent 343

promastigotes is due to remodeling of the surface membrane such that MSP is retained in 344

association with lipids. We cannot rule out the additional possibility that there may also be 345

recycling and degradation of MSP as a means of decreasing cellular levels of MSP, but this has 346

yet to be tested. 347

In addition to the above evidence that MSP release by virulent promastigotes requires a 348

specific membrane lipid composition, we approached the mechanisms by which Leishmania spp. 349

promastigotes release MSP using a model of in vivo conditions. The Matrigel™ matrix contains 350

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laminin, collagen IV, entacin, heparin sulfate proteoglycan, growth factors, collagenases, and 351

other undefined components. We demonstrated here that a combination of this matrix and 352

mammalian body temperature is sufficient to stimulate internal MSP release. We suggest that 353

the mechanism of MSP release in mammalian tissue differs from release in promastigote axenic 354

culture. Whether this reflects differences between MSP trafficking in the sand fly versus the 355

mammalian hosts is not clear. 356

The goal of the current study was to address how the three MSP subpopulations (surface, 357

internal and released) are regulated during metacyclogenesis, and in response to the mammalian 358

host environment. A model for MSP regulation in the different promastigote environments is 359

illustrated in Fig. 5. In this model MSP is abundantly released by the dividing, procyclic 360

promastigotes in the sand fly gut, as simulated by logarithmic growth of L. chagasi in culture. 361

This released MSP might be related to the nutrient requirements of Leishmania in the insect gut 362

environment, where residual mammalian blood from the sand fly meal is a main source of 363

nutrients. Indeed, it has been shown that down-regulation of MSP in L. amazonensis reduces the 364

parasites’ early development in sand flies (16). As procyclic promastigotes develop to 365

metacyclic promastigotes, the rate of released surface-localized MSP decreases and the 366

abundance of surface-localized MSP increases (47). Our data suggest this increase is due to 367

association of metacyclic MSP with lipid-containing membrane domains. Internal MSP is not 368

released during metacyclogenesis. However, after inoculation into mammalian subcutaneous 369

tissue by a sand fly vector, metacyclic promastigotes encounter a temperature increase, host 370

extracellular matrix, and innate immune mechanisms such as complement, antimicrobial peptides 371

and phagocytotic cells. In response to these stimuli, promastigotes could release internal MSP 372

into mammalian tissue. It is thus logical to consider the possibility that the surface-localized 373

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MSP plays a role in the promastigotes’ evasion of complement-mediated killing and their 374

phagocytosis/internalization by macrophages and other cells. Internal MSP, on the other hand, 375

may play a role in the degradation of extracellular matrix components such as collagen IV and 376

fibronectin, as suggested in a prior report on an L. amazonensis (25). As such, it could facilitate 377

promastigote migration toward cells such as macrophages, dendritic cells and fibroblasts that are 378

favorable for parasite entry and long-term survival. Thus, it is likely that the many isoforms of 379

MSP protease facilitate parasite survival through different mechanisms in the diverse host and 380

vector environments encountered by the parasite. 381

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Acknowledgments 382

CY was supported by a Veterans’ Affairs Merit Review Entry grant and a Veterans’ 383

Affairs Merit Review grant. Other support for this work included NIH R01 grants AI32135 and 384

AI059451 (MEW and JED), NIH R01 grants AI045540, AI048822 and AI067874 (MEW), and 385

Merit Review and Gulf War RFA grants from the Department of Veterans’ Affairs (MEW). 386

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Parasitol. 139:173-183. 541

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Parasitol. 135:171-183. 544

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Figure Legends 548

Figure 1. Homogeneity of MSP expression in clonal lines of L. chagasi. Single clones were 549

selected from M199 plates prepared from virulent L. chagasi promastigote populations. After 550

expanding, total cell lysates of stationary phase promastigote clones were subjected to IEF (A) or 551

2-DE (B) and transfer to nitrocellulose membranes. MSP proteins were detected by 552

immunoblotting. (A) immunoblot of total cellular proteins separated by IEF in a lysate of 553

stationary phase cells from the uncloned parental population (lane 1) or from four representatives 554

from a total of 124 clones (lanes 2-5). (B) a representative of the 2-DE MSP profiles conducted 555

on two dozen individual clones. Eleven previously described MSP isoforms from uncloned 556

promastigotes are labeled for reference (47, 48). 557

Figure 2. Surface-localized MSPs of attenuated versus virulent L. chagasi are differentially 558

regulated. (A) Example of a pulse-chase experiment demonstrating the rates of surface or 559

internal MSP loss from virulent L. chagasi promastigotes. Cultured promastigotes on day 3 (L) 560

or day 7 (S) of in vitro growth were metabolically labeled with [35S]-methionine + -cysteine and 561

surface biotinylated, followed by a chase without radioactivity for up to 72 h. Biotinylated 562

surface proteins were separated from the nonbiotinylated internal proteins using streptavidin 563

beads, and internal MSP was immunoprecipitated from bead-cleared fractions. Autoradiograms 564

are shown. The 66-kDa isoform seen at 0 h in the nonbiotinylated samples is most likely MSP 565

containing its propeptide (23). The T½ of the 63-kDa surface biotinylated MSP was determined 566

by a linear regression of f(x)=a+bx, using densitometric analysis of the bands. Graphic results 567

shown are the average ± SD of densitometric measurements of four independent experiments. 568

*P<0.01 by paired student t-test. (B) Rates of MSP synthesis in attenuated L5 versus virulent L. 569

chagasi were measured during logarithmic (L) or stationary (S) phase growth were determined 570

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by metabolically labeling promastigote proteins with [35S]-methionine + -cysteine and 571

immunoprecipitation with polyclonal antisera to MSP or P36. The ratio of MSP to P36 was 572

standardized to virulent stationary phase promastigotes, which was at one arbitrary unit. Data 573

shown are representative of two independent experiments. (C) Release of surface-localized MSP 574

into the extracellular medium. Attenuated L5 or virulent L. chagasi promastigotes during 575

logarithmic (L) or stationary (S) phase growth were surface biotinylated and resuspended in 576

fresh medium. Extracellular medium was collected 48 h later. MSP was detected in the 577

streptavidin-bead pull-down fraction by western blotting. Shown is the relative MSP abundance 578

standardized to stationary phase virulent promastigotes. One of two independent experiments is 579

shown. 580

Figure 3. MβCD enhances a dose-dependent release of surface-localized MSP. (A and B) 581

Virulent promastigotes on different days of growth were treated with the indicated concentration 582

of MβCD (0-15 mM) for 48 h. Filtered supernatants and cells were subjected to SDS-583

polyacrylamide gel electrophoresis. MSP was detected by immunoblotting. One of three 584

independent experiments, each of which was conducted in triplicate, is shown. (A) Extracellular 585

(E) and cellular (C) MSP content in parasites incubated with the indicated MβCD concentration 586

are shown. (B) The abundance of extracellular MSP in the medium of cells shown in panel (A) 587

was quantified by densitometric analysis. Values represent the percentage ([extracellular/total 588

MSP] × 100%) for each concentration. Statistical analysis on the 0 versus 10 or 15 mM MβCD: 589

*, p<0.05; **, p<0.01. (C) MβCD effect on total incorporation of [35S]-methionine + -cysteine 590

into newly synthesized proteins. MβCD-treated cells were metabolically labeled and newly 591

synthesized total protein was monitored as described in Materials and Methods. Values 592

represent the total incorporation of radioisotope into the newly synthesized proteins of treated 593

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parasites relative to the controls (0 mM MβCD). One of two independent experiments, with 594

three replicate conditions, is shown. (D) MβCD treatment enhances release of surface-localized 595

MSP into the extracellular medium. Stationary phase virulent L. chagasi promastigotes were 596

surface biotinylated in HBSS and incubated for 3 h at RT in the absence (0 mM) or presence (15 597

mM) of MβCD, as described in Materials and Methods. Biotinylated (S) and nonbiotinylated (I) 598

MSPs were isolated from filtered spent-medium with streptavidin beads and 599

immunoprecipitation, respectively. MSP was detected by immunoblotting. To monitor for cell 600

lysis, the cytosolic protein P36 was measured in the cleared fraction post biotin-streptavidin 601

affinity and MSP immunoprecipitation by immunoblot. Total cell lysates (cell) were included. 602

One of three independent replicate experiments is shown. 603

Figure 4. Release of internal MSP. (A) Stationary phase promastigotes in the first passage after 604

isolation from hamsters were incubated in HBSS (control, 3 h) or the Matrigel matrix for 1 or 605

3 h after surface-biotinylation at either 37oC or RT. After transfer to 4oC, parasites were 606

removed by centrifugation. Streptavidin beads were used to isolate biotinylated surface MSP 607

from both the whole cell lysate and the extracellular medium. The internal MSP was purified 608

from streptavidin-bead cleared fractions by immunoprecipitation, and was detected by western 609

blotting. Each condition was performed in triplicate. (B-C) Quantitation of released and internal 610

MSP. The band intensity of the extracellular (B) and internal (C) fractions for each experiment 611

in (A) was determined by densitometric analysis, and the average and standard deviation of the 612

triplicate samples were determined. Results from one of three experiments are presented. 613

Figure 5. A model of MSP release and possible functions during metacyclogenesis in the sand 614

fly vector and inoculation into the mammalian hosts. Solid and open circles represent surface-615

localized and internal MSP, respectively. Arrows show MSP being released into the 616

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extracellular environment. The width of the arrow is proportional to the amount of released 617

MSP. In the sand fly panel, the upper diagram depicts MSP release from procyclic (logarithmic 618

growth phase) promastigotes and the lower diagram depicts release from their metacyclic 619

(stationary phase) counterparts. In the mammalian host, a metacyclic promastigote is depicted. 620

MØ: macrophage. 621

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