Original Article |
Address correspondence to Alfredo Toraño, Servicio de Inmunología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, E-28220 Madrid, Spain. Phone: 34-91-509-7973; Fax: 34-91-509-7966; E-mail: atorano{at}isciii.es
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Abstract |
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Key Words: Trypanosomatids Leishmania complement opsonization promastigote lysis human serum
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Introduction |
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During Leishmania host invasion, complement-mediated promastigote killing can compromise parasite survival. Identification of promastigote opsonization by host serum is thus essential to understanding Leishmania infection strategy. Pioneering studies on promastigote opsonization in normal human serum (NHS) indicated that IgM anti-Leishmania antibodies were responsible for promastigote agglutination, classical complement pathway (CP) activation, and parasite killing (5, 6). Despite these data, understanding of the promastigote opsonization mechanism has to date been dominated by the concept that Leishmania spp. promastigotes activate complement in NHS through the alternative pathway (AP), thus lacking antibody involvement (79). Exceptions to this rule have been reported for Leishmania donovani promastigotes (8) and axenic metacyclic peanut agglutinin-negative forms of Leishmania major (10), but the view prevails that leishmanias activate complement via the AP (11, 12).
In addition to promastigote-C3 opsonization by the classical and alternative routes, it is also reported that two specific carbohydrate-binding proteins in serum, mannan-binding lectin (MBL) and the acute phase protein C-reactive protein (CRP), bind Leishmania parasites (1315); they thus could initiate promastigote opsonization through a novel antibody- and C1-independent mechanism, the lectin-mediated pathway.
To clarify this issue, we performed a comprehensive quantitative and kinetic analysis of promastigote opsonization in NHS in near-physiological conditions using promastigote cell binding assays, high opsonizing serum concentrations (25100%), and short incubation times (3 min). The results indicate that binding of natural IgM anti-Leishmania antibodies (NAb) to conserved trypanosomatid epitopes triggers C3 parasite opsonization, and that serum collectins (MBL) and pentraxins (CRP) do not participate significantly in complement activation. In NHS, promastigotes activate complement CP and AP simultaneously, but >85% of promastigote-bound C3 is generated through the CP, indicating that physiological C3 opsonization of Leishmania is activated through the CP in a natural infection. In the early infection period, promastigote lysis by complement parallels the course of C3 deposition (2). As real-time data on this mechanism were lacking, we measured real-time kinetics of promastigote killing in 50% NHS, and show that from 8595% of stationary culture promastigotes become permeable to propidium iodide in <3 min after serum contact. Human infection by Leishmania is thus an extremely rapid process, and promastigotes must display evasion strategies immediately after inoculation to avoid lysis by complement.
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Materials and Methods |
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Antibodies and Human Sera.
Blood from healthy donors was allowed to clot in siliconized glass tubes (20°C, 30 min), and serum aliquots stored in liquid nitrogen. Clinical and genetic data of sera from patients with hereditary deficiencies in complement factors C1q (C1qDS) and C2 (C2DS) have been reported elsewhere (16, 17). Antihuman C3 chain mAb SIM2749, IgG2b, developed in our laboratory, was purified from mouse ascites fluid by HiTrap-protein A treatment. NHS was adsorbed (Ads-NHS) for 30 min on ice with L. donovani or L. amazonensis promastigotes at a ratio of 1 ml of 50% PBS-diluted NHS:109 pelleted promastigotes. Ads-NHS was centrifuged (11,000 g, 3 min), and the supernatant readsorbed in two additional cycles; finally, serum was filtered through a 0.22-µm pore membrane to remove aggregates and used in functional assays. IgM was isolated from NHS by affinity chromatography on a protamine-Sepharose CL4B column by end-over-end mixing of 20 ml 50% H20-diluted NHS with 10 ml packed protamine-Sepharose beads (4 h, 20°C). After incubation, the column was washed and eluted as described (18). The IgM-enriched fraction was dialyzed against PBS, filtered to remove aggregates, stored at 4°C, and used within 24 h. NHS IgG was isolated on a HiTrap Protein G column (Amersham Pharmacia Biotech). Protein content was determined by BCA assay (19). SIM2749 (25 µg) was labeled with 5 µl of Na 125iodine (carrier-free, 105.36 mCi/ml; Dupont/NEN Life Science Products) in iodogen (Pierce Chemical Co.)-coated tubes as described (2). The [125I]SIM2749 immune reactive fraction (IRF) was measured by titrating a fixed, limiting amount of radiolabeled antibody against increasing concentrations of NHS-opsonized Leishmania promastigotes, until promastigote-bound C3 epitopes greatly exceeded [125I]SIM2749 paratopes. The antibody IRF was calculated by direct linear plot (20).
Kinetics of Complement Activation by Leishmania, Crithidia, and Phytomonas Promastigotes in NHS and Mg-EGTA-treated NHS.
50 µl of a 2 x 108/ml promastigote suspension were mixed with 50 µl of 50% NHS or 50% NHS-adjusted to 10 mM EGTA, 7 mM MgCl2 (Mg-EGTA-NHS), and incubated for varying time periods. The reaction was terminated by adding cold PBS containing 2.5% FCS and 0.05% NaN3 (PFS) and the parasites washed twice by centrifugation (11,000 g, 1 min). The cell pellet was resuspended in 200 µl of PFS containing 2 x 105 cpm of [125I]SIM2749 (specific activity 107 cpm/µg) and incubated 1 h on ice. Promastigotes were then washed twice by centrifugation as above, and bound [125I]SIM2749 cpm determined. A t50 index (time required for each species to reach 50% maximum C3 binding) was calculated from a plot of percent-normalized promastigote-C3 binding against incubation time.
Complement Activation by Leishmania in C1q- and C2-deficient Sera.
Duplicate 50 µl samples of a 2 x 108/ml L. donovani promastigote suspension were incubated at 37°C with 50 µl of 50% diluted NHS, 50% diluted Mg-EGTA-treated NHS, C1qDS, or C2DS, for 0, 0.5, 3, 5, 7, and 10 min. Conditions for L. amazonensis assay were identical, but to compensate for activity loss during long-term storage, C2DS was used at 60% concentration. Purified C1q (125 µg/ml) and C2 (47,500 CH50 units) were supplemented. After incubation, assay conditions were as described above for complement activation kinetics. Finally, bound [125I]SIM2749 cpm were determined.
Complement Activation by Leishmania in Ig-supplemented Ads-NHS.
IgM and IgG preparations devoid of complement activity were purified as described above. All IgM and IgG preparations used showed promastigote binding 100% of that observed for 25% NHS. L. donovani and L. amazonesis promastigote triggering of complement activation was analyzed in a two-step C3 binding assay. Pelleted L. donovani promastigotes (107) were resuspended in 100 µl of 25% NHS adjusted to 10 mM EDTA (NHS-EDTA), purified IgM adjusted to 10 mM EDTA (IgM-EDTA), or purified IgG adjusted to 10 mM EDTA (IgG-EDTA), and incubated (37°C, 30 s). Promastigotes were then washed twice by centrifugation (11,000 g, 1 min). Promastigote pellets preincubated in NHS-EDTA were resuspended in 100 µl of 25% NHS (positive control) or 25% L. donovani Ads-NHS; promastigotes preincubated in IgM-EDTA or IgG-EDTA were resuspended in 100 µl of 25% of L. donovani Ads-NHS. Tubes were incubated (2 min, 37°C), followed by two centrifugation washes (11,000 g, 1 min). Promastigotes were resuspended in 0.2 ml PFS containing 2 x 105 [125I]SIM2749 cpm and incubated (1 h, on ice). After two further washes, bound cpm were determined. Control samples for classical and alternative complement pathway activation were not preincubated. To study CP activation, 107 pelleted L. donovani promastigotes were resuspended in 100 µl of 25% NHS, 25% Mg-EGTA-NHS (NHS-EGTA), 25% purified IgM, 25% purified IgG, or 25% L. donovani-Ads-NHS, and incubated (2 min, 37°C). For AP activation, promastigotes were resuspended in 100 µl of 25% NHS, 25% NHS-EGTA, or 25% L. donovani-Ads-NHS, and incubated (15 min, 37°C). Samples were washed twice by centrifugation, promastigotes resuspended in 0.2 ml of PFS containing 2 x 105 cpm [125I]SIM2749, incubated 1 h on ice, and processed as above. NHS complement activation by L. amazonensis was measured similarly using L. amazonensis-Ads-NHS.
Quantitation of CP-activated Promastigote-C3 Binding during In Vitro Metacyclogenesis.
Cultures were seeded with 105 L. amazonensis promastigotes/ml and cultured (27°C, 17 d). Promastigotes were sampled at days 3, 4, 5, 6, 7, 10, 11, 14, and 17, washed twice by centrifugation in PBS, and opsonized (108 promastigotes/ml in 25% PBS-diluted NHS) at 37°C for 2.5 min. Promastigotes were washed, added to duplicate tubes at concentrations of 0.625, 1.25, 2.5, 5, and 10 x 106, adjusted to 107/0.1 ml with nonopsonized promastigotes, and incubated (2 h, 0°C) with a limiting amount of [125I]SIM2749. After incubation, promastigotes were washed twice by centrifugation and bound [125I]SIM2749 cpm determined. Background cpm ([125I]SIM2749 cpm bound to nonopsonized promastigotes) were subtracted. A single lot of [125I]SIM2749 (specific activity, 6 x 106 cpm/µg) was used for all experiments. Kd values were calculated by direct linear plot from data of three independent cultures.
To measure CP-activated C3 binding to stationary promastigotes of Leishmania and Crithidia, parasites (108 cells/ml) were incubated in 25% PBS-diluted NHS (37°C, 3 min). The reaction terminated by dilution with PFS (4°C), and C3-opsonized promastigotes washed twice by centrifugation (1,500 g, 15 min). Two duplicate series of tubes containing 105 and 2 x 105 C3-promastigotes were adjusted to 5 x 106 promastigotes/tube with nonopsonized promastigotes. A third series of control tubes contained 5 x 106 nonopsonized promastigotes. Leishmanias were pelleted and resuspended in 0.2 ml PFS containing increasing concentrations of [125I]SIM2749, until a paratope/ligand ratio of 10 was obtained. Samples were incubated on ice to equilibrium (
3 h). After reaction, parasites were washed twice by centrifugation (11,000 g, 1 min) in PFS and C3-bound [125I]SIM2749 determined. Functional [125I]SIM2749 was calculated as input cpm x IRF. SIM2749 Kd and the number of C3 molecules/promastigote were calculated by direct linear plot and Scatchard analysis (21, 22).
Real-time Kinetics of Leishmania Promastigote Lysis in NHS.
Promastigote cultures of L. major, L. amazonensis, L. donovani, and L. infantum were seeded in triplicate at 106 cells/ml, and cell growth registered daily. Parasites were sampled at mid-log and early stationary growth phase, i.e., 2 d after the end of log-phase growth, and real-time promastigote lysis in 50% pooled NHS was analyzed by measuring propidium iodide (PI) uptake by killed promastigotes in a FACSCaliburTM flow cytometer (Becton Dickinson). The reaction was initiated by addition of 100 µl of undiluted 0.22 µm porefiltered NHS into a tube containing 105 promastigotes, 2 µl PI (0.5 mg/ml; Sigma-Aldrich) and PBS (200 µl final reaction volume), and incubated in a 37°C waterbath during the data acquisition period. Promastigotes were identified and gated at a forward-angle light scatter versus side-angle light scatter. PI emission was collected in the FL2 detector through a 585/42 nm band pass filter. Detector amplification was set to include untreated promastigotes (negative control) between 100101 intensity. Promastigotes were acquired at a ratio of 250 events over a period of 204.8 s, with a data acquisition interval of 200 ms, and analyzed with CELLQuestTM software (Becton Dickinson). Promastigote PI uptake kinetics was analyzed in a FL2 versus time dot-plot divided into thirteen 15.98 s regions. The percentage of promastigotes incorporating PI over incubation time was quantitated for each region as number of events emitting PI fluorescence >101/total event number (PI emitting plus nonemitting promastigotes). Controls included (a) maximum cell lysis in promastigotes treated (30 min) with acetone:methanol (1:1), (b) pooled NHS, and (c) nonspecific promastigote lysis in 10 mM EDTA-chelated 50% NHS.
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Results |
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Complement Activation Mechanism by Leishmania Promastigotes in NHS.
Complement activation by the CP is Ca2+ and Mg2+ ion-dependent, whereas AP activation requires only Mg2+. Differential Ca2+ chelation in Mg-EGTA-treated NHS thus permits identification of the activating pathway (23). To confirm that rapid and slow promastigote-C3 deposition kinetics in NHS are triggered by the CP and the AP, respectively, the C3 binding rate to L. donovani and L. amazonensis promastigotes in NHS or Mg-EGTAtreated NHS was compared with that in sera of individuals congenitally deficient in C1q or C2 early CP factors. L. donovani is a very slow AP activator, allowing clear separation between the CP and AP C3 binding courses (Fig. 1 E). In NHS, C3 deposition on L. donovani promastigotes reaches a plateau after 3 min (Fig. 2 A), whereas the C3 binding rate is slower in C1qDS and C2DS, similar to the course followed in Mg-EGTAtreated NHS. After 3 min, the percentage of promastigote-C3 binding in C1qDS and C2DS is only 15.6 and 10.5%, respectively, of that observed in NHS. In contrast, promastigotes incubated in C1qDS and C2DS supplemented with purified C1q and C2 factors, respectively, recover CP C3 binding kinetics and deposit 70.5% (in C1q-supplemented C1qDS) and 72.4% (C2-supplemented C2DS) of total C3 bound in NHS. L. amazonensis is a rapid AP-activating species, and the course of CP and AP C3 binding are closer to each other in time than in L. donovani (Fig. 1 D). Analysis of L. amazonensis promastigote-C3 deposition kinetics in C2DS, and after C2DS supplementation with C2 factor, shows results similar to those in L. donovani (Fig. 2 B). These data indicate that irrespective of the species analyzed, Leishmania promastigotes require early CP factor activity for rapid C3 binding kinetics in NHS.
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Real-time Kinetics of Leishmania Promastigote Lysis in NHS.
Promastigotes of four Leishmania species were obtained from axenic cultures in log phase and the early stationary phase, and kinetic data on complement-mediated lysis determined by measuring PI-stained cells. Dot plots show real-time kinetics of log phase (Fig. 3 A, ad) and early stationary phase (Fig. 3 A, eh) promastigote killing in 50% NHS, as well as controls (Fig. 3 A, ik). Log phase leishmania begin to incorporate PI within 30 s of serum contact, and all cells are PI-labeled by 60 s (Fig. 3 B, top panel). Log phase promastigotes are thus highly susceptible to human complement. In contrast, PI uptake kinetics by early stationary phase promastigotes is slower; it begins within 3060 s of serum contact, and is complete by 2.5 min (Fig. 3 B, bottom panel). Stationary phase promastigotes show greater interspecies variability in PI uptake kinetics and percent killing (Fig. 3 B, bottom panel) than log phase promastigotes. In stationary phase L. major and L. amazonensis promastigotes, 15 and 10% of the population, respectively, show a degree of complement resistance (Fig. 3 A, ef). The precise size of this population is difficult to calculate as, in the presence of NHS, promastigote cell volume and refractile properties are altered, blurring the distinction between promastigotes, cell debris, and NHS background signal. Stationary phase L. donovani and L. infantum promastigotes appear to be more complement susceptible (Fig. 3 A, g and h), and no motile promastigotes were observed under the microscope. Lysis of log and early stationary phase promastigotes is gradual in all four species (Fig. 3 A, ag); the lytic mechanism differs for L. infantum stationary phase promastigotes in that the entire promastigote population undergoes sudden, simultaneous death, with no intermediate stages (Fig. 3 A, h). These results indicate that in NHS, Leishmania promastigote lysis is an extremely rapid reaction.
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Discussion |
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In 25% NHS, promastigotes of all trypanosomatid species studied activate classical and alternative complement pathways simultaneously (Fig. 1). Promastigote-C3 deposition is extremely rapid; the reaction is complete after 2 to 3 min, at which time 8693% of fixed C3 has been activated through the CP. The contribution of the AP to promastigote opsonization is much smaller, ranging from 7.113.7%. Promastigote C3 binding is traditionally measured after 15 min incubation (9, 10) or longer (3060 min) (7, 8, 11, 12, 28). CP C3 deposition can be detected only in a narrow time window that lasts but a few minutes (Fig. 1); after this time, classical and alternative pathway kinetics merge, and only AP-dependent activity is observed. This may explain why promastigote CP C3 deposition has passed unnoticed.
AP-mediated C3 deposition has a slower time course than the CP (Fig. 1). In AP triggering, no specific recognition mechanism is involved, and both the kinetics and the amount of C3 bound are directly related to the structure of the promastigote surface. The AP pathway permits differentiation of trypanosomatids by their C3 binding kinetics and APt50 values, disclosing the large instraspecies variability that exists within the subgenus Leishmania. Crithidia and Leishmania are genetically more distant than amphibians and mammals (29), yet larger AP kinetic differences are found among Leishmania species than among parasites of Leishmania, Crithidia and Phytomonas.
To identify the mechanism that triggers Leishmania opsonization in NHS, we used sera congenitally deficient in C1q or C2 complement factors. Promastigote CP C3 binding kinetics in C1q- or C2-deficient sera is restored by addition of purified C1q or C2, respectively (Fig. 2); this result supports the involvement of early CP factors in promastigote C3 opsonization. Parasite killing in C4- or C2-deficient sera (8, 12), or in normal serum depleted of natural anti-Leishmania antibodies (9, 11), has been taken as proof that this reaction is AP mediated. In these studies, earliest parasite lysis was measured after 15-min incubation, when AP-mediated promastigote killing is fully active. These experiments consequently provide no information on the role of the CP in the lytic mechanism, for which promastigote killing must be recorded in the early opsonization period (Fig. 1).
Candidate molecules in NHS to initiate parasite activation of the CP include anti-Leishmania NAb (2, 5, 6), MBL (13), and CRP (14, 15). Complement triggering was analyzed in a two-stage incubation assay that measures the ability of promastigote-bound IgM or IgG to initiate CP activity in Ads-NHS; this preparation lacks the ability to trigger CP C3 deposition. Promastigotes preincubated in NHS-EDTA or IgM-EDTA bind natural IgM anti-Leishmania antibodies; after a second incubation in Ads-NHS, they respectively deposit 88.8 and 88.6% of total C3 deposited in NHS (Table I). In contrast, promastigotes preincubated with IgG-EDTA fix only 17.8% of total C3 bound (Table I). Triggering of CP C3 deposition by calcium-chelated NHS and protamine-isolated IgM fractions (Table I) indicates that MBL and CRP do not participate in the reaction, as their activity is calcium-dependent (14, 30). A basic requirement for the physiological Leishmania opsonization mechanism in NHS is that C3 deposition must be complete within 23 min of incubation at 37°C. MBL and CRP are reported to bind to the Leishmania surface (1315), although these assays were done in far from physiological conditions (1 h in the cold). Average human plasma concentrations of MBL and CRP are 1,000- (10-10 M [31]) and 200-fold (
0.5 x 10-9 M [14]) lower, respectively, than that of natural IgM anti-Leishmania antibodies (
1.6 x 10-7 M [2]). It thus appears unlikely that they mediate the promastigote opsonization kinetics shown in Fig. 1. In chronic infections, MBL concentrations can increase 2- to 3-fold (32) and those of CRP up to 300-fold (33); MBL may thus act as an opsonin (34) and modulate disease progression (35).
Given sufficient time and suitable temperature conditions in vitro, Leishmania promastigotes can probably activate human complement via classical, alternative, and lectin pathways. Under quasi-physiological conditions, however, Leishmania opsonization is extremely rapid, and it is unlikely that MBL or CRP can account for this mechanism. We thus maintain that in NHS, natural IgM anti-Leishmania antibodies are the main trigger of CP activation and promastigote opsonization.
It has been proposed that parasite infective success relies on their capacity to establish multiple interactions with host cell receptors (36). The velocity of parasite opsonization and the massive C3 deposition suggest that promastigote-bound C3 has a pivotal role in the parasitehost interaction. Leishmania C3 binding is a prerequisite for promastigote IA (2, 3), receptor-mediated parasite binding to macrophages and endocytosis (3739), and intracellular Leishmania survival (40). To correlate C3 deposition levels with parasite infectivity, we analyzed promastigote-C3 binding during the L. amazonensis life cycle, and found that promastigotes maintain a stable, CP-mediated C3 binding capacity throughout in vitro metacyclogenenesis. Similar data, although measuring AP-mediated C3 deposition, have been reported for L. donovani, L. major, and L. amazonensis (911), although others found higher C3 binding to stationary promastigotes (37). Leishmania promastigotes in axenic cultures are believed to recapitulate the developmental sequence in the insect vector (41); promastigotes in the vector gut may thus behave as do the axenic promastigotes, and maintain invariant C3 binding capacity. Assuming that promastigote-bound C3 is responsible for receptor-mediated parasite binding to host cells and endocytosis, it is plausible that all C3-opsonized promastigotes egested from the sandfly gut into the blood pool have a similar probability of invading host leukocytes. In this case, the presence of infective metacyclic promastigotes would not be a necessary condition for infectivity.
Despite the abundant literature on promastigote killing by complement, time course studies of CP-mediated promastigote lysis at near-physiological conditions are lacking. Here we show real-time kinetic analyses of this mechanism (Fig. 3). Three aspects to be emphasized are the extreme velocity of the promastigote lytic reaction, the striking sensitivity of log phase Leishmania spp. populations to complement, and the differential complement susceptibility among stationary phase Leishmania spp promastigote species. In all four species tested, PI uptake by stationary phase promastigotes begins within 3060 s of serum contact and is complete by 2.5 min (Fig. 3 B, bottom panel), confirming previous results (2). L. major promastigotes incorporate PI more rapidly (Fig. 3 A, e), although this does not correlate with greater complement sensitivity. Using flagellar motility as a viability criterion, 15% (L. major) and 10% (L. amazonensis) of the promastigotes were viable after 2.5 min in 50% NHS. The data thus indicate that these species show a degree of complement resistance, and support the idea (24) that axenic Leishmania cultures give rise to developmental parasite forms that are resistant to human complement within the physiological infection period.
In contrast, L. donovani and L. infantum are highly sensitive to complement, which is also the case for promastigotes of all species tested in exponential growth (Fig. 3 B, top panel). From these data, we estimate that from 85% (stationary phase) to 100% (log phase) Leishmania spp. promastigotes are killed by complement after 2.5 min in human blood. To survive, Leishmania promastigotes must invade host cells within this period. Assuming a sandfly blood meal of 0.3 µl, and sandfly thoracic midgut promastigote body volume of 4 µm3 (42), each sandfly bite would inoculate at most 75 parasites. Estimating that at least 90% will be killed within 2.5 min, and that human blood phagocytes engulf promastigotes at a granulocyte:monocyte ratio of 2:1, in each bite, <4% of promastigotes inoculated (5 promastigotes) would enter a safe monocyte haven. It is thus clear that, during early stages of leishmaniosis transmission, serum opsonins exert very strong selective pressure on Leishmania, a mechanism that has presumably contributed to shaping the parasite's host evasion strategies.
Leishmania has been called a "parasite of paradox" for its ability to dwell in macrophages, the cell committed to its destruction (43); we would further justify this appellation. Complement protects vertebrates against protozoan invasion of the blood through the dual mechanisms of IA opsonic activity and the lytic cascade. Members of the Muridae family, such as the mouse and the hamster, have very low complement lytic activity against Leishmania, and may rely on IA opsonic activity to fend off infection (our unpublished data). In these genera, IA opsonic activity enhances promastigote phagocytosis and macrophage infection, which may explain the reservoir role of these rodents for Leishmania. In contrast, humans have a strong complement system and a potent IA mechanism; after inoculation, promastigote survival is linked to IA and endocytosis. We suggest that appropriation of IA-mediated opsonophagocytosis is the principal Leishmania strategy to evade the innate host response. Its capacity to transform the vertebrate innate protective response into the key to host invasion provides an additional motive for the sobriquet "parasite of paradox."
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Acknowledgments |
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This work was supported by grants 08.2/0006/97 from the Comunidad Autónoma de Madrid, PM99-0012 from the Programa Nacional de Salud (Ministerio de Educación y Cultura), and institutional funds from the Centro Nacional de Microbiología, Instituto de Salud Carlos III.
Submitted: July 31, 2001
Revised: December 11, 2001
Accepted: January 4, 2002
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Footnotes |
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References |
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