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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,1 Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 405362
Received 31 May 2007/ Accepted 12 June 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-PO4)]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2– L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2–, instead resembling the L. major lipophosphoglycan-deficient lpg1– mutant. Metacyclic lpg5A–/lpg5B– promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A–/lpg5B– mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2– Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leishmania synthesizes a variety of abundant glycoconjugates implicated in various steps of the infectious cycle (17, 39). These include lipophosphoglycan (LPG); glycosylphosphatidylinositol (GPI)-anchored proteins, including proteophosphoglycan (PPG) and glycoprotein 63 (gp63) (leishmanolysin); glycosylinositolphospholipids (GIPLs); and inositolphosphoceramide (21, 56, 63, 66). Notably these glycoconjugates share many structural motifs or domains (Fig. 1A). For example, the GPI anchors are common to LPG, proteins, and small surface glycolipids (Fig. 1A) (12). Additionally, the phosphoglycan [Gal(ß1,4)Man(1-PO4)] disaccharide-phosphate repeating units (PG repeats) modify a variety of surface and secreted proteins and comprise the major portion of LPG, which contains 15 to 30 PG repeats (21, 56, 60). Thus, while studies carried out on purified PGs point to important roles such as modulating host signal transduction, inhibiting phagolysosomal fusion, and mediating oxidant resistance (reviewed by in reference 8), the structural similarity among these complex molecules often leads to imprecision in our understanding of their unique and/or overlapping roles in vivo.
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However, studies of the lpg2– mutant of L. mexicana, which is similarly deficient in PG synthesis but otherwise unaltered, showed that it retained amastigote virulence and the ability to induce acute pathology (22). The lpg2– L. mexicana phenotype raised the possibility that Leishmania species differed in their reliance upon PGs for virulence or that an LPG2-dependent molecule other than PGs played a critical role in L. major but not L. mexicana virulence (57). Studies of Man biosynthetic enzymes pointed to a role of Man-containing glycoconjugates in L. mexicana virulence, although the broad effects of these mutants through LPG2-independent routes, including dolichol-mediated protein N glycosylation (Fig. 1B), made it problematic to attribute the effects to any specific "virulence" glycoconjugate (14, 54). However, recent data have more directly implicated abundant cytoplasmic mannans, synthesized via gluconeogenesis, in Leishmania amastigote survival (38, 44). Due to their cytoplasmic localization, mannans are unlikely to be affected by the loss of L. major LPG2, a supposition confirmed in this work.
Given the complexity and involvement of the Man synthetic pathway in general glycoconjugate synthesis, we turned our focus to galactose, the second monosaccharide within the basic PG repeating unit (Fig. 1A). While in Leishmania galactose (Gal) can be obtained by salvage or through the epimerization of UDP-Glc in the glycosome (46, 58), our interest in the PG assembly and secretion via the Golgi apparatus prompted a strategy centered on this compartment. We recently described the characterization of the family of 12 Leishmania NSTs and functional studies of the LPG5A and LPG5B genes, which encode UDP-Gal transporters whose functions partially overlap (3). Notably, an lpg5A–/lpg5B– double gene mutant completely abrogated UDP-Gal uptake into the Golgi apparatus, as this mutant lacked LPG and protein-linked PGs. Thus, the lpg5A–/lpg5B– mutant provides an independent perspective from which to study the role of PGs in L. major (Fig. 1B). The data presented here confirm a role for PGs in the initial establishment phase of infection of vertebrate macrophages, probably reflecting the loss of LPG (50), but in neither amastigote-mediated virulence nor acute pathology. These data lend support to the existence of a vital LPG2-dependent molecule, unrelated to PGs, required for amastigote virulence in L. major.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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lpg5A::HYG/lpg5A::PAC (designated lpg5A–), 
pg5B::BSD/pg5B::NEO (designated lpg5B–), lpg5A::HYG/lpg5A::PAC/pg5B::BSD/pg5B::NEO (designated lpg5A–/lpg5B–), lpg5A–/lpg5B– [pIR1SAT-LPG5B-LPG5A] (designated lpg5A–/lpg5B–/+LPG5B+LPG5A), pg1::HYG/pg1/::PAC (designated lpg1–), lpg2::HYG/lpg2::HYG (designated (lpg2–), and lpg2–/Rev mutants were described previously (3, 50, 52, 53). A second "add-back" line was generated by transformation of the lpg5A–/lpg5B– line with SwaI-digested pIR1-SAT-LPG5B-LPG5A (strain B5081); this results in integration of the construct within the gene encoding the rRNA small subunit, which confers stable, strong, and uniform expression. The formal name of this line is lpg5A–/lpg5B–/SSU::IR1SAT-LPG5B-LPG5A. Prior to study, all lines were passed through mice once by injecting hind footpads of BALB/c mice (Charles River Laboratories, Wilmington, MA) with a large inoculum (1 x 107 to 5 x 107) of stationary-phase parasites and recovering parasites by needle aspiration of the footpad regardless of pathology 4 to 6 weeks afterwards. Cultures were identified by the number of times that they had been inoculated into mice (M1, M2, etc.) and the number of times passed in vitro after recovery from infected animals (P1, P2, etc.). As L. major may lose virulence during in vitro culture, parasites were passed no more than six times prior to use. (e.g., M1P6). Dulbecco modified Eagle medium (DMEM) was purchased from Gibco BRL (under Invitrogen, Carlsbad, CA). Anti-mouse immunoglobulin G (IgG):fluorescein isothiocyanate (FITC) and anti-mouse IgM:Texas red antibodies were from Jackson Immunoresearch (West Grove, PA). Hygromycin B was from Calbiochem (San Diego, CA), puromycin was from Sigma (St. Louis, MO), G418 powder was from BioWhittaker (now under Cambrex Bio Science, Walkersville, MD), phleomycin was from InvivoGen (San Diego, CA), and nourseothricin was from Werner BioAgents (Jena, Germany). Hoechst 33342 nucleic acid dye was purchased from Molecular Probes (now under Invitrogen, Carlsbad, CA). Other reagents were purchased from Sigma or Fisher.
Mouse infections.
Female BALB/c mice (6 to 10 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). In a typical experiment, five mice per group were inoculated subcutaneously in the left hind footpad with 106 metacyclic or 105 amastigote stage parasites. Lesion thickness was measured using a Vernier caliper (Mitutoyo) and defined as the average difference in thickness between infected and uninfected hind footpads for each group of mice (55). Metacyclic promastigotes were prepared using the Ficoll gradient enrichment method (49), and lesion-derived amastigotes were recovered from infected lesions (
2-mm thickness) as described previously (53). Limiting-dilution assays were performed as described previously (26).
Macrophage infections. Starch-elicited peritoneal macrophages were recovered from BALB/c mice and then plated on glass coverslips (43, 50). Metacyclic parasites were opsonized with C5-deficient serum, resuspended in DMEM containing 10% fetal calf serum, and infected at multiplicity of infection of 10. Lesion-derived amastigotes were infected at a multiplicity of infection of 3 in DMEM containing 10% fetal calf serum (53). After 2 hours, the cells were washed extensively and overlaid with fresh medium, and thereafter medium was changed daily. At 2 hours, 1 day, 2 days, and 5 days postinfection, cells were fixed in 3.7% (vol/vol) formaldehyde in phosphate-buffered saline (PBS) and stained in 2 to 2.5 µg/ml Hoechst 33342 (in PBS) prior to scoring for intracellular parasites.
Western analysis. Western analysis was done as described previously (50). Briefly, 1 x 106 cells (or serial twofold dilutions thereof) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4% polyacrylamide stacking gel and 12.5% resolving gel) and transferred to nitrocellulose membranes. WIC79.3 antibody, which recognizes Gal-modified PG repeating units, was used at a dilution of 1:500 (6). The anti-gp63 antibody 235 was used at a 1:1,000 dilution (5). A polyclonal gp46 antibody (a kind gift from D. McMahon-Pratt) was used at a 1:20,000 dilution (31). Where necessary, blots were treated with Western Re-Probe (Genotech, St. Louis, MO) and probed with a monoclonal antitubulin antibody (Sigma) at a dilution of 1:50,000. Anti-mouse IgG:horseradish peroxidase and anti-rabbit IgG:horseradish peroxidase antibodies were from Amersham (now part of GE Healthcare, Piscataway, NJ), and chemiluminescence reagents were from Perkin-Elmer (Wellesley, MA).
Indirect immunofluorescence assay. TAT-1 antibody, which recognizes trypanosome tubulin, was a kind gift from K. Gull (61). Macrophages infected for 2 days with lesion-derived amastigotes were fixed for 1 minute in 3.7% formaldehyde in PBS, followed by permeabilization with ice-cold ethanol for 15 min on ice. The cells were rehydrated for 10 min in PBS, followed by sequential incubations with TAT-1 at a 1:2 dilution, anti-mouse IgG:TR at a 1:100 dilution, WIC79.3 at a 1:500 dilution, and anti-mouse IgG:FITC at a 1:100 dilution. Cells were washed with PBS between incubations. Cells were mounted in 50% (vol/vol) glycerol in PBS, sealed, and visualized on an Olympus AX70 fluorescence microscope.
Mannan extraction and analysis. Parasites were harvested at densities of 0.7 x 107 to 2.0 x 107 cells/ml and extracted for mannans as described elsewhere (44). Briefly, cells (2 x 109 to 5 x 109) were extracted in chloroform-methanol-water (1:2:0.8) for 2 h with sonication. The samples were centrifuged at 15,000 x g for 5 min, and the supernatant was collected, dried under a stream of nitrogen, and partitioned with water-saturated 1-butanol. The water phase (containing the mannans) was desalted by passage through a 1-ml column of AG50-X12 (H+) layered over AG1-X8 (OH–). The desalted mannans were dried with a Speedvac drier and then fluorophore labeled at the reducing ends with 8-aminonaphthalene-1,3,6-trisulfate and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) according to manufacturer's specifications (Glyko Inc., Novato, CA).
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RESULTS |
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Limiting-dilution assays were performed to assess whether the initial delay in lesion pathology reflected decreased parasite numbers (Fig. 4). These experiments showed that indeed parasite numbers were much less than WT parasite numbers for the lpg5A–/lpg5B– mutant, from 18- to 270-fold less when measured after 10 or 28 days, respectively (Fig. 4).
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lpg5A–/lpg5B– amastigotes are fully virulent. The ability of the lpg5A–/lpg5B– parasites to induce pathology following an initial delay could be explained by the need for these gene products during the initial establishment of macrophages, such as PGs (including LPG and/or PPGs), but not for survival as amastigotes thereafter, as seen previously for LPG with the lpg1– mutant (50). To test this idea, we purified lpg5A–/lpg5B– amastigotes from visibly progressing lesions (such as those shown in Fig. 2B or 3A) and used them directly in macrophage or mouse infections (Fig. 4).
In macrophage infections, WT amastigotes entered and replicated rapidly thereafter without any delay (Fig. 5A). Similar results were obtained with lpg5A–/lpg5B– amastigotes and with lpg1– amastigotes, used for comparison (Fig. 5A). In mouse infections, the lpg5A–/lpg5B– amastigotes induced lesions which appeared and progressed at the same rate as those induced by the WT (Fig. 5B). These data argued that like lpg1– and unlike lpg2– L. major (50, 51), lpg5A–/lpg5B– amastigotes are as virulent as WT amastigotes.
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To test this, macrophages were infected with amastigotes purified from lesions as described above and, after 2 days, were examined by indirect immunofluorescence with anti-PG monoclonal antibodies. Macrophages and parasites were visualized by phase microscopy and reactivity with an -tubulin antiserum (Fig. 6A to F). In macrophages infected by WT amastigotes, PGs were readily detected, not only on the parasite itself but also shed into the macrophage, as seen previously (Fig. 6G) (19, 50). In contrast, lpg5A–/lpg5B– amastigotes were devoid of anti-PG reactivity (Fig. 6H). As a control, we performed parallel tests with amastigotes from the lpg2–/Rev mutant, which similarly survives within macrophages despite the absence of PGs (Fig. 6I). Thus, the survival of the lpg5A–/lpg5B– amastigotes cannot be explained by activation of an alternative NST or other pathway leading to PG synthesis.
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Total cellular mannans were extracted from WT and lpg2– L. major and L. mexicana, fluorophore derivatized, and analyzed by FACE analysis (Fig. 8). WT L. mexicana mannans were highly polymerized, ranging upwards of 10 mannose residues, and were absent in a null mutant (gdmp–) lacking cytoplasmic GDP-Man pyrophosphorylase (Fig. 8A) (44). Notably, mannan levels were unaltered in the L. mexicana or L. major lpg2– mutants relative to the WT (Fig. 8A and B). Similar results were obtained with WT L. donovani and lpg2– mutants (data not shown). Interestingly, the degree of mannan polymerization in L. major (<10) was less than that in L. mexicana upon entry into stationary phase, and it declined further after 5 days in stationary phase (Fig. 7B). This would be consistent with a role for mannan in energy metabolism in the metacyclic as well as amastigote stage of Leishmania parasites (44). Regardless, the equivalence of mannan levels in WT and lpg2– comparisons within both Leishmania species makes it unlikely that mannans contribute to LPG2-dependent virulence effects.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We ruled out the possibility that the amastigote virulence phenotype arose through second-site alterations not involving the LPG5A and LPG5B genes (Fig. 2B) or that the lpg5A–/lpg5B– parasites possessed amastigote-specific UDP-Gal NSTs or other alternate pathways leading to PG synthesis that could potentially bypass the lpg5A–/lpg5B– mutant (Fig. 6) (3). Thus, in contrast to the situation for promastigote virulence, we conclude that PGs are not essential for amastigote virulence in L. major, as suggested previously for L. mexicana (22).
Our findings were also consistent with previous work on a second-site revertant of L. major lpg2–, lpg2–/Rev, which, like the lpg5A–/lpg5B– mutant, lacked PGs and showed initial macrophage establishment phase defects as promastigotes but otherwise retained amastigote virulence in mouse infections (53). As yet the nature of the second-site mutation in the lpg2–/Rev mutant and its consequences for glycoconjugate synthesis (if any) have not been determined.
These data suggest that there is a PG-independent, LPG2-dependent pathway required for amastigote virulence specifically in L. major. Given the known role of LPG2 in Golgi nucleotide sugar transport, it seems most likely that a deficiency of some LPG2-dependent glycoconjugate underlies the virulence phenotype. Since we have now ruled out all of the known major glycoconjugate candidates of L. major, it seems likely that an uncharacterized, and most likely nonabundant, glycoconjugate is responsible. The intense study that the Man-containing glycoconjugate pathway has received experimentally could be viewed as arguing against the involvement of a Man-containing glycoconjugate (14, 44). Nonetheless, when new methods and approaches are applied, new glycoconjugates may emerge, as exemplified by the recent discovery of the abundant cytoplasmic mannan that may play an important role in amastigote virulence (44). However, we have shown here that mannan levels exhibit no LPG2-dependent changes in either L. major or L. mexicana (Fig. 7), as anticipated, eliminating this cytoplasmic molecule as a candidate (44). While GDP-Man synthetic mutants of L. mexicana are avirulent, their lack of general dolichol-mediated protein N glycosylation (Fig. 1B) and structural abnormalities make it difficult to attribute virulence defects to any specific glycoconjugate (14, 54). Thus, the possibility that undiscovered, less-abundant LPG2-dependent Man-containing glycoconjugates essential for L. major amastigote virulence exist cannot be excluded.
LPG2 is a multispecific GDP-sugar transporter with specificities for both GDP-D-Arap and GDP-L-Fuc in addition to GDP-Man (20, 28). Potentially the critical role of LPG2 involves these sugars, most likely arabinose (Ara), since Fuc has not been reported in L. major (48). In L. major, two D-Arap transferases mediating terminal arabinosylation of Gal-modified PG repeats of metacyclic promastigotes have been identified, SCA1 and SCA2 (10). The terminal D-Arap substitutions disrupt binding of LPG to the sand fly midgut lectin, allowing detachment of L. major promastigotes and subsequent transmission (23, 30, 41). We believe that the general lack of PG dependency suggests that it is unlikely that Ara-containing or other modifications of the PG repeating unit account for LPG2-dependent amastigote virulence, although arabinosylation of other, as-yet-unknown glycoconjugates remains a possibility. Interestingly, Ara-containing glycolipids have been reported previously in L. donovani (62); however, their role in virulence has not been investigated.
In recent work we have shown that lpg2– L. donovani (16) also shows decreased virulence in both mouse and hamster infections (M. Wilson and S. M. Beverley, unpublished data). These findings raise the important question as to why L. mexicana does not show the same LPG2 dependency for amastigote virulence as in L. major and L. donovani, despite the extensive similarities in the structures of known LPG2-dependent glycoconjugates. Potentially L. mexicana may synthesize novel glycoconjugates and/or other molecules that fulfill this role in the absence of LPG2. Alternatively, the molecules required for amastigote virulence may differ between the species due to differences in the parasite biology, with one example being the natures of the parasitophorous vacuoles formed by L. mexicana versus L. donovani and L. major (4). The availability of well-characterized mutants of all three species should greatly facilitate the resolution of this important question in the future.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant AI 31078 to S.M.B. and S.J.T.
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FOOTNOTES |
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Published ahead of print on 2 July 2007.
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Glc-6-P
), and lpg5B– (
) promastigotes. (B) Promastigotes that had been differentiated from lpg5A–/lpg5B– lesion amastigotes (M2P4) (
) were used to infect naïve mice and compared to the WT (•) and the lpg5A–/lpg5B– mutant (M1P3) (
) infection showing amastigote survival. Peritoneal macrophages were infected with three amastigotes/macrophage, and survival was monitored over time as described in Materials and Methods. Symbols correspond to WT (•), lpg1– (
), and lpg5A–/lpg5B– (
