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Leishmania Major Infections in Mammalian Hosts

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Leishmania Major Infections in Mammalian Hosts The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts Gerald F. Spa¨ th*†, L. A. Garraway‡, Salvatore J. Turco§, and Stephen M. Beverley*‡¶ *Department of Molecular Microbiology, Washington University Medical School, St. Louis, MO 63110; ‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and §Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536 Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved June 6, 2003 (received for review January 30, 2003) The abundant cell surface glycolipid lipophosphoglycan (LPG) was the core GPI anchor domains have similarities with those present implicated in many steps of the Leishmania infectious cycle by in both GIPLs and GPI-anchored proteins (1, 5, 6). biochemical tests. The presence of other abundant surface or After vertebrate infection, infective metacyclic Leishmania secreted glycoconjugates sharing LPG domains, however, has led must resist the action of complement, attach and enter macro- to uncertainty about the relative contribution of LPG in vivo. Here phages, resist host defenses such as oxidants and hydrolytic we used an Leishmania major lpg1؊ mutant, which lacks LPG alone enzymes, inhibit macrophage activation, and differentiate to the and shows attenuated virulence, to dissect the role of LPG in the amastigote stage, which is adapted for long-term survival and establishment of macrophage infections in vivo. lpg1؊ was highly replication within an acidified phagolysosome. LPG has been susceptible to human complement, had lost the ability to inhibit implicated in many steps required for establishment of macro- phagolysosomal fusion transiently, and was oxidant sensitive. phage infections and for survival in the insect vector (3, 4, 7–10). Studies of mouse mutants defective in relevant defense mecha- LPG does not play a role in the amastigote stage, however, nisms confirmed the role of LPG in oxidant resistance but called because this parasite stage synthesizes little or no LPG and does into question the importance of transient inhibition of phagoly- not require LPG for virulence. However, amastigotes continue sosomal fusion for Leishmania macrophage survival. Moreover, the to make structurally related glycoconjugates (3, 4, 7, 9, 11). limited lytic activity of mouse complement appears to be an Typically, LPG roles were studied with purified LPG and ineffective pathogen defense mechanism in vitro and in vivo, sophisticated biochemical and cellular assays. However, con- unlike human hosts. In contrast, lpg1؊ parasites bound C3b and cerns have been raised: LPG may be applied in routes and resisted low pH and proteases normally, entered macrophages amounts that may not be physiologically relevant, and the sharing efficiently and silently, and continued to inhibit host-signaling or similarity of LPG domains to those of other parasite mole- pathways. These studies illustrate the value of mechanistic ap- cules discussed above raises the issue of specificity and the proaches focusing on both parasite and host defense pathways in possibility of cross-activity. For example, many of the functions dissecting the specific biological roles of complex virulence factors attributed to LPG above have been ascribed also to PPG, GIPLs, such as LPG. and͞or GPI-anchored proteins (3, 6, 9, 12–14). Thus, teasing out the specific contributions of LPG within the complex milieu of phosphoglycans ͉ trypanosomatid protozoan parasite ͉ oxidant the parasite glycocalyx remains a significant challenge. resistance ͉ inhibition of macrophage activation ͉ adhesin Here we describe studies of a Leishmania major mutant specifically lacking LPG, which is defective in its ability to infect sand flies, mice, and macrophages (11, 15). The lpg1Ϫ mutant he protozoan parasite Leishmania is an intracellular patho- was obtained by targeted inactivation of a putative galacto- gen that resides within an acidified phagolysosome of ver- T furanosyltransferase necessary for synthesis of the LPG glycan tebrate host macrophages and is transmitted by biting phleboto- Ϫ core; the lpg1 mutant was otherwise normal in PG, GPI- mine sand flies. Within the phagolysosome, the parasite must anchored proteins, GIPLs, and metacyclic gene expression (11, resist the hydrolytic environment and avoid macrophage activa- 16, 17). We also made use of mouse mutants defective in relevant tion. People infected with Leishmania can develop diseases host defenses to extend and confirm our findings on the role(s) ranging from mild to disfiguring to fatal, depending on the of LPG. Overall, the studies establish a role for LPG in many but species of parasite and the host factors. Current chemotherapy not all of the steps previously identified in macrophage invasion is inadequate, and although vaccination is thought to be feasible, and survival and, in some cases, raise questions about the no clinically effective vaccine exists. relevance of several aspects of the interaction of Leishmania with Leishmania promastigotes are covered with a dense surface its hosts assumed previously to be important. glycocalyx, composed largely of molecules attached by glyco- sylphosphatidylinositol (GPI) anchors (1). These GPI-anchored Materials and Methods molecules include proteins such as the parasite surface protease Leishmania Culture. L. major LV39 clone 5 promastigotes (Rho͞ gp63 and proteophosphoglycans (PPGs), as well as short GPI- SU͞59͞P) (18) were grown in medium 199 (M199) (19). The anchored glycosylinositolphospholipids (GIPLs). The most abundant constituent is a large GPI-anchored phosphoglycan called lipophosphoglycan (LPG) (2–4). In all Leishmania spe- This paper was submitted directly (Track II) to the PNAS office. cies, the GPI anchor of LPG is composed of a 1-O-alkyl-2- Abbreviations: GIPLs, glycosylinositolphospholipids; GPI, glycosylphosphatidylinositol; lysophosphatidylinositol lipid anchor and a heptasaccharide LPG, lipophosphoglycan; LPS, lipopolysaccharide; moi, multiplicity of infection; NMMA, core, to which is joined a long phosphoglycan (PG) polymer NG-monomethyl-L-arginine; PEMs, peritoneal exudate macrophages; PG, phosphoglycan; ␤ ␣ PPGs, proteophosphoglycans; RT, room temperature. composed of 15–30 [Gal 1,4Man 1-PO4] repeating units (sub- †Present address: Department of Medical and Molecular Parasitology, New York University stituted with other sugars in some species) and which is termi- School of Medicine, New York, NY 10010. nated by a capping oligosaccharide. These domains are shared ¶To whom correspondence should be addressed at: Department of Molecular Microbiol- with other molecules; the PG repeating units and caps occur on ogy, Washington University Medical School, Campus Box 8230, 660 South Euclid Avenue, secreted proteins such as PPG or secreted acid phosphatase, and St. Louis, MO 63110. E-mail: [email protected]. 9536–9541 ͉ PNAS ͉ August 5, 2003 ͉ vol. 100 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1530604100 Downloaded by guest on September 24, 2021 LPG1 mutant (LPG1::HYG͞LPG1::PAC), referred to as lpg1Ϫ, and its LPG1-restored derivative (LPG1::HYG͞LPG1::PAC [pSNBR-LPG1]) referred to as lpg1Ϫ͞ϩLPG1 or the LPG1 ‘‘add-back,’’ were grown briefly in the absence of selective agents before use (11). Metacyclics were prepared from day 4 station- ary-phase cultures by an LPG-independent density-gradient centrifugation method (17). Mouse Strains. BALB͞c mice were obtained from Charles River Breeding Laboratories. C57BL͞6-Cybbtm1 null mutant (phoxϪ), C57BL͞6-Nos2tm1Lau inducible NO synthase null mutant, C57BL͞6, C5-deficient B10.D2-H2d H2-T18c Hc0͞oSnJ, and C5-sufficient control B10.D2-H2d H2-T18c Hc1͞nSnJ mice were obtained from The Jackson Laboratory. Mouse Infections. Virulence was assessed after inoculation of mouse footpads (20). Groups were injected s.c. into the footpad with 105 metacyclic parasites per mouse. Infections were mon- Fig. 1. lpg1Ϫ metacyclic promastigotes are sensitive to human but not mouse itored by comparing the thickness of the injected and uninjected complement. (A) Flow cytometric analysis of lysis by human serum. Metacyclic footpads with a Vernier caliper. Parasites were enumerated in WT, lpg1Ϫ, and lpg1Ϫ͞ϩLPG1 lines were incubated in the presence (shaded) or the infected tissue by a limiting-dilution assay (21). absence (open) of 2% human serum for 30 min in the presence of propidium iodide and then subjected to flow cytometry. (B) Infections of normal and Macrophage Infections. Infections of mouse peritoneal exudate C5-deficient mice. WT (E)orlpg1Ϫ (‚) metacyclics (105) were inoculated into macrophages (PEMs) were performed and relative survival was the footpad of isogenic WT (Upper) or C5-deficient (Lower) mice, and lesion calculated by normalizing values to the initial parasite burden at formation was followed. The average and standard deviation are shown for 2 h postinfection (11, 17, 22). Typically 104 macrophages were groups of five mice; this experiment is representative of three independent seeded in 24-well plates and incubated with 105 parasites in 500 tests. ␮l. For synchronous infections, parasites were incubated at 4°C MICROBIOLOGY for 20 min for attachment, free parasites were removed by lpg1Ϫ low- and high-infectivity infections, respectively] washing, and cultures were shifted to 37°C. Macrophages were was achieved as described above. Fusogenic FITC-positive activated with 100 ng͞ml lipopolysaccharide (LPS; Escherichia phagosomes
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