Abstract
Lipophosphoglycan (LPG) is the major surface glycoconjugate of metacyclic Leishmania promastigotes and is associated with virulence in various species of this parasite. Here, we generated a LPG-deficient mutant of Leishmania infantum, the foremost etiologic agent of visceral leishmaniasis in Brazil. The L. infantum LPG-deficient mutant (Δlpg1) was obtained by homologous recombination and complemented via episomal expression of LPG1 (Δlpg1 + LPG1). Deletion of LPG1 had no observable effect on parasite morphology or on the presence of subcellular organelles, such as lipid droplets. While both wild-type and add-back parasites reached late phase in axenic cultures, the growth of Δlpg1 parasites was delayed. Additionally, the deletion of LPG1 impaired the outcome of infection in murine bone marrow-derived macrophages. Although no significant differences were observed in parasite load after 4 h of infection, survival of Δlpg1 parasites was significantly reduced at 72 h post-infection. Interestingly, L. infantum LPG-deficient mutants induced a strong NF-κB-dependent activation of the inducible nitric oxide synthase (iNOS) promoter compared to wild type and Δlpg1 + LPG1 parasites. In conclusion, the L. infantum Δlpg1 mutant constitutes a powerful tool to investigate the role(s) played by LPG in host cell-parasite interactions.
Highlights
Lipophosphoglycan (LPG) is one of the most abundant components of Leishmania membranes (Turco and Descoteaux, 1992)
Area Under the Curve (AUC) analysis of the growth curve revealed a significant difference only when comparing WT and lpg1 parasites (p < 0.05), yet no differences were observed between WT and lpg1 + LPG1 mutants (Figure 2B)
To assess the impact of LPG on the expression of inducible nitric oxide synthase by host cells, we first analyzed iNOS transcript levels in RAW 264.7 cells infected with WT or transgenic parasites
Summary
Lipophosphoglycan (LPG) is one of the most abundant components of Leishmania membranes (Turco and Descoteaux, 1992). Purified LPG has been considered as a pathogen-associated molecular pattern molecule (PAMP) that triggers Toll-like receptors (TLR) and is known to interfere with pro-inflammatory and signaling pathways in host cells (Descoteaux et al, 1991; Descoteaux and Turco, 1993; Becker et al, 2003; de Veer et al, 2003; Kavoosi et al, 2009; Rojas-Bernabé et al, 2014; Tavares et al, 2014; Lima et al, 2017) This complex glycolipid is organized in four domains: a conserved 1-O-alkyl-2-lyso-phosphatidyl(myo)inositol membrane anchor, a conserved diphosphoheptasaccharide core structure, a polymer of repeating phosphodisaccharide units (phosphoglycan or PG) carrying species-specific side chains and variable, often mannose-rich cap structures (Turco and Descoteaux, 1992; McConville and Ferguson, 1993). The biosynthesis of LPG has attracted considerable interest, to date only few enzymes and transporters involved in this process have been identified either biochemically, genetically, or both (Ryan et al, 1993; Descoteaux et al, 1995, 1998, 2002)
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