Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability
TL;DR: The role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes, as its activity is focused on molecules implicated in virulence rather than viability.
Abstract: Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide-phosphate repeat unit Galbeta1,4Manalpha1-PO4, shared by other developmentally regulated molecules implicated in parasite virulence. Functional complementation of a Leishmania donovani LPG-defective mutant (OB1) accumulating a truncated LPG containing only the Manalpha1-PO4 residue of the first repeat unit identified LPG3, the Leishmania homolog of the mammalian endoplasmic reticulum (ER) chaperone GRP94. LPG3 resembles GRP94, as it localizes to the parasite ER, and lpg3(-) mutants show defects including down-regulation of surface GPI-anchored proteins and mild effects on other glycoconjugates. LPG3 binds cellular proteins and its Leishmania infantum GRP94 ortholog is highly immunogenic, suggesting a potential role in directing the immune response. However, null lpg3(-) mutants grow normally, are completely defective in the synthesis of phosphoglycans, and the LPG3 mRNA is regulated developmentally but not by stress or heat. Thus the role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes. Like the other glycoconjugate synthetic pathways in this parasite, its activity is focused on molecules implicated in virulence rather than viability.
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TL;DR: Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins and the basis for this selectivity remains obscure.
335 citations
TL;DR: This analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal, and as an alternative, L.Donovani appears to use multiple nonclassical secretion pathways, including the release of exosome-like microvesicles.
Abstract: Background: Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1α, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis. Results: Analysis of leishmania conditioned medium resulted in the identification of 151 proteins apparently secreted by L. donovani. Ratios reflecting the relative amounts of each leishmania protein secreted, as compared to that remaining cell associated, revealed a hierarchy of protein secretion, with some proteins secreted to a greater extent than others. Comparison with an in silico approach defining proteins potentially exported along the classic eukaryotic secretion pathway suggested that few leishmania proteins are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. Unexpectedly, a large majority of known eukaryotic exosomal proteins was detected in leishmania conditioned medium, suggesting a vesicle-based secretion system. Conclusion: This analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal. As an alternative, L. donovani appears to use multiple nonclassical secretion pathways, including the release of exosomelike microvesicles.
286 citations
Cites background from "Leishmania LPG3 encodes a GRP94 hom..."
...Promastigote surface coat constituents have been the focus of considerable interest [8-10], and many of these - including glycoproteins, proteoglycans, and glycolipids - have been shown to play protective roles [8,11,12]....
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TL;DR: It is demonstrated that leishmania exosomes are predominantly immunosuppressive, and the first evidence to suggest that changes in the protein cargo of exosome may influence the impact of these vesicles on myeloid cell function is suggested.
Abstract: We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.
278 citations
TL;DR: The in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line and should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.
219 citations
Cites background from "Leishmania LPG3 encodes a GRP94 hom..."
..., 2000), lipophosphoglycan biosynthesis, structure and function (Beverley and Turco, 1995; Descoteaux et al., 2002) and surface membrane transporters (Vasudevan et al....
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TL;DR: Recent structural and mechanistic progress in defining the function of organelle-specific and cytosolic Hsp90 is summarized, including the impact of individual cochaperones on the maturation of specific clients and complexes with clients as well as ways of exploiting HSp90 as a drug target.
Abstract: Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the maturation of a plethora of substrates ("clients"), including protein kinases, transcription factors, and E3 ubiquitin ligases, positioning Hsp90 as a central regulator of cellular proteostasis. Hsp90 undergoes large conformational changes during its ATPase cycle. The processing of clients by cytosolic Hsp90 is assisted by a cohort of cochaperones that affect client recruitment, Hsp90 ATPase function or conformational rearrangements in Hsp90. Because of the importance of Hsp90 in regulating central cellular pathways, strategies for the pharmacological inhibition of the Hsp90 machinery in diseases such as cancer and neurodegeneration are being developed. In this review, we summarize recent structural and mechanistic progress in defining the function of organelle-specific and cytosolic Hsp90, including the impact of individual cochaperones on the maturation of specific clients and complexes with clients as well as ways of exploiting Hsp90 as a drug target.
157 citations
References
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TL;DR: It is proposed that the KDEL sequence marks proteins that are to be retained in the ER and discuss possible retention mechanisms.
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851 citations
"Leishmania LPG3 encodes a GRP94 hom..." refers background in this paper
...LPG has been implicated in many important steps in the Leishmania life cycle (Turco and Descoteaux, 1992; McConville and Ferguson, 1993; Handman, 1999), and a Leishmania major mutant lacking LPG alone in an otherwise virulent background is attenuated in sand ̄y, mouse and macrophage infections (Sacks et al....
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