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Leishmania donovani lipophosphoglycan: effects on actin and phagosomal maturation
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Leishmania donovani promastigotes survive intracellularly in macrophages by inhibiting phagosomal maturation. This ability is conferred by surface lipophosphoglycan (LPG), which is transferred to the host-cell plasma and phagosomal membranes during phagocytosis. LPG modulates the biophysical properties of membranes and has several effects on the host cell, including inhibition of protein kinase C alpha (PKCα)-mediated signaling. Our studies were focused on molecular mechanisms operating in the establishment of L. donovani infection, especially effects on host-cell F-actin.

We found that L. donovani promastigotes induced accumulation of periphagosomal F-actin, an effect directly dependent on LPG. The F-actin accumulation correlated to inhibition of phagosomal maturation. Cortical F-actin was increased as well. Macrophages overexpressing dominant-negative (DN) PKCα also displayed elevated cortical F-actin, decreased phagocytic capacity, elevated periphagosomal F-actin, and defective phagosomal maturation, effects similar to those seen when exposing the cells to LPG. LPG colocalized with lipid rafts in the host-cell membrane, and lipid rafts were necessary both for translocation of activated PKCα to the membrane, and for the effects of LPG on host cell actin dynamics and phagosomal maturation. Introduction of constitutively active Rac1 and Cdc42 into the host macrophage mimicked the effects of LPG on actin dynamics and phagosomal maturation while DN Rac1 and Cdc42 abrogated LPG's effects on actin.

Taken together, our results show that LPG partitions into lipid rafts in macrophages and induces an accumulation of periphagosomal F-actin, which is correlated to inhibition of phagosomal maturation. The effect of LPG on actin involves inhibition of PKCα and depends on active Rac1 and Cdc42.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 106 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 800
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26655Local ID: 11220ISBN: 91-7373-489-6 (print)OAI: oai:DiVA.org:liu-26655DiVA: diva2:247204
Public defence
2003-09-18, Aulan, Hälsans Hus, Hälsouniversitet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-11Bibliographically approved
List of papers
1. Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation
Open this publication in new window or tab >>Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation
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2001 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 3, no 7, 439-447 p.Article in journal (Refereed) Published
Abstract [en]

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13848 (URN)10.1046/j.1462-5822.2001.00127.x (DOI)
Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2017-12-13Bibliographically approved
2. Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages
Open this publication in new window or tab >>Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages
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2003 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 302, no 4, 653-658 p.Article in journal (Refereed) Published
Abstract [en]

Protein kinase C α (PKCα) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the dissassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKCα. To investigate a possible connection between PKCα and LPG’s effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PKCα (DN PKCα). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKCα-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKCα is involved in F-actin turnover in macrophages and that PKCα-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKCα by LPG.

Keyword
PKCα, Actin, Phagocytosis, Macrophage, Lipophosphoglycan
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13850 (URN)10.1016/S0006-291X(03)00231-6 (DOI)
Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2017-12-13Bibliographically approved
3. Lipid rafts are required for the effects of Leishmania donovani lipophosphoglycan on periphagosomal F-actin and phagosomal maturation
Open this publication in new window or tab >>Lipid rafts are required for the effects of Leishmania donovani lipophosphoglycan on periphagosomal F-actin and phagosomal maturation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lipophosphoglycan (LPG) is the major surface glycoconjugate on Leishmania donovani promastigotes, and is cmcial for pro mastigote survival following phagocytosis by macrophages. LPG consists of a chain of repeating phosphodisaccharides anchored to the parasite membrane by a lysophosphatidylinositol lipid anchor with an unusually long saturated fatty acid residue. During phagocytosis, LPG transfers from the parasite surface to the plasma membrane of the host macrophage. The presence of LPG alters the biophysical properties of the host cell membrane and the signaling capacity of the macrophage. LPG induces accumulation ofF-actin around the phagosome, and inhibits phagosome maturation. The effects of LPG on the host ce!l include inhibition of PKCα, a PKC isoenzyme involved in F-actin tumover.

The biophysical properties of the LPG lipid anchor suggest that it partitions into caveolae or lipid rafts, which are cholesterol-rich plasma membrane microdomains central for signal transduction. Since PKCa is enriched in caveolae/lipid rafts in other cell types, we investigated if lipid rafts constitute a platform for the interaction of LPG and PKCα. We found that the plasma membrane of human monocyte-derived macrophages were rich in lipid rafts, but did not contain caveolae. LPG colocalized with lipid raft markers after interaction with WT L. donovani promastigotes. The presence of LPG inhibited the translocation of PKCα to the plasma membrane. Destruction of lipid rafts by cholesterol depletion lead to a complete eradication of LPG's effects on periphagosomal F-actin and phagosomal maturation. We also found that cholesterol depletion reduced uptake of WT L. donovani promastigotes, while uptake of an LPG-defective mutant was not affected.

We conclude that LPG partitions to lipid rafts in the plasma membrane of human macrophages and inhibits the translocation of PKCα to the membrane. The presence of lipid rafts is a prerequisite for LPG to exert its effects on host cell actin and phagosomal maturation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84524 (URN)
Available from: 2012-10-11 Created: 2012-10-11 Last updated: 2012-10-11Bibliographically approved
4. Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycan
Open this publication in new window or tab >>Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycan
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The intracellular parasite Leishmania donovani survives inside macrophage phagosomes by inhibiting phagosornal maturation. Its main surface glycoconjugate, lipophosphoglycan (LPG), is crucial for survival and essential for the build-up of a coat of F-actin surrounding the phagosome. Previous studies have shown that inhibition of PKCα by LPG is partly responsible for the elevated levels of F-actin around the phagosome (1, 2). This study shows that simultaneous inhibition of Cdc42 and Rac1, members of the Rho family of small GTPases, prevented the accumulation of F-actin around L. donovani containing phagosomes in murine macrophages. Moreover, an LPG-defective L. donovani mutant normally not capable of accumulating F-actin around it's phagosome, displayed elevated amounts of periphagosomal F-actin in cells pre-treated with permanently active forms of Cdc42 and Rac. The lysosomal marker LAMP1 did not translocate normally to phagosomes in these cells, indicating defective phagosomal maturation. We conclude that Cdc42 and Rac are activated by L. donovani in an LPG-dependent manner, and that this activation contributes to the accumulation of periphagosomal F-actin around L. donovani phagosomes. Our results also indicate a direct link between the build-up of periphagosomal F-actinand inhibition of phagosomal mahuation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84526 (URN)
Available from: 2012-10-11 Created: 2012-10-11 Last updated: 2012-10-11Bibliographically approved

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Holm, Åsa

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