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Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
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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.

Place, publisher, year, edition, pages
2001. Vol. 3, no 7, 439-447 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13848DOI: 10.1046/j.1462-5822.2001.00127.xOAI: oai:DiVA.org:liu-13848DiVA: diva2:21859
Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Leishmania donovani Lipophosphoglycan: Modulation of Macrophage and Dendritic Cell Function
Open this publication in new window or tab >>Leishmania donovani Lipophosphoglycan: Modulation of Macrophage and Dendritic Cell Function
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Leishmania donovani is a blood-borne tropicial parasite, which infects humans through bites by Phlebotomus sandflies. The parasite survives and multiplies inside macrophages in inner organs, and causes the deadly disease visceral leishmaniasis (Kala-Azar).

Macrophages and dendritic cells (DC) are professional antigen-presenting cells involved in the initiation of immune responses. Immature DC are present in all tissues where they internalise and process antigen, in response to which they migrate from tissue, into draining lymphoid organs, undergo maturation and present antigens to lymphocytes. Control measures for leishmaniasis include testing of new diagnostics and development of affordable and effective vaccines for humans.

Lipophosphoglycan (LPG) is the major surface component of Leishmania donovani promastigotes. LPG comprises a membrane-anchoring lysophosphatidylinositol part and an extracellular chain of disaccharide phosphates. These repetitions are crucial for parasite survival inside macrophages following phagocytosis. LPG has several specific effects on the host cell including inhibition of protein kinase C (PKC) activity, and inhibition of phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin.

Confocal microscopy and image analysis were used to follow F-actin dynamics in single macrophages during phagocytosis of L. donovani promastigotes and LPG-coated particles. F-actin did not depolymerize, but instead progressively polymerized around phagosomes with LPG-containing prey. This correlated with reduced translocation of PKCα to the phagosome and blocked phagosomal maturation. LPG also inhibited cortical actin turnover, which could be the underlying cause of the reduced uptake of LPG-containing prey. Extracellular- and intracellular calcium was necessary for phagocytosis, periphagosomal F-actin breakdown and phagosomal maturation in macrophages interacting with unopsonized prey,and for the action of LPG.

We also studied F-actin turnover in macrophages overexpressing dominant-negative (DN) PKCα. DN PKCα macrophages showed increased amounts of cortical F-actin, decreased phagocytic capacity, inhibition of periphagosomal F-actin breakdown and defective phagosomal maturation. When DN PKCα macrophages interacted with LPG-containing prey, phagocytosis was almost completely blocked.

Moreover, we found that Leishmania promastigotes and particularly LPG inhibit DC maturation and detachment from distinct surfaces. Thus, LPG from Leishmania donovani could directly inhibit DC migration to lymphoid organs, antigen-presentation and development of immunity.

Place, publisher, year, edition, pages
Institutionen för molekylär och klinisk medicin, 2006
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 946
Series
Keyword
Lipophosphoglycan, Leishmania donovani, macrophage, actin, phagocytosis, PKC alpha, dendritic cell
National Category
Pathobiology
Identifiers
urn:nbn:se:liu:diva-6527 (URN)91-85497-84-3 (ISBN)
Public defence
2006-06-01, Linden, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2009-06-05
2. Leishmania donovani lipophosphoglycan: effects on actin and phagosomal maturation
Open this publication in new window or tab >>Leishmania donovani lipophosphoglycan: effects on actin and phagosomal maturation
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:nbn:se:liu:diva-26655 (URN)11220 (Local ID)91-7373-489-6 (ISBN)11220 (Archive number)11220 (OAI)
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
3. Effects of lipophosphoglycan on macrophage actin dynamics
Open this publication in new window or tab >>Effects of lipophosphoglycan on macrophage actin dynamics
2002 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Leishmania donovani is a blood-borne tropicial parasite, which infects humans through bites by Phlebotomine sandflies. The parasite survives and multiplies inside macrophages in inner organs, and causes the deadly disease visceral leishmaniasis (Kala-Azar). Lipophosphoglycan (LPG) is the major surface component of Leishmania donovani promastigotes. LPG comprises a membrane-anchoring

lysophosphatidylinositol moiety and an extracellular chain of repeating disaccharide phosphates. The repeats are crucial for parasite survival inside macrophages following phagocytosis. LPG has several effects on the host cell including inhibition of protein kinase C (PKC) activity, and inhibition of phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. Confocal microscopy and image analysis was used to follow F-actin dynamics in single macrophages during phagocytosis of L. donovani promastigotes and LPG-coated particles. F-actin did not depolymerize, but instead progressively polymerized around phagosomes with LPG-containing prey. This directly correlated with reduced translocation of PKCα to the phagosome and blocked phagosomal maturation. It was also found that LPG inhibited cortical actin turnover, which may be the underlying cause of the reduced uptake of LPG-containing prey. Free calcium was necessary for phagocytosis, periphagosomal F-actin breakdown and phagosomal maturation in macrophages interacting with unopsonized prey. LPG required free calcium to exert its inhibitory effects on these processes. We also studied F -actin turnover in macrophages overexpressing dominant-negative (DN) PKCα. DN PKCα macrophages showed increased amounts of cortical F-actin, decreased phagocytic capacity, inhibition of periphagosomal F-actin breakdown and defective phagosomal maturation. When DN PKCa macrophages interacted with LPG-containing prey, phagocytosis was almost completely blocked.

Together, our results indicate that blockage of F-actin breakdown through inhibition of PKCa by LPG reduces phagocytosis and is instrumental for the inhibition of phagosomal maturation induced by L. donovani promastigotes.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2002. 42 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 58
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26683 (URN)11250 (Local ID)91-7373-190-0 (ISBN)11250 (Archive number)11250 (OAI)
Presentation
2002-12-10, Hälsouniversitetet, Linköping, 10:15 (Swedish)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-09-18

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Holm, ÅsaTejle, KatarinaMagnusson, Karl-EricRasmusson, Birgitta

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