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Effects of lipophosphoglycan on macrophage actin dynamics
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-26683Local ID: 11250ISBN: 91-7373-190-0 (print)OAI: oai:DiVA.org:liu-26683DiVA: diva2:247233
Presentation
2002-12-10, Hälsouniversitetet, Linköping, 10:15 (Swedish)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-09-18
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. Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with un-opsonized prey
Open this publication in new window or tab >>Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with un-opsonized prey
2002 (English)In: Bioscience Reports, ISSN 0144-8463, Vol. 22, no 5-6, 529-540 p.Article in journal (Refereed) Published
Abstract [en]

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.

Keyword
Macrophage, phagocytosis, calcium, actin, phagosome maturation, lipophosphoglycan
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13849 (URN)10.1023/A:1022025903688 (DOI)
Available from: 2006-05-23 Created: 2006-05-23 Last updated: 2013-09-18
3. Protein C α regulates phagocytosis actin dynamics and phagosomal maturation in macrophages
Open this publication in new window or tab >>Protein C α regulates phagocytosis actin dynamics and phagosomal maturation in macrophages
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Protein kinase C α (PKCα) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Promastigotes of the protozoan parasite Leishmania donovani cause an accumulation of periphagosomal F-actin instead of the normal decrease seen with other prey [1]. This accumulation is dependent on promastigote lipophosphoglycan (LPG), which has several detrimental effects on the cell including inhibition of PKCα activity.

To directly address the role of PKCα and LPG for actin remodeling in macrophages, we investigated F-actin dynamics in RAW 264.7 macrophages overexpressing a dominant-negative mutant of PKCα (DN PKCα). We found that DN PKCα-overexpressing cells displayed increased levels of cortical F-actin and decreased phagocytic capacity, which was augmented when the cells were subjected to LPG-coated prey. The DN PKCα-overexpressing cells also showed defective breakdown of periphagosomal F-actin and inhibition of phagosomal maturation. The level of periphagosomal F-actin was similar to that of controls subjected to LPG-coated prey. Our results show that PKCα regulates phagocytosis and F-actin turnover in macrophages, and that PKCα-dependent breakdown of periphagosomal F-actin is required for normal phagosomal maturation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-97635 (URN)
Available from: 2013-09-18 Created: 2013-09-18 Last updated: 2013-09-18

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