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The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking.

Franco IS, Shohdy N, Shuman HA - PLoS Pathog. (2012)

Bottom Line: When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets.Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization.These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA. irinafranco@hotmail.com

ABSTRACT
Legionella pneumophila, the causative agent of Legionnaires' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.

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Localization of translocated VipA and VipA-1 during infection of THP-1 macrophages.THP-1 macrophages were infected with L. pneumophila strains (JR32, ΔvipA, ΔvipA pMMB207c-Ptac-vipA or ΔvipA pMMB207c-Ptac-vipA-1) with an MOI = 50 for 8 hours. Immunofluorescence was carried out as described (Materials and Methods). DNA was stained with DAPI and VipA with a purified polyclonal anti-VipA antibody. A. and F-actin was stained with Rhodamine-phalloidin. B. Representative confocal microscopy images are shown. C. Immunoblot of samples containing lysates of THP-1 macrophages infected for 3 hr with the indicated L. pneumophila strains at an MOI = 50 (see Material and Methods). P, lysate pellet; SN, lysate supernatant.
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ppat-1002546-g006: Localization of translocated VipA and VipA-1 during infection of THP-1 macrophages.THP-1 macrophages were infected with L. pneumophila strains (JR32, ΔvipA, ΔvipA pMMB207c-Ptac-vipA or ΔvipA pMMB207c-Ptac-vipA-1) with an MOI = 50 for 8 hours. Immunofluorescence was carried out as described (Materials and Methods). DNA was stained with DAPI and VipA with a purified polyclonal anti-VipA antibody. A. and F-actin was stained with Rhodamine-phalloidin. B. Representative confocal microscopy images are shown. C. Immunoblot of samples containing lysates of THP-1 macrophages infected for 3 hr with the indicated L. pneumophila strains at an MOI = 50 (see Material and Methods). P, lysate pellet; SN, lysate supernatant.

Mentions: In order to assess the subcellular localization of VipA in host cells under physiological conditions, infection of THP-1 monocyte-like cells was carried out, cells fixed at several time points post-infection, processed for immunofluorescence using a polyclonal anti-VipA antibody and analysed using Laser Scanning Confocal Microscopy. After infection with wild-type strain L. pneumophila JR32, VipA was found in diverse structures inside the host cell, which varied in size from puncta to larger formations (Figures 6 and 7). VipA was not associated with the LCV at any time point from 30 min to 14 hr after uptake (Figure 6A and B; data not shown). To assess the localization of the VipA-1 mutant, L. pneumophila vipA -mutant background strains were constructed carrying plasmids encoding IPTG-inducible copies of either the wild-type or the mutant vipA allele (respectively, ΔvipA pMMB207c-Ptac-vipA+ or ΔvipA pMMB207c-Ptac-vipA-1). Infection with the strain carrying Ptac-vipA+ led to a similar distribution of the effector as observed in JR32, although the protein was observed in the host cell earlier after uptake. This localization was lost in the vipA-1 mutant, in which the effector was translocated in levels similar to the wild-type (Figure 6C) and homogeneously distributed in the host cell cytosol (Figures 6A and 7A).


The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking.

Franco IS, Shohdy N, Shuman HA - PLoS Pathog. (2012)

Localization of translocated VipA and VipA-1 during infection of THP-1 macrophages.THP-1 macrophages were infected with L. pneumophila strains (JR32, ΔvipA, ΔvipA pMMB207c-Ptac-vipA or ΔvipA pMMB207c-Ptac-vipA-1) with an MOI = 50 for 8 hours. Immunofluorescence was carried out as described (Materials and Methods). DNA was stained with DAPI and VipA with a purified polyclonal anti-VipA antibody. A. and F-actin was stained with Rhodamine-phalloidin. B. Representative confocal microscopy images are shown. C. Immunoblot of samples containing lysates of THP-1 macrophages infected for 3 hr with the indicated L. pneumophila strains at an MOI = 50 (see Material and Methods). P, lysate pellet; SN, lysate supernatant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285593&req=5

ppat-1002546-g006: Localization of translocated VipA and VipA-1 during infection of THP-1 macrophages.THP-1 macrophages were infected with L. pneumophila strains (JR32, ΔvipA, ΔvipA pMMB207c-Ptac-vipA or ΔvipA pMMB207c-Ptac-vipA-1) with an MOI = 50 for 8 hours. Immunofluorescence was carried out as described (Materials and Methods). DNA was stained with DAPI and VipA with a purified polyclonal anti-VipA antibody. A. and F-actin was stained with Rhodamine-phalloidin. B. Representative confocal microscopy images are shown. C. Immunoblot of samples containing lysates of THP-1 macrophages infected for 3 hr with the indicated L. pneumophila strains at an MOI = 50 (see Material and Methods). P, lysate pellet; SN, lysate supernatant.
Mentions: In order to assess the subcellular localization of VipA in host cells under physiological conditions, infection of THP-1 monocyte-like cells was carried out, cells fixed at several time points post-infection, processed for immunofluorescence using a polyclonal anti-VipA antibody and analysed using Laser Scanning Confocal Microscopy. After infection with wild-type strain L. pneumophila JR32, VipA was found in diverse structures inside the host cell, which varied in size from puncta to larger formations (Figures 6 and 7). VipA was not associated with the LCV at any time point from 30 min to 14 hr after uptake (Figure 6A and B; data not shown). To assess the localization of the VipA-1 mutant, L. pneumophila vipA -mutant background strains were constructed carrying plasmids encoding IPTG-inducible copies of either the wild-type or the mutant vipA allele (respectively, ΔvipA pMMB207c-Ptac-vipA+ or ΔvipA pMMB207c-Ptac-vipA-1). Infection with the strain carrying Ptac-vipA+ led to a similar distribution of the effector as observed in JR32, although the protein was observed in the host cell earlier after uptake. This localization was lost in the vipA-1 mutant, in which the effector was translocated in levels similar to the wild-type (Figure 6C) and homogeneously distributed in the host cell cytosol (Figures 6A and 7A).

Bottom Line: When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets.Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization.These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA. irinafranco@hotmail.com

ABSTRACT
Legionella pneumophila, the causative agent of Legionnaires' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.

Show MeSH
Related in: MedlinePlus