scholarly journals Yersinia pestis Can Reside in Autophagosomes and Avoid Xenophagy in Murine Macrophages by Preventing Vacuole Acidification

2009 ◽  
Vol 77 (6) ◽  
pp. 2251-2261 ◽  
Author(s):  
Céline Pujol ◽  
Kathryn A. Klein ◽  
Galina A. Romanov ◽  
Lance E. Palmer ◽  
Carol Cirota ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Fluorescent Protein ◽  
Viable Count ◽  
Activated Macrophages ◽  
Intracellular Replication ◽  
Microscopic Imaging ◽  
Murine Macrophages ◽  
Late Endosomes ◽  
Autophagic Process ◽  
Green Fluorescent

ABSTRACT Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that ∼40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (∼30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.

scholarly journals Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

2002 ◽  
Vol 156 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Pierre Barbero ◽  
Lenka Bittova ◽  
Suzanne R. Pfeffer
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Live Cells ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

scholarly journals Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Lambertus H. J. Aarts ◽  
Onno Roovers ◽  
Alister C. Ward ◽  
Ivo P. Touw
Keyword(s):  
Fluorescent Protein ◽  
Surface Expression ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Serine Threonine Kinase ◽  
Late Endosomes ◽  
Dileucine Motif ◽  
Internalization Kinetics ◽  
Green Fluorescent ◽  
Kinetics Of

Abstract We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

scholarly journals Skp1p Regulates Soi3p/Rav1p Association with Endosomal Membranes but Is Not Required for Vacuolar ATPase Assembly

2006 ◽  
Vol 5 (12) ◽  
pp. 2104-2113 ◽  
Author(s):  
E. J. Brace ◽  
Leah P. Parkinson ◽  
Robert S. Fuller
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Sole Source ◽  
Vacuolar Atpase ◽  
Cellular Functions ◽  
Yeast Protein ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Green Fluorescent

ABSTRACT Skp1p is an essential component of SCF-type E3 ubiquitin ligase complexes and associates with these through binding to F-box proteins. Skp1p also binds F-box proteins in a number of non-SCF complexes. The Skp1p-associated yeast protein Soi3p/Rav1p (hereafter referred to as Rav1p) is a component of the RAVE complex required for regulated assembly of vacuolar ATPase (V-ATPase). Rav1p is also involved in transport of TGN proteins and endocytic cargo between early and late endosomes. To evaluate the role of Skp1p in the RAVE complex, we made use of the fact that overexpression of Rav1p is toxic because it sequesters Skp1p from essential interactions. We isolated a separation of function allele of SKP1, skp1(Asn108Tyr), that completely abrogated the Rav1p interaction but allowed Skp1p to perform other essential cellular functions. Cells containing the skp1(Asn108Tyr) allele as the sole source of Skp1p exhibited normal V-ATPase assembly and activity. However, in the skp1(Asn108Tyr) mutant strain, the membrane-associated pool of Rav1-green fluorescent protein was increased, suggesting that Skp1p is important for the release of Rav1p from endosomal membranes where it functions in V-ATPase assembly. Thus, although part of the RAVE complex, Skp1p does not appear to be involved in V-ATPase assembly but instead in the cycling of the complex off membranes. This work also provides a generalizable approach to defining the roles of interactions of Skp1p with individual F-box proteins through the isolation of special alleles of SKP1.

scholarly journals Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils

2005 ◽  
Vol 73 (11) ◽  
pp. 7142-7150 ◽  
Author(s):  
Roman A. Lukaszewski ◽  
Dermot J. Kenny ◽  
Rosa Taylor ◽  
D. G. Cerys Rees ◽  
M. Gill Hartley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Fluorescent Protein ◽  
Virulent Strain ◽  
Late Phase ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Intracellular Bacteria ◽  
Necrosis Factor Alpha ◽  
Green Fluorescent

ABSTRACT The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103 to 104 CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

Delayed accumulation of activated macrophages and inhibition of remyelination after spinal cord injury in an adult rodent model

2008 ◽  
Vol 8 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Masaaki Imai ◽  
Masahiko Watanabe ◽  
Kaori Suyama ◽  
Takahiro Osada ◽  
Daisuke Sakai ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Bone Marrow ◽  
Spinal Cord Injury ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Activated Macrophages ◽  
Cord Injury ◽  
Myelin Debris ◽  
Chemotactic Factors ◽  
Green Fluorescent

Object Inhibition of remyelination is part of the complex problem of persistent dysfunction after spinal cord injury (SCI), and residual myelin debris may be a factor that inhibits remyelination. Phagocytosis by microglial cells and by macrophages that migrate from blood vessels plays a major role in the clearance of myelin debris. The object of this study was to investigate the mechanisms underlying the failure of significant remyelination after SCI. Methods The authors investigated macrophage recruitment and related factors in rats by comparing a contusion model (representing contusive SCI with residual myelin debris and failure of remyelination) with a model consisting of chemical demyelination by lysophosphatidylcholine (representing multiple sclerosis with early clearance of myelin debris and remyelination). The origin of infiltrating macrophages was investigated using mice transplanted with bone marrow cells from green fluorescent protein–transfected mice. The changes in levels of residual myelin debris and the infiltration of activated macrophages in demyelinated lesions were investigated by immunostaining at 2, 4, and 7 days postinjury. To investigate various factors that might be involved, the authors also investigated gene expression of macrophage chemotactic factors and adhesion factors. Results Activated macrophages coexpressing green fluorescent protein constituted the major cell population in the lesions, indicating that the macrophages in both models were mainly derived from the bone marrow, and that very few were derived from the intrinsic microglia. Immunostaining showed that in the contusion model, myelin debris persisted for a long period, and the infiltration of macrophages was significantly delayed. Among the chemotactic factors, the levels of monocyte chemoattractant protein–1 and granulocyte–macrophage colony-stimulating factor were lower in the contusion model at 2 and 4 days postinjury. Conclusions The results suggest that the delayed infiltration of activated macrophages is related to persistence of myelin debris after contusive SCI, resulting in the inhibition of remyelination.

scholarly journals The Ability To Replicate in Macrophages Is Conserved between Yersinia pestis and Yersinia pseudotuberculosis

2003 ◽  
Vol 71 (10) ◽  
pp. 5892-5899 ◽  
Author(s):  
Céline Pujol ◽  
James B. Bliska
Keyword(s):  
Yersinia Pestis ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Type Iii Secretion System ◽  
Evolutionary Process ◽  
Secretion System ◽  
Intracellular Replication ◽  
Murine Macrophages ◽  
Type Iii ◽  
Chromosomal Type

ABSTRACT Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10+) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p−) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.

scholarly journals Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae

2006 ◽  
Vol 5 (2) ◽  
pp. 411-421 ◽  
Author(s):  
Jun-ya Shoji ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Filamentous Fungi ◽  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Membrane Dynamics ◽  
Late Endosomes ◽  
Staining Methods ◽  
Green Fluorescent ◽  
Pyridinium Dibromide ◽  
Egfp Fluorescence

ABSTRACT Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Δvam3 and Δpep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.

scholarly journals The ADP-Ribosylation Domain of Pseudomonas aeruginosa ExoS Is Required for Membrane Bleb Niche Formation and Bacterial Survival within Epithelial Cells

2010 ◽  
Vol 78 (11) ◽  
pp. 4500-4510 ◽  
Author(s):  
Annette A. Angus ◽  
David J. Evans ◽  
Joseph T. Barbieri ◽  
Suzanne M. J. Fleiszig
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Annexin V ◽  
Rab Gtpase ◽  
Bacterial Survival ◽  
Intracellular Replication ◽  
Contrast Imaging ◽  
Niche Formation ◽  
Green Fluorescent

ABSTRACT Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.

Sign in / Sign up

Export Citation Format

Share Document