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Cellular Microbiology Apr 2020Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate...
Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.
Topics: Animals; Bacterial Proteins; Biological Transport; Cytosol; Endocytosis; Female; Flavoproteins; Host-Pathogen Interactions; Legionella pneumophila; Macrophages; Mice; Protein Transport; RAW 264.7 Cells; RNA Interference; Vacuoles
PubMed: 32096265
DOI: 10.1111/cmi.13151 -
The Journal of Infectious Diseases Mar 2022The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small...
The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.
Topics: Bacterial Proteins; Cytoplasm; Cytosol; Hemolysin Proteins; Humans; Listeria monocytogenes; Listeriosis; Vacuoles
PubMed: 32582947
DOI: 10.1093/infdis/jiaa367 -
Current Opinion in Microbiology Dec 2017The intracellular parasite Toxoplasma gondii resides inside a vacuole, which shields it from the host's intracellular defense mechanisms. The cytokine interferon gamma... (Review)
Review
The intracellular parasite Toxoplasma gondii resides inside a vacuole, which shields it from the host's intracellular defense mechanisms. The cytokine interferon gamma (IFNγ) upregulates host cell effector pathways that are able to destroy the vacuole, restrict parasite growth and induce host cell death. Interferon-inducible GTPases such as the Guanylate Binding Proteins (GBPs), autophagy proteins and ubiquitin-driven mechanisms play important roles in Toxoplasma control in mice and partly also in humans. The host inflammasome is regulated by GBPs in response to bacterial infection in murine cells and may also respond to Toxoplasma infection. Elucidation of murine Toxoplasma defense mechanisms are guiding studies on human cells, while inevitably leading to the discovery of human-specific pathways that often function in a cell type-dependent manner.
Topics: Animals; Autophagy; GTP-Binding Proteins; Humans; Toxoplasma; Toxoplasmosis; Vacuoles
PubMed: 29141239
DOI: 10.1016/j.mib.2017.10.021 -
Microbiology Spectrum Aug 2016This review is devoted to a consideration of the way in which the NADPH oxidase of neutrophils, NOX2, functions to enable the efficient killing of bacteria and fungi. It... (Review)
Review
This review is devoted to a consideration of the way in which the NADPH oxidase of neutrophils, NOX2, functions to enable the efficient killing of bacteria and fungi. It includes a critical examination of the current dogma that its primary purpose is the generation of hydrogen peroxide as substrate for myeloperoxidase-catalyzed generation of hypochlorite. Instead, it is demonstrated that NADPH oxidase functions to optimize the ionic and pH conditions within the vacuole for the solubilization and optimal activity of the proteins released into this compartment from the cytoplasmic granules, which kill and digest the microbes. The general role of other NOX systems as electrochemical generators to alter the pH and ionic composition in compartments on either side of a membrane in plants and animals will also be examined.
Topics: Animals; Bacteria; Fungi; Humans; Hydrogen-Ion Concentration; Microbial Viability; NADPH Oxidases; Neutrophils; Osmolar Concentration; Peroxidase; Vacuoles
PubMed: 27726789
DOI: 10.1128/microbiolspec.MCHD-0018-2015 -
Microbiology Spectrum Dec 2022Soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play conserved roles in membrane fusion events in eukaryotes and have been...
Soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play conserved roles in membrane fusion events in eukaryotes and have been documented to be involved in fungal growth and pathogenesis. However, little is known about the roles of SNAREs in trap morphogenesis in nematode-trapping fungi (NTF). , one of the constricting ring-forming NTF, captures free-living nematodes via rapid ring cell inflation. Here, we characterized DdVam7 of , a homolog of the yeast SNARE protein Vam7p. Deletion of significantly suppressed vegetative growth and conidiation. The mutation significantly impaired trap formation and ring cell inflation, resulting in a markedly decreased nematode-trapping ability. A large vacuole could develop in ring cells within ~2.5 s after instant inflation in . In the Δ mutant, the vacuoles were small and fragmented in hyphae and uninflated ring cells, and the large vacuole failed to form in inflated ring cells. The localization of DdVam7 in vacuoles suggests its involvement in vacuole fusion. In summary, our results suggest that DdVam7 regulates vegetative growth, conidiation, and the predatory process by mediating vacuole assembly in , and this provides a basis for studying mechanisms of SNAREs in NTF and ring cell rapid inflation. is a nematode-trapping fungus that can capture nematodes through a constricting ring, the most sophisticated trapping device. It is amazing that constricting ring cells can inflate to triple their size within seconds to capture a nematode. A large centrally located vacuole is a unique signature associated with inflated ring cells. However, the mechanism underpinning trap morphogenesis, especially vacuole dynamics during ring cell inflation, remains unclear. Here, we documented the dynamics of vacuole assembly during ring cell inflation via time-lapse imaging for the first time. We characterized a SNARE protein in (DdVam7) that was involved in vacuole assembly in hyphae and ring cells and played important roles in vegetative growth, conidiation, trap morphogenesis, and ring cell inflation. Overall, this study expands our understanding of biological functions of the SNARE proteins and vacuole assembly in NTF trap morphogenesis and provides a foundation for further study of ring cell rapid inflation mechanisms.
Topics: Animals; Ascomycota; Fungal Proteins; Nematoda; SNARE Proteins; Vacuoles
PubMed: 36287065
DOI: 10.1128/spectrum.01872-22 -
The New Phytologist Nov 2021Phosphorus (P) is an essential element for plant growth and development. Vacuoles play a fundamental role in the storage and remobilization of P in plants, while our... (Review)
Review
Phosphorus (P) is an essential element for plant growth and development. Vacuoles play a fundamental role in the storage and remobilization of P in plants, while our understanding of the evolutionary mechanisms of creating and reusing P stores are limited. Besides, we also know very little about the coordination of intercellular P translocation, neither the inorganic phosphate (Pi) signaling nor the Pi transport patterns. Here we summarize recent advances in understanding the core elements involved in cellular and/or subcellular P homeostasis and signaling in unicellular green algae and multicellular land plants. We also propose further work that might help to uncover the high-resolution intracellular and intercellular landscape of Pi distribution and signaling in plants.
Topics: Biological Transport; Chlorophyta; Embryophyta; Phosphates; Vacuoles
PubMed: 34482553
DOI: 10.1111/nph.17716 -
Pathogens and Disease Feb 2021Chlamydia trachomatis is the leading cause of infectious blindness and a sexually transmitted infection. All chlamydiae are obligate intracellular bacteria that... (Review)
Review
Chlamydia trachomatis is the leading cause of infectious blindness and a sexually transmitted infection. All chlamydiae are obligate intracellular bacteria that replicate within a membrane-bound vacuole termed the inclusion. From the confines of the inclusion, the bacteria must interact with many host organelles to acquire key nutrients necessary for replication, all while promoting host cell viability and subverting host defense mechanisms. To achieve these feats, C. trachomatis delivers an arsenal of virulence factors into the eukaryotic cell via a type 3 secretion system (T3SS) that facilitates invasion, manipulation of host vesicular trafficking, subversion of host defense mechanisms and promotes bacteria egress at the conclusion of the developmental cycle. A subset of these proteins intercalate into the inclusion and are thus referred to as inclusion membrane proteins. Whereas others, referred to as conventional T3SS effectors, are released into the host cell where they localize to various eukaryotic organelles or remain in the cytosol. Here, we discuss the functions of T3SS effector proteins with a focus on how advances in chlamydial genetics have facilitated the identification and molecular characterization of these important factors.
Topics: Bacterial Proteins; Chlamydia Infections; Chlamydia trachomatis; HeLa Cells; Host-Pathogen Interactions; Humans; Inclusion Bodies; Protein Transport; Type III Secretion Systems; Vacuoles; Virulence Factors
PubMed: 33512479
DOI: 10.1093/femspd/ftaa078 -
The Yale Journal of Biology and Medicine Sep 2019The parasitophorous vacuole membrane (LPVM) sits at the interface of the parasite and its host. Evidence shows that molecules from the endocytic pathway as well as... (Review)
Review
The parasitophorous vacuole membrane (LPVM) sits at the interface of the parasite and its host. Evidence shows that molecules from the endocytic pathway as well as molecules from the secretory pathway are localized in the LPV and displayed on LPVM. In the review, we discuss our current understanding of the composition of the LPVM.
Topics: Animals; Autophagy; Host-Parasite Interactions; Humans; Intracellular Membranes; Leishmania; Lipids; Vacuoles
PubMed: 31543712
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2017Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been...
Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.
Topics: Biological Transport; Fluorescence Resonance Energy Transfer; Listeria monocytogenes; Microscopy, Electron; Microscopy, Fluorescence; Vacuoles
PubMed: 27914079
DOI: 10.1007/978-1-4939-6673-8_11 -
Toxicology and Applied Pharmacology Feb 2012"Lysosomotropic" cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw... (Review)
Review
"Lysosomotropic" cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration.
Topics: Animals; Autophagy; Biological Transport; Cations; Endocytosis; Humans; Lysosomes; Mitosis; Myocytes, Smooth Muscle; Pharmaceutical Preparations; Vacuolar Proton-Translocating ATPases; Vacuoles
PubMed: 22198553
DOI: 10.1016/j.taap.2011.12.004