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Infection, Genetics and Evolution :... Jun 2021Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse...
Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse eukaryotic cells. Since several bacteria can enter into yeast cells, including Candida albicans, the aims of this work were to evaluate if L. monocytogenes was able to harbor, retaining its viability, within C. albicans cells and to evaluate the effect of temperature and an antibiotic as stressing factors in its rate of entry into yeast cells. Both microorganisms were co-incubated in BHI broth during 48 h and the entry of bacteria into yeast cells was evaluated at different times. Then, yeasts free of extracellular bacteria were obtained seeding samples of the co-culture on YGC agar, which contains chloramphenicol, to obtain extracellular bacteria-free yeasts. These extracellular bacteria free yeasts were used to search for bacterial DNA in total yeast DNA and to evaluate the viability of intra-yeast bacteria. Finally, the effect of temperature and of chloramphenicol as inducers of stress on the rate of bacterial entry into yeast cells were investigated. After co-culturing both microorganisms, wet mount optical microscopy showed the presence of moving bacteria within yeasts and transmission electron microscopy confirmed the presence of intra-yeast bacteria. PCR allowed to amplify L. monocytogenes iap gene in C. albicans total DNA obtained from yeasts free of extracellular bacteria. Moreover, the SYTO 9 green fluorescence observed in bacterial cells within vacuoles of yeasts suggests that intra-yeast bacteria remain viable. Furthermore, the entry of L. monocytogenes into yeasts cells was favored by the presence of stressing factors (chloramphenicol and temperature). Therefore, yeasts may be reservoirs of viable L. monocytogenes and might spread them to the following generations of yeasts.
Topics: Candida albicans; DNA, Bacterial; Disease Reservoirs; Listeria monocytogenes; Vacuoles
PubMed: 33639305
DOI: 10.1016/j.meegid.2021.104779 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2023As a generally-recognized-as-safe microorganism, is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology.... (Review)
Review
As a generally-recognized-as-safe microorganism, is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in . by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, . has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in . are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Golgi Apparatus; Metabolic Engineering; Vacuoles
PubMed: 37401597
DOI: 10.13345/j.cjb.221030 -
Plant Cell Reports Nov 2022Selective autophagy functions as a regulatory mechanism by targeting native and functional proteins to ensure their proper levels and activities in plant adaptive... (Review)
Review
Selective autophagy functions as a regulatory mechanism by targeting native and functional proteins to ensure their proper levels and activities in plant adaptive responses. Autophagy is a cellular degradation and recycling pathway with a key role in cellular homeostasis and metabolism. Autophagy is initiated with the biogenesis of autophagosomes, which fuse with the lysosomes or vacuoles to release their contents for degradation. Under nutrient starvation or other adverse environmental conditions, autophagy usually targets unwanted or damaged proteins, organelles and other cellular components for degradation and recycling to promote cell survival. Over the past decade, however, a substantial number of studies have reported that autophagy in plants also functions as a regulatory mechanism by targeting enzymes, structural and regulatory proteins that are not necessarily damaged or dysfunctional to ensure their proper abundance and function to facilitate cellular changes required for response to endogenous and environmental conditions. During plant-pathogen interactions in particular, selective autophagy targets specific pathogen components as a defense mechanism and pathogens also utilize autophagy to target functional host factors to suppress defense mechanisms. Autophagy also targets native and functional protein regulators of plant heat stress memory, hormone signaling, and vesicle trafficking associated with plant responses to abiotic and other conditions. In this review, we discuss advances in the regulatory roles of selective autophagy through targeting of native proteins in plant adaptive responses, what questions remain and how further progress in the analysis of these special regulatory roles of autophagy can help understand biological processes important to plants.
Topics: Autophagy; Vacuoles; Plants; Homeostasis; Signal Transduction
PubMed: 35922498
DOI: 10.1007/s00299-022-02910-w -
ACS Chemical Biology Jun 2022Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method...
Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in . Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the endoplasmic reticulum-vacuole-tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.
Topics: Cholesterol; Intermediate Filament Proteins; Lipid Metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sphingolipids; Vacuoles
PubMed: 35667650
DOI: 10.1021/acschembio.2c00120 -
Protoplasma Nov 2020Eukaryotic organisms share many common features in terms of endomembrane trafficking. This fact has helped plant scientists to propose testable hypotheses on how plant...
Eukaryotic organisms share many common features in terms of endomembrane trafficking. This fact has helped plant scientists to propose testable hypotheses on how plant intracellular membrane trafficking is achieved and regulated based on knowledge from yeast and mammals. However, when a new compartment has been identified in a plant cell that has a vesicle tethering complex located at a position which is completely different to its counterpart in yeast and mammalian cells, caution is demanded when interpreting possible interactions with other trafficking elements. This is exemplified by the recently discovered EMAC (ER and microtubule-associated compartment). It has been postulated that this compartment is the recipient of vacuolar sorting receptors (VSRs) transported retrogradely via "retromer vesicles" from a post-Golgi location. Unfortunately, this suggestion was based entirely on our knowledge of retromer from yeast and mammalian cells, and did not take into account the available literature on the composition, localization, and function of the plant retromer. It also lacked reference to recent contradictory findings on VSR trafficking. In this short article, we have tried to rectify this situation, pointing out that plant retromer may not function as a pentameric complex of two subunits: the retromer core and the sorting nexins.
Topics: Biological Transport; Golgi Apparatus; Sorting Nexins; Vacuoles
PubMed: 32780164
DOI: 10.1007/s00709-020-01543-8 -
Autophagy Sep 2023Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and...
Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and fusing with lysosomes/vacuoles, after which the materials are degraded for recycling purposes. Autophagy is associated with increased cell survival under different stress conditions. AP-lysosome/vacuole fusion is a critical step in autophagy. Some mutant cells can accumulate phagophores under autophagy-induction conditions. Autophagy is interrupted when accumulated phagophores cannot fuse with lysosomes/vacuoles, resulting in a significant decrease in cell survivability. However, phagophore-lysosome/vacuole fusion has been reported in related mammalian cells and yeast mutant cells. This observation indicates that it is possible to restore a partial autophagy process after interruption. Furthermore, these findings indicate that phagophore closure is not a prerequisite for its fusion with the lysosome/vacuole in the mutant cells. The phagophore-lysosome/vacuole fusion strategy can significantly rescue defective autophagy due to failed phagophore closure. This commentary discusses the fusion of phagophores and lysosomes/vacuoles and implications of such fusion events.: AB: autophagic body; AL: autolysosome; AP: autophagosome; ATG: autophagy related; EM: electron microscopy; ESCRT: endosomal sorting complex required for transport; ET: electron tomography; FIB: focus ion beam; IM: inner membrane; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; OM; outer membrane; STX17: syntaxin 17; TEM: transmission electron microscopy; TM: transmembrane domain; Vps: vacuolar protein sorting; WT: wild-type.
Topics: Animals; Autophagosomes; Vacuoles; Saccharomyces cerevisiae; Autophagy; Lysosomes; Membrane Fusion; Mammals
PubMed: 37083184
DOI: 10.1080/15548627.2023.2205272 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061 -
Development (Cambridge, England) Jun 2023Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs)...
Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical hematopoietic cells, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in avian HECs, and that aquaporin 1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence to indicate that morphological segregation of hematopoietic cells from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses.
Topics: Vacuoles; Cell Adhesion; Cell Differentiation; Hemangioblasts; Morphogenesis; Aquaporins; Hematopoiesis
PubMed: 37272531
DOI: 10.1242/dev.201275 -
Physiologia Plantarum Apr 2021Chloride channels (CLCs), member of anion transporting proteins, are present ubiquitously in all life forms. Diverging from its name, the CLC family includes both...
Chloride channels (CLCs), member of anion transporting proteins, are present ubiquitously in all life forms. Diverging from its name, the CLC family includes both channel and exchanger (proton-coupled) proteins; nevertheless, they share conserved structural organization. They are engaged in diverse indispensable functions such as acid and fluoride tolerance in prokaryotes to muscle stabilization, transepithelial transport, and neuronal development in mammals. Mutations in genes encoding CLCs lead to several physiological disorders in different organisms, including severe diseases in humans. Even in plants, loss of CLC protein function severely impairs various cellular processes critical for normal growth and development. These proteins sequester Cl into the vacuole, thus, making them an attractive target for improving salinity tolerance in plants caused by high abundance of salts, primarily NaCl. Besides, some CLCs are involved in NO transport and storage function in plants, thus, influencing their nitrogen use efficiency. However, despite their high significance, not many studies have been carried out in plants. Here, we have attempted to concisely highlight the basic structure of CLC proteins and critical residues essential for their function and classification. We also present the diverse functions of CLCs in plants from their first cloning back in 1996 to the knowledge acquired as of now. We stress the need for carrying out more in-depth studies on CLCs in plants, for they may have future applications towards crop improvement.
Topics: Biological Transport; Chloride Channels; Protons; Salt Tolerance; Vacuoles
PubMed: 33034380
DOI: 10.1111/ppl.13240 -
The Journal of Biological Chemistry Dec 2021Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and... (Review)
Review
Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.
Topics: Animals; Bacterial Proteins; Homeostasis; Host-Pathogen Interactions; Humans; Inflammation; Legionella pneumophila; Legionnaires' Disease; Type IV Secretion Systems; Ubiquitination; Vacuoles
PubMed: 34695417
DOI: 10.1016/j.jbc.2021.101340