-
MBio Oct 2023Inflammasomes are essential for host defense against intracellular bacterial pathogens like , as they activate caspases, which promote cytokine release and cell death to...
Inflammasomes are essential for host defense against intracellular bacterial pathogens like , as they activate caspases, which promote cytokine release and cell death to control infection. In mice, interferon (IFN) signaling promotes inflammasome responses against bacteria by inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the -containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed . In contrast, the functions of IFN and GBPs in human inflammasome responses to are poorly understood. We show that IFN-γ enhances inflammasome responses to in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with and/or LCVs in a type IV secretion system (T4SS)-dependent manner and promotes damage to the LCV, which leads to increased exposure of the bacteria to the host cell cytosol. Thus, our findings reveal species- and pathogen-specific differences in how GBPs function to promote inflammasome responses.
Topics: Humans; Animals; Mice; Inflammasomes; Legionella; Vacuoles; Carrier Proteins; Signal Transduction; Legionella pneumophila; GTP-Binding Proteins
PubMed: 37737612
DOI: 10.1128/mbio.01707-23 -
The New Phytologist Sep 2023Pollen tubes have dynamic tubular vacuoles. Functional loss of AP-3, a regulator of one vacuolar trafficking route, reduces pollen tube growth. However, the role of...
Pollen tubes have dynamic tubular vacuoles. Functional loss of AP-3, a regulator of one vacuolar trafficking route, reduces pollen tube growth. However, the role of canonical Rab5 GTPases that are responsible for two other vacuolar trafficking routes in Arabidopsis pollen tubes is obscure. By using genomic editing, confocal microscopy, pollen tube growth assays, and transmission electron microscopy, we demonstrate that functional loss of canonical Rab5s in Arabidopsis, RHA1 and ARA7, causes the failure of pollen tubes to grow through style and thus impairs male transmission. Functional loss of canonical Rab5s compromises vacuolar trafficking of tonoplast proteins, vacuolar biogenesis, and turgor regulation. However, rha1;ara7 pollen tubes are comparable to those of wild-type in growing through narrow passages by microfluidic assays. We demonstrate that functional loss of canonical Rab5s compromises endocytic and secretory trafficking at the plasma membrane (PM), whereas the targeting of PM-associated ATPases is largely unaffected. Despite that, rha1;ara7 pollen tubes contain a reduced cytosolic pH and disrupted actin microfilaments, correlating with the mis-targeting of vacuolar ATPases (VHA). These results imply a key role of vacuoles in maintaining cytoplasmic proton homeostasis and in pollen tube penetrative growth through style.
Topics: Arabidopsis; Pollen Tube; Arabidopsis Proteins; GTP Phosphohydrolases; Adenosine Triphosphatases
PubMed: 37301984
DOI: 10.1111/nph.19059 -
Nature Communications Sep 2023Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for...
Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.
Topics: Actins; Vacuoles; Actin Cytoskeleton; Cell Physiological Phenomena; Osmosis
PubMed: 37730720
DOI: 10.1038/s41467-023-41337-z -
Advanced Science (Weinheim,... Aug 2023Demyelinating disorders are among the most common and debilitating diseases in neurology. Canavan disease (CD) is a lethal demyelinating disease caused by mutation of...
Demyelinating disorders are among the most common and debilitating diseases in neurology. Canavan disease (CD) is a lethal demyelinating disease caused by mutation of the aspartoacylase (ASPA) gene, which leads to the accumulation of its substrate N-acetyl-l-aspartate (NAA), and consequently demyelination and vacuolation in the brain. In this study, hypoimmunogenic human induced pluripotent stem cell (iPSC)-derived oligodendrocyte progenitor cells (OPC) are developed from a healthy donor as an "off-the-shelf" cell therapy. Hypoimmunogenic iPSCs are generated through CRISPR/Cas9 editing of the human leukocyte antigen (HLA) molecules in healthy donor-derived iPSCs and differentiated into OPCs. The OPCs are engrafted into the brains of CD (nur7) mice and exhibit widespread distribution in the brain. The engrafted OPCs mature into oligodendrocytes that express the endogenous wildtype ASPA gene. Consequently, the transplanted mice exhibit elevated human ASPA expression and enzymatic activity and reduced NAA level in the brain. The transplanted OPCs are able to rescue major pathological features of CD, including defective myelination, extensive vacuolation, and motor function deficits. Moreover, the hypoimmunogenic OPCs exhibit low immunogenicity both in vitro and in vivo. The hypoimmunogenic OPCs can be used as "off-the-shelf" universal donor cells to treat various CD patients and many other demyelinating disorders, especially autoimmune demyelinating diseases, such as multiple sclerosis.
Topics: Humans; Mice; Animals; Myelin Sheath; Induced Pluripotent Stem Cells; Oligodendrocyte Precursor Cells; Oligodendroglia; Multiple Sclerosis; Canavan Disease
PubMed: 37271923
DOI: 10.1002/advs.202206910 -
The EMBO Journal Jul 2023The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic...
The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.
Topics: Brucella abortus; Mitophagy; Vacuoles; Endoplasmic Reticulum; Mitochondria
PubMed: 37232029
DOI: 10.15252/embj.2022112817 -
PLoS Pathogens Aug 2023Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms...
Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.
Topics: Humans; Sandfly fever Naples virus; Endocytosis; Endosomes; Vacuoles; Virus Internalization; Hydrogen-Ion Concentration
PubMed: 37578957
DOI: 10.1371/journal.ppat.1011562 -
Molecular Therapy : the Journal of the... May 2024Sialidosis (mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the...
Sialidosis (mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the disease is deficiency of the lysosomal sialidase NEU1, resulting in accumulation of sialylated glycoproteins/oligosaccharides in tissues and body fluids. Neu1 mice recapitulate the severe, early-onset forms of the disease, affecting visceral organs, muscles, and the nervous system, with widespread lysosomal vacuolization evident in most cell types. Sialidosis is considered an orphan disorder with no therapy currently available. Here, we assessed the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis. Neu1 mice were co-injected with two scAAV2/8 vectors, expressing human NEU1 and its chaperone PPCA. Treated mice were phenotypically indistinguishable from their WT controls. NEU1 activity was restored to different extent in most tissues, including the brain, heart, muscle, and visceral organs. This resulted in diminished/absent lysosomal vacuolization in multiple cell types and reversal of sialyl-oligosacchariduria. Lastly, normalization of lysosomal exocytosis in the cerebrospinal fluids and serum of treated mice, coupled to diminished neuroinflammation, were measures of therapeutic efficacy. These findings point to AAV-mediated gene therapy as a suitable treatment for sialidosis and possibly other diseases, associated with low NEU1 expression.
PubMed: 38796704
DOI: 10.1016/j.ymthe.2024.05.029 -
IScience Jul 2023Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and...
Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.
PubMed: 37456828
DOI: 10.1016/j.isci.2023.107218 -
Journal of Clinical Medicine Feb 2024VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently recognized systemic autoinflammatory disease caused by somatic mutations in...
VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently recognized systemic autoinflammatory disease caused by somatic mutations in hematopoietic progenitor cells. This case series of four patients with VEXAS syndrome and comorbid myelodysplastic syndrome (MDS) aims to describe clinical, imaging, and hematologic disease presentations as well as response to therapy. Four patients with VEXAS syndrome and MDS are described. A detailed analysis of imaging features, hemato-oncological presentation including bone marrow microscopy and clinical-rheumatological disease features and treatment outcomes is given. All patients were male; ages ranged between 64 and 81 years; all were diagnosed with MDS. CT imaging was available for three patients, all of whom exhibited pulmonary infiltrates of varying severity, resembling COVID-19 or hypersensitivity pneumonitis without traces of scarring. Bone marrow microscopy showed maturation-disordered erythropoiesis and pathognomonic vacuolation. Somatic mutation in the codon 41 were found in all patients by next-generation sequencing. Therapy regimes included glucocorticoids, JAK1/2-inhibitors, nucleoside analogues, as well as IL-1 and IL-6 receptor antagonists. No fatalities occurred (observation period from symptom onset: 18-68 months). Given the potential underreporting of VEXAS syndrome, we highly recommend contemporary screening for mutations in patients presenting with ambiguous signs of systemic autoinflammatory symptoms which persist over 18 months despite treatment. The emergence of cytopenia, especially macrocytic hyperchromic anemia, should prompt early testing for mutations. Notably conspicuous, pulmonary alterations in CT imaging of patients with therapy-resistant systemic autoinflammatory symptoms should be discussed in interdisciplinary medical teams (Rheumatology, Hematology, Radiology and further specialist departments) to facilitate timely diagnosis during the clinical course of the disease.
PubMed: 38398362
DOI: 10.3390/jcm13041049 -
Archives of Microbiology Oct 2023Anhydrobiosis is a state of living organisms during which their metabolism is reversibly delayed or suspended due to a high degree of dehydration. Yeast cells, which are... (Review)
Review
Anhydrobiosis is a state of living organisms during which their metabolism is reversibly delayed or suspended due to a high degree of dehydration. Yeast cells, which are widely used in the food industry, may be induced into this state. The degree of viability of yeast cells undergoing the drying process also depends on rehydration. In an attempt to explain the essence of the state of anhydrobiosis and clarify the mechanisms responsible for its course, scientists have described various cellular compounds and structures that are responsible for it. The structures discussed in this work include the cell wall and plasma membrane, vacuoles, mitochondria, and lysosomes, among others, while the most important compounds include trehalose, glycogen, glutathione, and lipid droplets. Various proteins (Stf2p; Sip18p; Hsp12p and Hsp70p) and genes (STF2; Nsip18; TRX2; TPS1 and TPS2) are also responsible for the process of anhydrobiosis. Each factor has a specific function and is irreplaceable, detailed information is presented in this overview.
Topics: Saccharomyces cerevisiae; Gene Expression Regulation; Cell Membrane; Lipid Droplets; Vacuoles
PubMed: 37782422
DOI: 10.1007/s00203-023-03683-w