Vacuolar Rupture Research Articles

Rational design and discovery of novel hydrazide derivatives as potent succinate dehydrogenase inhibitors inspired by natural d/l-camphor.

Succinate dehydrogenase inhibitors (SDHIs) have rapidly become one of the fastest-growing categories of fungicides used against plant pathogenic fungi. Recent research advancements have emphasized that structural modifications of SDHIs using naturally sourced scaffolds represent an innovative strategy for developing new, highly effective, broad-spectrum fungicides. A novel series of d/l-camphorhydrazide derivatives potentially targeting fungal succinate dehydrogenase (SDH) were designed, synthesized and evaluated for their antifungal effects against Rhizoctonia solani, Fusarium graminearum, Valsa mali and Botrytis cinerea. Amongst them, compounds A1-7 (d-camphor) and A2-7 (l-camphor) displayed excellent in vitro activity against R. solani with median effective concentration (EC50) values of 0.38 and 0.48 μg mL-1, which were obviously superior to that of boscalid (0.87 μg mL-1). A2-5 (l-camphor, EC50 = 3.27 μg mL-1) exhibited good activity against V. mali. A2-7 (2.13 μg mL-1), A2-21 (5.2 μg mL-1) and A1-5 (5.15 μg mL-1) showed good antifungal activity against F. graminearum with EC50 values below that of boscalid (5.85 μg mL-1). Preliminary mechanistic studies, using scanning and transmission electron microscopy, indicated that compound A1-7 induced disordered entanglement of hyphae, shrinkage of hyphal surfaces, and vacuole swelling and rupture, which disrupted normal hyphal growth. Additionally, compound A1-7 induced the production and accumulation of reactive oxygen species, disrupted mitochondrial membrane potential, and effectively inhibited the germination and formation of sclerotia in R. solani. Moreover, the molecular docking results and SDH enzyme assays yielded promising outcomes. In this study, the designed and optimized compounds A1-7 and A2-7 emerged as promising candidates for SDH-targeting fungicides, demonstrating strong antifungal activity. These compounds hold potential as new antifungal agents for further research. © 2024 Society of Chemical Industry.

Development and Holocrine Secretion of Resin Ducts in Kielmeyera appariciana (Calophyllaceae).

The modes of formation and release of secretion are complex processes that occur in secretory ducts and their description has great divergence in some species. The use of modern techniques to detect hydrolytic enzymes, cytoskeleton arrangement and indicators of programmed cell death may help clarify the processes involved during the ontogeny of that gland. The goal of our study was to analyze subcellular changes during schizogenous formation and secretion production and release into the lumen in resin ducts of Kielmeyera appariciana. Our results demonstrate the participation of pectinase through the loosening of the central cells of the rosette, which subsequently split from each other through polarized growth mediated by a rearrangement of the microtubules. The resin is mainly synthesized in plastids and endoplasmic reticulum and is observed inside vesicles and small vacuoles. The secretion release is holocrine and occurs through programmed cell death related to the release of reactive oxygen species, causing cytoplasm darkening, chromatin condensation, vacuole rupture and plastid and mitochondria degeneration. Cellulase activity was identified prior to the rupture of the cell wall, causing the release of secretion into the lumen of the duct. The participation of the cytoskeleton was observed for the first time during schizogeny of ducts as well as programmed cell death as part of the process of the release of holocrine secretion. This type of secretion release may be a key innovation in Kielmeyera since it has not been observed in ducts of any other plant thus far.

Novel 5-Sulfonyl-1,3,4-thiadiazole-Substituted Flavonoids as Potential Bactericides and Fungicides: Design, Synthesis, Three-Dimensional Quantitative Structure-Activity Relationship Studies.

Flavonoids, ubiquitous natural products, provide sources for drug discovery owing to their structural diversity, broad-spectrum pharmacological activity, and excellent environmental compatibility. To develop antibacterial and antifungal agents with novel mechanisms of action and innovative structures, a series of novel 5-sulfonyl-1,3,4-thiadiazole-substituted flavonoids were designed and synthesized, and their biological activities against seven agriculturally common phytopathogenic microorganisms were evaluated. The results of the antimicrobial bioassay showed that most of the target compounds displayed excellent inhibitory effects against Xanthomonas oryzae, Rhizoctonia solani, and Colletotrichum orbiculare. Compounds 1, 3, 7, 9, 13, and 14 exhibited remarkable antibacterial activity against X. oryzae pv. oryzae with EC50 values below 10 μg/mL, which were superior to bismerthiazol (70.89 μg/mL). Compound 2 (EC50 = 0.41 μg/mL) displayed the most effective inhibitory potency against R. solani in vivo, comparable protective effects with the positive control carbendizam. Preliminary mechanistic studies indicated that compound 2 induced disordered entanglement of hyphae, shrinkage of hyphal surfaces, extravasation of cellular contents, and vacuole swelling and rupture, which disrupted normal hyphal growth. Subsequently, compounds 35-53 with good antifungal activity were designed and synthesized based on reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) models. Compound 49 showed high efficacy and superior antifungal activity against R. solani, with an EC50 value of 0.28 μg/mL and a half-maximal effective concentration of 0.46 μg/mL.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Human GBP1 facilitates the rupture of the Legionella-containing vacuole and inflammasome activation.

Inflammasomes are essential for host defense against intracellular bacterial pathogens like Legionella, 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 Legionella-containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed Legionella. In contrast, the functions of IFN and GBPs in human inflammasome responses to Legionella are poorly understood. We show that IFN-γ enhances inflammasome responses to Legionella in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with Legionella 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.

Involvement of Vacuolar Processing Enzyme CgVPE1 in Vacuole Rupture in the Programmed Cell Death during the Development of the Secretory Cavity in Citrus grandis 'Tomentosa' Fruits.

Vacuolar processing enzymes (VPEs) with caspase-1-like activity are closely associated with vacuole rupture. The destruction of vacuoles is one of the characteristics of programmed cell death (PCD) in plants. However, whether VPE is involved in the vacuole destruction of cells during secretory cavity formation in Citrus plants remains unclear. This research identified a CgVPE1 gene that encoded the VPE and utilized cytology and molecular biology techniques to explore its temporal and spatial expression characteristics during the PCD process of secretory cavity cells in the Citrus grandis 'Tomentosa' fruit. The results showed that CgVPE1 is an enzyme with VPE and caspase-1-like activity that can self-cleave into a mature enzyme in an acidic environment. CgVPE1 is specifically expressed in the epithelial cells of secretory cavities. In addition, it mainly accumulates in vacuoles before it is ruptured in the secretory cavity cells. The spatial and temporal immunolocalization of CgVPE1 showed a strong relationship with the change in vacuole structure during PCD in secretory cavity cells. In addition, the change in the two types of VPE proteins from proenzymes to mature enzymes was closely related to the change in CgVPE1 localization. Our results indicate that CgVPE1 plays a vital role in PCD, causing vacuole rupture in cells during the development of the secretory cavity in C. grandis 'Tomentosa' fruits.

Localization of CgVPE1 in secondary cell wall formation during tracheary element differentiation in the pericarp of Citrus grandis 'Tomentosa' fruits.

CgVPE1 is important in the differentiation of TE cells in C. grandis 'Tomentosa' fruits as it may directly affects secondary cell wall construction while participating in PCD. The vacuolar processing enzyme (VPE) plays an important role in both developmental and environmentally inducible programmed cell death (PCD); it was originally identified as a cysteine protease localized in the vacuole to activate and mature vacuolar proteins in plants. Interestingly, we found a VPE called CgVPE1 to be associated with deposition of the secondary cell wall in tracheary element (TE) cells in the pericarp of Citrus grandis 'Tomentosa' fruits. We then used ultrathin sections and the TUNEL assay to verify that PCD is involved in TE development. Furthermore, CgVPE1 was found to be mainly expressed in secretory cavities and TEs in the pericarp of Citrus grandis 'Tomentosa' fruits. Immunolocalization of CgVPE1 in the pericarp indicated that CgVPE1 is mainly distributed in the central large vacuole, endoplasmic reticulum, Golgi vesicles, cytosol, and secondary wall before TE maturation. CgVPE1 appeared earlier in the endoplasmic reticulum and Golgi vesicles of TEs cells. The vesicles containing CgVPE1 near the large central vacuole and secondary wall were observed, respectively. CgVPE1 proteins content in the cytoplasm decreased sharply, while the CgVPE1 content in the secondary cell wall did not change significantly after vacuole rupture. CgVPE1 protein contents in the secondary cell wall were significantly reduced until the TE cells developed into hollow thick-walled cells. Furthermore, labeling of VPE homologues in Arabidopsis thaliana using immunoelectron microscopy with anti-CgVPE1 antibody revealed that VPE homologues were specifically distributed in the secondary cell wall of stem TEs. Overall, these results suggested that CgVPE1 is not only involved PCD during TE cell development; furthermore, it may directly participate in the construction of plant secondary cell walls.

Low Temperature Heating-Induced Death and Vacuole Injury in Cladosporium sphaerospermum Conidia.

The mechanism of thermal death of mold conidia has not been understood in detail. The purpose of this study is to analyze the death kinetics of heated conidia of Cladosporium sphaerospermum and to ascertain the expectant cell injury responsible for the death. The death of the dormant (resting) conidia of Cladosporium sphaerospermum was examined at temperatures of between 43 and 54℃ with the conventional colony count method. The death reaction apparently followed the first order kinetics, but the Arrhenius plot of the death rate constant demonstrated seemingly a break. The linearity at temperatures higher than that at the break was lost at lower temperatures, suggesting the involvement of an unusual mechanism in the latter temperatures. In the cell morphology, we observed with quinacrine staining the vacuole rupture at a lower temperature but not at a high temperature. Interestingly, the vacuole rupture by low-temperature heating was found to correlate with the viability loss. Furthermore, active protease originally locating in vacuoles was detected in the cytoplasm of the conidia after heated at a low temperature. The results obtained suggest the involvement of potent autophagic cell death induced by low temperature heating of C. sphaerospermum conidia.

Disruption of the Interfacial Membrane Leads to Magnaporthe oryzae Effector Re-location and Lifestyle Switch During Rice Blast Disease.

To cause the devastating rice blast disease, the hemibiotrophic fungus Magnaporthe oryzae produces invasive hyphae (IH) that are enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that the disruption of the EIHM correlates with the hyphal growth stage in first-invaded susceptible rice cells. Our approach utilized GFP that was secreted from IH as an EIHM integrity reporter. Secreted GFP (sec-GFP) accumulated in the EIHM compartment but appeared in the host cytoplasm when the integrity of the EIHM was compromised. Live-cell imaging coupled with sec-GFP and various fluorescent reporters revealed that the loss of EIHM integrity preceded shrinkage and eventual rupture of the rice vacuole. The vacuole rupture coincided with host cell death, which was limited to the invaded cell with presumed closure of plasmodesmata. We report that EIHM disruption and host cell death are landmarks that delineate three distinct infection phases (early biotrophic, late biotrophic, and transient necrotrophic phases) within the first-invaded cell before reestablishment of biotrophy in second-invaded cells. M. oryzae effectors exhibited infection phase-specific localizations, including entry of the apoplastic effector Bas4 into the host cytoplasm through the disrupted EIHM during the late biotrophic phase. Understanding how infection phase-specific cellular dynamics are regulated and linked to host susceptibility will offer potential targets that can be exploited to control blast disease.

Impact of mitochondrial aldehyde dehydrogenase 2 on cognitive impairment in the AD model mouse.

Alzheimer's disease (AD) is one of the major life-threatening diseases for the elderly because neither pathogenesis nor effective treatment is available. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) has been shown to reduce the cell-damaging aldehydes in response to reactive oxygen species (ROS). However, whether it plays a role in AD remains elusive. In the present study, we found that ALDH2 overexpression significantly improved the cognitive function of the AD mouse. Behavioral analyses of ALDH2-overexpressing APP/PS1 AD mice showed that the learning and cognitive abilities were significantly higher in these mice than in the control group APP/PS1 mice. Further open-field behavior experiments showed the same results. At the cellular level, ALDH2 protects nerve cells. HT22 cells were challenged with Aβ to establish an AD cell model, in the presence or absence of the ALDH2 activator Alda-1 and ALDH2 inhibitor Daidzin. Incubation with 50 μM Aβ for 24 h significantly reduced HT22 cell survival and cell viability, the effects of which were attenuated by the ALDH2 activator Alda-1 (50 μM). Aβ challenge promoted apoptosis and upregulated caspase3 level but suppressed Bcl-2 level, and the upregulated caspase3 level was reversed by the ALDH-2 agonist Alda-1. Aβ-induced clonal ball abnormal was reversed by Alda-1. Aβ altered the mitochondria geometry evidenced by vacuolar degeneration and membrane rupture, whereas Alda-1 changed the Aβ-induced mitochondria geometry anomalies. Moreover, superoxide anion and toxic 4-hydroxy-nonanal (4-HNE) and ROS increased by Aβ challenge were reversed by Alda-1. Meanwhile, Aβ-induced ATP reduction was reversed by Alda-1. Taken together, ALDH2 overexpression significantly improves the cognitive function of the AD mice. Furthermore, our results suggested that ALDH2 protects against Aβ hippocampal neuronal toxicity possibly through alleviating toxic aldehydes and ROS, as well as increasing ATP production to preserve mitochondrial integrity and reduce neuronal apoptosis.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

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.

Processes controlling programmed cell death of root velamen radicum in an epiphytic orchid.

Development of the velamen radicum on the outer surface of the root epidermis is an important characteristic for water uptake and retention in some plant families, particularly epiphytic orchids, for survival under water-limited environments. Velamen radicum cells derive from the primary root meristem; however, following this development, velamen radicum cells die by incompletely understood processes of programmed cell death (PCD). We combined the use of transmission electron microscopy, X-ray micro-tomography and transcriptome methods to characterize the major anatomical and molecular changes that occur during the development and death of velamen radicum cells of Cymbidium tracyanum, a typical epiphytic orchid, to determine how PCD occurs. Typical changes of PCD in anatomy and gene expression were observed in the development of velamen radicum cells. During the initiation of PCD, we found that both cell and vacuole size increased, and several genes involved in brassinosteroid and ethylene pathways were upregulated. In the stage of secondary cell wall formation, significant anatomical changes included DNA degradation, cytoplasm thinning, organelle decrease, vacuole rupture and cell wall thickening. Changes were found in the expression of genes related to the biosynthesis of cellulose and lignin, which are instrumental in the formation of secondary cell walls, and are regulated by cytoskeleton-related factors and phenylalanine ammonia-lyase. In the final stage of PCD, cell autolysis was terminated from the outside to the inside of the velamen radicum. The regulation of genes related to autophagy, vacuolar processing enzyme, cysteine proteases and metacaspase was involved in the final execution of cell death and autolysis. Our results showed that the development of the root velamen radicum in an epiphytic orchid was controlled by the process of PCD, which included initiation of PCD, followed by formation of the secondary cell wall, and execution of autolysis following cell death.

Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination.

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.

Vacuolar processing enzymes in the plant life cycle.

Vacuolar processing enzyme (VPE) is a cysteine-type endopeptidase that has a substrate-specificity for asparagine or aspartic acid residues and cleaves peptide bonds at their carboxyl-terminal side. Various vacuolar proteins are synthesized as larger proprotein precursors, and VPE is an important initiator of maturation and activation of these proteins. It mediates programmed cell death (PCD) by provoking vacuolar rupture and initiating the proteolytic cascade leading to PCD. Vacuolar processing enzyme also possesses a peptide ligation activity, which is responsible for producing cyclic peptides in several plant species. These unique functions of VPE support developmental and environmental responses in plants. The number of VPE homologues is higher in angiosperm species, indicating that there has been differentiation and specialization of VPE function over the course of evolution. Angiosperm VPEs are separated into two major types: the γ-type VPEs, which are expressed mainly in vegetative organs, and the β-type VPEs, whose expression occurs mainly in storage organs; in eudicots, the δ-type VPEs are further separated within γ-type VPEs. This review also considers the importance of processing and peptide ligation by VPE in vacuolar protein maturation.

Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants.

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.

Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing.

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses

SUMMARYLegionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition,endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/ STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.

Intracellular rupture, exocytosis and actin interaction of endocytic vacuoles in pancreatic acinar cells: initiating events in acute pancreatitis.

Key points Giant trypsin‐containing endocytic vacuoles are formed in pancreatic acinar cells stimulated with inducers of acute pancreatitis.F‐actin envelops endocytic vacuoles and regulates their properties.Endocytic vacuoles can rupture and release their content into the cytosol of acinar cells.Endocytic vacuoles can fuse with the plasma membrane of acinar cells and exocytose their content. Intrapancreatic activation of trypsinogen is an early event in and hallmark of the development of acute pancreatitis. Endocytic vacuoles, which form by disconnection and transport of large post‐exocytic structures, are the only resolvable sites of the trypsin activity in live pancreatic acinar cells. In the present study, we characterized the dynamics of endocytic vacuole formation induced by physiological and pathophysiological stimuli and visualized a prominent actin coat that completely or partially surrounded endocytic vacuoles. An inducer of acute pancreatitis taurolithocholic acid 3‐sulphate and supramaximal concentrations of cholecystokinin triggered the formation of giant (more than 2.5 μm in diameter) endocytic vacuoles. We discovered and characterized the intracellular rupture of endocytic vacuoles and the fusion of endocytic vacuoles with basal and apical regions of the plasma membrane. Experiments with specific protease inhibitors suggest that the rupture of endocytic vacuoles is probably not induced by trypsin or cathepsin B. Perivacuolar filamentous actin (observed on the surface of ∼30% of endocytic vacuoles) may play a stabilizing role by preventing rupture of the vacuoles and fusion of the vacuoles with the plasma membrane. The rupture and fusion of endocytic vacuoles allow trypsin to escape the confinement of a membrane‐limited organelle, gain access to intracellular and extracellular targets, and initiate autodigestion of the pancreas, comprising a crucial pathophysiological event.

Changes in the morphology and functions of vacuoles during the death of ray parenchyma cells in Cryptomeria japonica

Changes in the morphology and functions of vacuoles provide useful information about the mechanism of cell death. In the present study, we monitored the morphology and contents of vacuoles during the death of ray parenchyma cells in the conifer Cryptomeria japonica. In differentiating xylem, ray parenchyma cells had large central vacuoles. In sapwood, vacuoles in ray parenchyma cells contained proteins, an indication that one of the main functions of these vacuoles might be protein storage. A dramatic decrease in the protein content of some vacuoles was detected in the intermediate wood before the initiation of vacuole rupture. Although vacuole rupture was detected from the intermediate wood to the outermost heartwood, some vacuoles were obviously enlarged in the inner intermediate wood. Condensed nuclei were first observed after the rupture of these large vacuoles in ray parenchyma cells. It seems plausible that the autolysis of the contents of ray parenchyma cells might be caused by the rupture of the enlarged vacuoles in the inner intermediate wood.

Imaging macropinosomes during Shigella infections.

Macropinocytosis is the uptake of extracellular fluid within vesicles of varying size that takes place during numerous cellular processes in a large variety of cells. A growing number of pathogens, including viruses, parasites, and bacteria are known to induce macropinocytosis during their entry into targeted host cells. We have recently discovered that the human enteroinvasive, bacterial pathogen Shigella causes in situ macropinosome formation during its entry into epithelial cells. These infection-associated macropinosomes are not generated to ingest the bacteria, but are instead involved in Shigella's intracellular niche formation. They make contacts with the phagocytosed shigellae to promote vacuolar membrane rupture and their cytosolic release. Here, we provide an overview of the different imaging approaches that are currently used to analyze macropinocytosis during infectious processes with a focus on Shigella entry. We detail the advantages and disadvantages of genetically encoded reporters as well as chemical probes to trace fluid phase uptake. In addition, we report how such reporters can be combined with ultrastructural approaches for correlative light electron microscopy either in thin sections or within large volumes. The combined imaging techniques introduced here provide a detailed characterization of macropinosomes during bacterial entry, which, apart from Shigella, are relevant for numerous other ones, including Salmonella, Brucella or Mycobacteria.