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Molecular Microbiology Mar 2021Control of the host cell is crucial to the Apicomplexan parasite, Toxoplasma gondii, while it grows intracellularly. To achieve this goal, these single-celled eukaryotes... (Review)
Review
Control of the host cell is crucial to the Apicomplexan parasite, Toxoplasma gondii, while it grows intracellularly. To achieve this goal, these single-celled eukaryotes export a series of effector proteins from organelles known as "dense granules" that interfere with normal cellular processes and responses to invasion. While some effectors are found attached to the outer surface of the parasitophorous vacuole (PV) in which Toxoplasma tachyzoites reside, others are found in the host cell's cytoplasm and yet others make their way into the host nucleus, where they alter host transcription. Among the processes that are severely altered are innate immune responses, host cell cycle, and association with host organelles. The ways in which these crucial processes are altered through the coordinated action of a large collection of effectors is as elegant as it is complex, and is the central focus of the following review; we also discuss the recent advances in our understanding of how dense granule effector proteins are trafficked out of the PV.
Topics: Animals; Host-Pathogen Interactions; Humans; Immunity, Innate; Organelles; Protein Transport; Protozoan Proteins; Toxoplasma; Vacuoles; Virulence Factors
PubMed: 33400323
DOI: 10.1111/mmi.14679 -
Intracellular replication of in epithelial cells requires suppression of the caspase-4 inflammasome.MSphere Oct 2023Pathogenesis of infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three...
Pathogenesis of infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular , comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular , not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of . IMPORTANCE can exhibit an intracellular lifestyle within epithelial cells and . The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when is intracellular. Results showed that intracellular lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular , and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.
Topics: Humans; HeLa Cells; Pseudomonas aeruginosa; Inflammasomes; Epithelial Cells; Vacuoles
PubMed: 37589460
DOI: 10.1128/msphere.00351-23 -
Autophagy Apr 2024During starvation in the yeast vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There...
During starvation in the yeast vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 ( cells) or Npc2 ( cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that and cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells. BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza--Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; , yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, -(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; , yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD, optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.
Topics: Saccharomyces cerevisiae; Vacuoles; Deep Learning; Saccharomyces cerevisiae Proteins; Microscopy, Fluorescence; Autophagy; Lipid Droplets; Tomography, X-Ray
PubMed: 37908116
DOI: 10.1080/15548627.2023.2270378 -
FEBS Letters Sep 2022Autophagy fulfills a crucial role in plant cellular homeostasis by recycling diverse cellular components ranging from protein complexes to whole organelles. Autophagy... (Review)
Review
Autophagy fulfills a crucial role in plant cellular homeostasis by recycling diverse cellular components ranging from protein complexes to whole organelles. Autophagy cargos are shuttled to the vacuole for degradation, thereby completing the recycling process. Canonical autophagy requires the lipidation and insertion of ATG8 proteins into double-membrane structures, termed autophagosomes, which engulf the cargo to be degraded. As such, the autophagy pathway actively contributes to intracellular membrane trafficking. Yet, the autophagic process is not fully considered a bona fide component of the canonical membrane trafficking pathway. However, recent findings have started to pinpoint the interconnection between classical membrane trafficking pathways and autophagy. This review details the latest advances in our comprehension of the interplay between these two pathways. Understanding the overlap between autophagy and canonical membrane trafficking pathways is important to illuminate the inner workings of both pathways in plant cells.
Topics: Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Plants; Vacuoles
PubMed: 35593306
DOI: 10.1002/1873-3468.14404 -
Proceedings of the National Academy of... May 2023Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8...
Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear-vacuole junction (NVJ) and with Atg13 to mediate cytoplasm-to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, we determined the X-ray crystal structure of the Vac8-Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8-Nvj1 and Vac8-Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17-bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8-Nvj1 and Vac8-Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.
Topics: Saccharomyces cerevisiae; Vesicular Transport Proteins; Saccharomyces cerevisiae Proteins; Vacuoles; Cytoplasm; Protein Transport; Autophagy; Autophagy-Related Proteins; Adaptor Proteins, Signal Transducing; Receptors, Cell Surface
PubMed: 37094131
DOI: 10.1073/pnas.2211501120 -
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 -
Plant Signaling & Behavior May 2016The Arabidopsis NHX antiporter family contains eight members that are divided into three subclasses: vacuolar, endosomal, and plasma membrane. While the plasma membrane...
The Arabidopsis NHX antiporter family contains eight members that are divided into three subclasses: vacuolar, endosomal, and plasma membrane. While the plasma membrane and vacuolar NHXs have been studied extensively, the activity and function of the endosomal NHXs are beginning to be discovered. AtNHX5 and AtNHX6 are endosomal Na(+),K(+)/H(+) antiporters that share high sequence similarity. They are localized in the Golgi, trans-Golgi network (TGN), and prevacuolear compartment (PVC). Studies have shown that AtNHX5 and AtNHX6 mediate K(+) and Na(+) transport, and regulate cellular pH homeostasis. Sequence alignment has found that AtNHX5 and AtNHX6 contain four conserved acidic amino acid residues in transmembrane domains that align with yeast and human NHXs. Three of these conserved acidic residues are critical for K(+) transport and seedling growth in Arabidopsis. Moreover, studies have shown that the precursors of the seed storage proteins are missorted to the apoplast in the nhx5 nhx6 knockout mutant, suggesting that AtNHX5 and AtNHX6 regulate protein transport into the vacuole. Further analysis found that AtNHX5 and AtNHX6 regulated the binding of VSR to its cargoes. Taken together, AtNHX5 and AtNHX6 play an important role in cellular ion and pH homeostasis, and are essential for protein transport into the vacuole.
Topics: Arabidopsis; Arabidopsis Proteins; Biological Transport; Conserved Sequence; Endosomes; Homeostasis; Hydrogen-Ion Concentration; Ions; Potassium; Sodium-Hydrogen Exchangers; Vacuoles
PubMed: 26890367
DOI: 10.1080/15592324.2016.1147643 -
MBio Jan 2024Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of...
Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens.
Topics: Animals; Humans; Toxoplasma; Parasites; Proteins; Vacuoles; Autophagy; Protozoan Proteins
PubMed: 38095418
DOI: 10.1128/mbio.02595-23 -
PLoS Pathogens Jul 2023Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM)....
Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.
Topics: Animals; Mice; Toxoplasma; Protozoan Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Vacuoles; Nutrients
PubMed: 37498952
DOI: 10.1371/journal.ppat.1011543 -
Journal of Cell Science Nov 2019Autophagy is initiated by the formation of a phagophore assembly site (PAS), the precursor of autophagosomes. In mammals, autophagosome formation sites form throughout...
Autophagy is initiated by the formation of a phagophore assembly site (PAS), the precursor of autophagosomes. In mammals, autophagosome formation sites form throughout the cytosol in specialized subdomains of the endoplasmic reticulum (ER). In yeast, the PAS is also generated close to the ER, but always in the vicinity of the vacuole. How the PAS is anchored to the vacuole and the functional significance of this localization are unknown. Here, we investigated the role of the PAS-vacuole connection for bulk autophagy in the yeast We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13. lacking Vac8 show inefficient autophagosome-vacuole fusion, and form fewer and smaller autophagosomes that often localize away from the vacuole. Thus, the stable PAS-vacuole connection established by Vac8 creates a confined space for autophagosome biogenesis between the ER and the vacuole, and allows spatial coordination of autophagosome formation and autophagosome-vacuole fusion. These findings reveal that the spatial regulation of autophagosome formation at the vacuole is required for efficient bulk autophagy.
Topics: Autophagosomes; Autophagy; Membrane Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles; Vesicular Transport Proteins
PubMed: 31649143
DOI: 10.1242/jcs.235002