Vacuolar Integrity Research Articles

PH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans.

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis.

A HOPS Protein, MoVps41, Is Crucially Important for Vacuolar Morphogenesis, Vegetative Growth, Reproduction and Virulence in Magnaporthe oryzae.

The homotypic fusion and protein sorting protein complex (HOPS) is the first known tether complex identified in the endocytic system that plays a key role in promoting homotypic vacuolar fusion, vacuolar biogenesis and trafficking in a wide range of organisms, including plant and fungi. However, the exact influence of the HOPS complex on growth, reproduction and pathogenicity of the economically destructive rice blast fungus has not been investigated. In this study, we identified M. oryzae vacuolar protein sorting 41 (MoVps41) an accessory subunit of HOPS complex and used targeted gene deletion approach to evaluate its contribution to growth, reproduction and infectious life cycle of the rice blast fungus. Corresponding results obtained from this study showed that MoVps41 is required for optimum vegetative development of M. oryzae and observed that MoVps41 deletion mutant displayed defective vegetative growth. Our investigation further showed that MoVps41 deletion triggered vacuolar fragmentation, compromised membrane integrity and pathogenesis of the ΔMovps41 mutant. Our studies also showed for the first time that MoVps41 plays an essential role in the regulation of sexual and asexual reproduction of M. oryzae. In summary, our study provides insight into how MoVps41 mediated vacuolar fusion and biogenesis influences reproduction, pathogenesis, and vacuolar integrity in M. oryzae and also underscores the need to holistically investigate the HOPS complex in rice blast pathogen.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Asymmetric Inheritance of Aggregated Proteins and Age Reset in Yeast Are Regulated by Vac17-Dependent Vacuolar Functions

SummaryAge can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one. This phenomenon requires asymmetry-generating genes (AGGs) that govern the asymmetrical inheritance of aggregated proteins. Using a genome-wide imaging screen to identify AGGs in Saccharomyces cerevisiae, we discovered a previously unknown role for endocytosis, vacuole fusion, and the myosin-dependent adaptor protein Vac17 in asymmetrical inheritance of misfolded proteins. Overproduction of Vac17 increases deposition of aggregates into cytoprotective vacuole-associated sites, counteracts age-related breakdown of endocytosis and vacuole integrity, and extends replicative lifespan. The link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles. We also show that the protein disaggregase Hsp104 interacts physically with endocytic vesicle-associated proteins, such as the dynamin-like protein, Vps1, which was also shown to be required for Vac17-dependent sequestration of protein aggregates. These data demonstrate that two physiognomies of aging—reduced endocytosis and protein aggregation—are interconnected and regulated by Vac17.

Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

Screening for Drugs Against the Plasmodium falciparum Digestive Vacuole by Imaging Flow Cytometry.

Phenotypic assays are increasingly employed to provide clues about drug mechanisms. In antimalarial drug screening, however, the majority of assays are designed to only measure parasite-killing activity. We describe here a high-content assay to detect drug-mediated perturbation of the digestive vacuole integrity in the trophozoite stage of Plasmodium falciparum, using the ImageStream imaging flow cytometer.

Self- and nonself recognition during hyphal interactions in Rosellinia necatrix

Hyphal interactions of 10 Rosellinia necatrix isolates were analyzed. Most incompatible pairings produced broad melanized barrage lines, with a few exceptions. The variation in barrage lines revealed using light microscopy might result from distinct frequencies of different hyphal interaction types. In incompatible pairings, hyphal anastomosis was rarely observed. Activated-charcoal treatment of oatmeal agar media prevented both incompatible and compatible reactions. FUN-1 staining showed vacuole dysfunction at hyphal interaction sites, and the localization of a Lifeact-GFP mutant revealed that actin dynamics was prominent in compatible pairings. These results suggested that self/nonself recognition commonly proceeded to vacuole dysfunction and that actin dynamics played a critical role in the restoration of vacuole integrity.

Mechanosensitive channels Msy1 and Msy2 are required for maintaining organelle integrity upon hypoosmotic shock in Schizosaccharomyces pombe.

The mechanosensitive channels, Mys1 and Msy2, in fission yeast are localized in the endoplasmic reticulum membrane and control cytoplasmic Ca(2+) levels in the hypoosmotic response. We here investigated changes in organellar structures with hypoosmotic shock using transmission electron microscopy. While msy1(-) and msy2(-) single mutant cells developed a number of swollen vacuoles following hypoosmotic shock, similar to wild-type cells, msy1(-) msy2(-) double mutant cells only had two abnormally large vacuoles and cracks between the inner and outer nuclear membranes. These results suggest that Msy1 and Msy2 may be involved in maintaining vacuole integrity and protecting the nuclear envelope upon hypoosmotic shock and also that these two channels are functionally complementary.

In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system.

In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S c₁₀-, S₇₀-, and S₇₅-haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions.

Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings. Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H2O2) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ. The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H2O2 synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole. The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.

Identification ofSalmonellaPathogenicity Island-2 Type III Secretion System Effectors Involved in Intramacrophage Replication of S. enterica Serovar Typhimurium: Implications for Rational Vaccine Design

ABSTRACTSalmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells.

Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Vacuolar pathogens value membrane integrity

Bacteria commonly encode virulence determinants that mediate the disruption of host cell membranes; however, vacuole lysis and invasion of the host cell cytosol is a strategy used by only a select few bacterial pathogens for intracellular replication (1). Many bacterial pathogens replicate in specialized vacuoles that must be actively maintained to prevent these organelles from fusing with lysosomes and to regulate acquisition of new membrane for vacuole expansion during bacterial replication (2). However, why is this, when, in theory, it should be easier for a bacterium to lyse the vacuole and replicate in the cytosol? A study in PNAS provides new data that demonstrate that vacuole integrity is important for the intracellular survival of Legionella pneumophila and illustrate how invasion of the host cytosol can play a role in countering bacterial infection (3).

V-ATPase, ScNhx1p and Yeast Vacuole Fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

The protein SdhA maintains the integrity of the Legionella -containing vacuole

Legionella pneumophila directs the formation of a specialized vacuole within host cells, dependent on protein substrates of the Icm/Dot translocation system. Survival of the host cell is essential for intracellular replication of L. pneumophila. Strains lacking the translocated substrate SdhA are defective for intracellular replication and activate host cell death pathways in primary macrophages. To understand how SdhA promotes evasion of death pathways, we performed a mutant hunt to identify bacterial suppressors of the ΔsdhA growth defect. We identified the secreted phospholipase PlaA as key to activation of death pathways by the ΔsdhA strain. Based on homology between PlaA and SseJ, a Salmonella protein associated with vacuole degradation, we determined the roles of SdhA and PlaA in controlling vacuole integrity. In the absence of sdhA, the Legionella-containing vacuole was unstable, resulting in access to the host cytosol. Both vacuole disruption and host cell death were largely dependent on PlaA. Consistent with these observations, the ΔsdhA strain colocalized with galectin-3, a marker of vacuole rupture, in a PlaA-dependent process. Access of ΔsdhA strains to the macrophage cytosol triggered multiple responses in the host cell, including degradation of bacteria, induction of the type I IFN response, and activation of inflammasomes. Therefore, we have demonstrated that the Legionella-containing vacuole is actively stabilized by the SdhA protein during intracellular replication. This vacuolar niche affords the bacterium protection from cytosolic host factors that degrade bacteria and initiate immune responses.

Multiple roles for membrane-associated protein trafficking and signaling in gravitropism

Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature.

Lactic-acid stress causes vacuolar fragmentation and impairs intracellular amino-acid homeostasis in Saccharomyces cerevisiae

To gain more insight into adaptation response to lactic-acid stress in yeast, a genome-wide screening for genes whose disruption caused hypersensitivity to 4.0% l-lactic acid (pH 2.8) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 107 genes that contributed significantly to the ability of yeast cells to adapt lactic-acid stress. More than 30% of the genes identified in this screening were newly identified to be involved in mechanisms for adaptation response to lactic acid. We found that protein urmylation by Uba4 and N-terminal acetylation by Nat3 were involved in lactic acid adaptation mechanisms. Functional categorization of the genes followed by microscopic analysis revealed that a variety of cellular functions were involved in adaptation response to lactic acid and function associated with vacuolar transport played important roles in adaptation response to lactic acid. We also found that vacuole fragmented immediately upon exposure to lactic- and hydrochloric-acid stress. In addition, our analysis revealed that lactic-acid stress significantly reduced the amount of intracellular amino acids. Amino acid supplementation recovered the adaptation deficiency to lactic acid, suggesting that intracellular amino-acid homeostasis plays important roles in adaptation response to lactic-acid stress. These data suggest that enhancing vacuolar integrity, as well as maintaining intracellular amino-acid homeostasis may be an efficient approach to confer resistance to lactic-acid stress.

The Chlamydia Protease CPAF Regulates Host and Bacterial Proteins to Maintain Pathogen Vacuole Integrity and Promote Virulence

The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole ("inclusion"). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a preinfected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive antichlamydial therapeutic strategy.

Sortin1-Hypersensitive Mutants Link Vacuolar-Trafficking Defects and Flavonoid Metabolism in Arabidopsis Vegetative Tissues

Sortin1 is a chemical genetic-hit molecule that causes specific mislocalization of plant and yeast-soluble and membrane vacuolar markers. To better understand its mode of action, we designed a Sortin1-hypersensitive screen and identified several Sortin1-hypersensitive and flavonoid-defective mutants. Mechanistically, Sortin1 mimics the effect of the glutathione inhibitor buthionine sulfoximine and alters the vacuolar accumulation of flavonoids, likely blocking their transport through vacuole-localized ABC transporters. Structure-activity relationship studies conducted in Arabidopsis revealed the structural requirements for Sortin1 bioactivity and demonstrated that overlapping Sortin1 substructures can be used to discriminate between vacuolar-flavonoid accumulations and vacuolar-biogenesis defects. We conclude that Sortin1 is a valuable probe for dissecting novel links among flavonoid transport, vacuolar integrity, and the trafficking of vacuolar targeted cargoes in Arabidopsis.