Bacterial pathogen manipulation of host membrane trafficking.

Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

A vacuolar hexose transport is required for xylem development in the inflorescence stem.

In Angiosperms, the development of the vascular system is controlled by a complex network of transcription factors. However, how nutrient availability in the vascular cells affects their development remains to be addressed. At the cellular level, cytosolic sugar availability is regulated mainly by sugar exchanges at the tonoplast through active and/or facilitated transport. In Arabidopsis (Arabidopsis thaliana), among the genes encoding tonoplastic transporters, SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER 16 (SWEET16) and SWEET17 expression has been previously detected in the vascular system. Here, using a reverse genetics approach, we propose that sugar exchanges at the tonoplast, regulated by SWEET16, are important for xylem cell division as revealed in particular by the decreased number of xylem cells in the swt16 mutant and the accumulation of SWEET16 at the procambium-xylem boundary. In addition, we demonstrate that transport of hexoses mediated by SWEET16 and/or SWEET17 is required to sustain the formation of the xylem secondary cell wall. This result is in line with a defect in the xylem cell wall composition as measured by Fourier-transformed infrared spectroscopy in the swt16swt17 double mutant and by upregulation of several genes involved in secondary cell wall synthesis. Our work therefore supports a model in which xylem development partially depends on the exchange of hexoses at the tonoplast of xylem-forming cells.

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.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

Murine Irgm Paralogs Regulate Nonredundant Functions To Execute Host Defense to Toxoplasma gondii.

Gamma interferon (IFN-γ)-induced immunity-related GTPases (IRGs) confer cell-autonomous immunity to the intracellular protozoan pathogen Toxoplasma gondii. Effector IRGs are loaded onto the Toxoplasma-containing parasitophorous vacuole (PV), where they recruit ubiquitin ligases, ubiquitin-binding proteins, and IFN-γ-inducible guanylate-binding proteins (Gbps), prompting PV lysis and parasite destruction. Host cells lacking the regulatory IRGs Irgm1 and Irgm3 fail to load effector IRGs, ubiquitin, and Gbps onto the PV and are consequently defective for cell-autonomous immunity to Toxoplasma. However, the role of the third regulatory IRG, Irgm2, in cell-autonomous immunity to Toxoplasma has remained unexplored. Here, we report that Irgm2 unexpectedly plays a limited role in the targeting of effector IRGs, ubiquitin, and Gbps to the Toxoplasma PV. Instead, Irgm2 is instrumental in the decoration of PVs with γ-aminobutyric acid receptor-associated protein-like 2 (GabarapL2). Cells lacking Irgm2 are as defective for cell-autonomous host defense to Toxoplasma as pan-Irgm-/- cells lacking all three Irgm proteins, and Irgm2-/- mice succumb to Toxoplasma infections as readily as pan-Irgm-/- mice. These findings demonstrate that, relative to Irgm1 and Irgm3, Irgm2 plays a distinct but critically important role in host resistance to Toxoplasma.

Apple ALMT9 Requires a Conserved C-Terminal Domain for Malate Transport Underlying Fruit Acidity.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.

Variations in the content of tonoplast lipids under abiotic stress.

MAIN CONCLUSION: The vacuolar membrane is an essential component in protecting the plant cell from stress factors. Different variations in the tonoplast lipid content, which depend on the type of stress, have been reviewed. The lipid content of vacuolar membranes of beet roots (Beta vulgaris L.) under hypoosmotic, hyperosmotic and oxidative types of stress has been studied. These types of stress induce variations in the content of almost all the classes of studied lipids (phospholipids, glycoglycerolipids, sterols and fatty acids). The variations, which are characteristic of a single stress, include the variations (i) in the content of individual glycoglycerolipids and in their total content, (ii) in the total content of sterols, and (iii) in the ratio of content of phosphatidylcholine/phosphatidylethanolamine in the scope of tonoplast phospholipids. Variations observed under all of the types of stress under scrutiny include (i) variations in the content of fatty acids of tonoplast lipids, (ii) some decrease in the content of phosphatidic acid and phosphatidylethanolamine, and (iii) variations in the content of individual sterols. Stigmasterol, campesterol, as well as the stigmasterol/sitosterol ratio increased in varying degrees under all of the types of stress. The most substantial variations have been observed in the content of sterols under abiotic stress. This is probably due to role of sterols in regulation of such membrane characteristics as permeability and microviscosity. In our opinion, sterols may represent one of the main components of tonoplast adaptive mechanisms.

Predicting the unexpected in stomatal gas exchange: not just an open-and-shut case.

Plant membrane transport, like transport across all eukaryotic membranes, is highly non-linear and leads to interactions with characteristics so complex that they defy intuitive understanding. The physiological behaviour of stomatal guard cells is a case in point in which, for example, mutations expected to influence stomatal closing have profound effects on stomatal opening and manipulating transport across the vacuolar membrane affects the plasma membrane. Quantitative mathematical modelling is an essential tool in these circumstances, both to integrate the knowledge of each transport process and to understand the consequences of their manipulation in vivo. Here, we outline the OnGuard modelling environment and its use as a guide to predicting the emergent properties arising from the interactions between non-linear transport processes. We summarise some of the recent insights arising from OnGuard, demonstrate its utility in interpreting stomatal behaviour, and suggest ways in which the OnGuard environment may facilitate 'reverse-engineering' of stomata to improve water use efficiency and carbon assimilation.

Rosette Colonies of Choanoflagellates (Salpingoeca rosetta) Show Increased Food Vacuole Formation Compared with Single Swimming Cells.

Choanoflagellates exist as both single-celled and colonial forms and filter-feed by generating water currents using a single apical flagellum. Hydrodynamic modeling studies have differed in predictions of whether single cells or colonies produce greater fluid flow to enhance feeding, and a recent study reported no increase in feeding efficiency of stalked colonies of choanoflagellates compared with single cells. We report that rosette colonies of Salpingoeca rosetta demonstrate higher rates of food vacuole formation compared with unicellular, slow swimmers.

The cell biology of cell-in-cell structures.

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.

Three Distinct Types of Microautophagy Based on Membrane Dynamics and Molecular Machineries.

Microautophagy is originally defined as lysosomal (vacuolar) membrane dynamics to directly enwrap and transport cytosolic components into the lumen of the lytic organelle. Molecular details of microautophagy had remained unknown until genetic studies in yeast identified a set of proteins required for the process. Subsequent studies with other experimental model organisms resulted in a series of discoveries that accompanied an expansion of the definition of microautophagy to also encompass endosomal membrane dynamics. These findings, however, still impose puzzling, non-integrated images as to the molecular mechanism of microautophagy. By reviewing recent studies on microautophagy in various experimental systems, we propose the classification of microautophagy into three types, as the basis for developing a comprehensive view of the process.

Autophagy and vacuolar biogenesis during the nectary development.

MAIN CONCLUSION: Different autophagy pathways are a driver of vacuolar biogenesis and are development stage specific during the extrafloral nectary development in Citharexylum myrianthum. Plant autophagy plays an important role in various developmental processes such as seed germination, pollen maturation and leaf senescence. However, studies that address the evidence of autophagy and its role in the development of plant glands are scarce and largely restricted to laticifers. Regarding nectary, studies have repeatedly pointed to signs of degradation associated with the end of the secretory cycle, without exploring autophagy. Likewise, the relationship between autophagy and biogenesis of vacuoles remains an unexplored issue. In this study, using conventional and microwave fixation in association with ultracytochemical methods for transmission electron microscopy, we investigated the occurrence of autophagy and its implication in the differentiation of extrafloral nectary in Citharexylum myrianthum (Verbenaceae) under natural conditions, focusing on the vacuole biogenesis. We described a variety of vacuole types associated with the stage of nectary epidermis development, which differs with respect to origin, function and nature of the products to be stored. Three distinct autophagy pathways were detected: macroautophagy, microautophagy (both restricted to the undifferentiated epidermal cells, at the presecretory stage) and megaautophagy (circumscribed to the differentiated epidermal cells, at the postsecretory stage). Our study clearly demonstrated that the vacuole variety and autophagy processes in the nectary epidermal cells are development specific. This study highlights the role of autophagy in vacuole biogenesis and its implications for the development of nectary and opens new venues for future studies on regulation mechanisms for autophagy in plant secretory structures under normal conditions.

Singlet Oxygen Plays an Essential Role in the Root's Response to Osmotic Stress.

The high osmotic potentials in plants subjected to drought stress can be mimicked by the application of high molecular weight polyethylene glycol. Here, we quantified the effects of exposure to polyethylene glycol on the growth of the main and lateral roots of Arabidopsis (Arabidopsis thaliana) seedlings. The effects on root growth were highly correlated with the appearance of singlet oxygen, as visualized using the singlet oxygen-specific probe singlet oxygen sensor green. The production of singlet oxygen was followed by cell death, as indicated by the intracellular accumulation of propidium iodide due to the loss of membrane integrity. Cell death began in the epidermal region of the root tip and spread in a dynamic manner to meristematic sections. In parallel, gene expression changes specific to the presence of singlet oxygen were observed. The accumulation of other reactive oxygen species, namely hydrogen, peroxide, nitric oxide, and superoxide, did not correlate with cell death. In addition, both the singlet oxygen scavenger His and the lipoxygenase inhibitor salicylhydroxamic acid specifically inhibited singlet oxygen accumulation and cell death. These results suggest a light-independent, type-I source of singlet oxygen production. Serpin-protease interactions were used as a model to assess the possibility of vacuolar-type cell death. Osmotic stress induced the accumulation of complexes between the cytoplasmic serpin AtSERPIN1 and its cognate vacuolar proteases, indicating that vacuolar integrity was compromised. These findings imply that singlet oxygen plays an essential role in conveying the root response to osmotic stress.

Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets.

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

A Transient Developmental Background Finding in the Retina Observed in Neonatal Dogs in Juvenile Toxicology Studies.

In a juvenile toxicology program, an unexpected finding of vacuolation of inner nuclear, ganglion cell, and nerve fiber layers of the retina was observed microscopically in routine Davidson's fixed and hematoxylin and eosin-stained tissue sections of eyes in beagle dogs at approximately 5 weeks of age. There was no necrosis or degeneration of the affected cells and no associated inflammation. Fluorescein angiography revealed no vascular leakage. Optical coherence tomography (OCT) indicated swollen cells in the same layers of the retina as observed at light microscopic examination. Transmission electron microscopy revealed that the retinal vacuolation likely was consistent with intracellular swelling of amacrine, horizontal, and/or bipolar cells of the inner nuclear layer as affected cells had an expanded cytoplasm but contained normal nucleus and organelles. As assessed by animal behavior and full-field electroretinography, the retinal vacuolation appeared to have no impact on visual function. Retinal vacuolation was seen in approximately 40% of dogs at 5 weeks of age using OCT and/or light microscopic examination. Because the change was transient and age related, did not result in degenerative retinal changes, and was not present in dogs older than 5 weeks of age, it was considered a background developmental observation in beagle dogs.

Discovery of gas vesicles in Streptomyces sp. CB03234-S and potential effects of gas vesicle gene overexpression on morphological and metabolic changes in streptomycetes.

Gas vesicles are usually found in aquatic microbes to help against adverse growth conditions and also present applied potentials in various biotechnological areas. Although gvp genes encoding gas vesicle proteins are occurred in many bacteria, the formation of gas vesicles in streptomycetes has not been reported yet. During the transcriptome analysis of Streptomyces sp. CB03234-S, a high producer of 10-membered enediynes tiancimycins (TNMs) generated in our previous studies, the notable activation of a gvp gene cluster (gvp3234) was detected, which may be related to the morphological change and yield increase in CB03234-S. Subsequent bioinformatic analysis revealed that gvp3234 possessed some unique features different from other gvp gene clusters, and its relative expression level was much higher than homologues in Streptomyces coelicolor. The overexpression of gvp3234 further improved the total titer of TNM-A and TNM-D from 12.3 ± 0.1 mg/L up to 21.4 ± 0.8 mg/L in CB03234-S, and TEM analysis clearly showed the emergence of gas vesicles, while the heterologous expression of gvp3234 in Streptomyces sp. CB09001 also resulted in the similar formation of fluffy mycelial pellets with long hyphae like in CB03234-S and enhanced the production of xanthone derivative. Our work reported a functionally expressed gvp gene cluster and the corresponding formation of gas vesicles in streptomycetes for the first time. The revealed potential effects of gas vesicles on morphological and metabolic changes shall provide an alternative strategy for strain improvement of industrial streptomycetes to obtain better performances in liquid cultivation and enhance production of secondary metabolites.

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. ABSTRACT: 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.

Specialized Vacuoles of Myrosin Cells: Chemical Defense Strategy in Brassicales Plants.

Plant vacuoles display many versatile functions. Vacuoles in vegetative tissues are generally involved in protein degradation, and are called lytic vacuoles. However, vegetative vacuoles in specialized cells can accumulate large concentrations of proteins, such as those in idioblast myrosin cells along veins in the order Brassicales, which store large amounts of myrosinases (thioglucoside glucohydrolase and thioglucoside glucohydrolase). Myrosinases cleave the bond between sulfur and glucose in sulfur-rich compounds (glucosinolates) to produce toxic compounds (isothiocyanates) when plants are damaged by pests. This defense strategy is called the myrosinase-glucosinolate system. Recent studies identified atypical myrosinases, PENETRATION 2 (PEN2) and PYK10, along with key components for development of myrosin cells. In this review, we discuss three topics in the myrosinase-glucosinolate system. First, we summarize the complexity and importance of the myrosinase-glucosinolate system, including classical myrosinases, atypical myrosinases and the system that counteracts the myrosinase-glucosinolate system. Secondly, we describe molecular machineries underlying myrosin cell development, including specific reporters, cell lineage, cell differentiation and cell fate determination. The master regulators for myrosin cell differentiation, FAMA and SCREAM, are key transcription factors involved in guard cell differentiation. This indicates that myrosin cells and guard cells share similar transcriptional networks. Finally, we hypothesize that the myrosinase-glucosinolate system may have originated in stomata of ancestral Brassicales plants and, after that, plants co-opted this defense strategy into idioblasts near veins at inner tissue layers.

Evidence for preservation of vacuolar compartments during foehn-induced chalky ring formation of Oryza sativa L.

MAIN CONCLUSION: Vacuolar compartments being sustained among the amyloplasts inadequately accumulated in rice endosperm cells are the main cause of chalky ring formation under dry wind conditions. Foehn-induced dry wind during the grain-filling stage induces shoot water deficit in rice (Oryza sativa L.) plants, which form a ring-shaped chalkiness in their endosperm that degrades milling quality and rice appearance. Air spaces formed in several inner cells cause significant transparency loss due to irregular light reflection. Although starch synthesis was suggested to be retarded by osmotic adjustment at foehn-induced moderately low water potential, the source of these air spaces remains unknown. We hypothesised that the preservation of vacuoles accompanied by a temporary reduction in starch biosynthesis in the inner cells leads to the chalky ring formation. Panicle water status measurement, light and transmission electron microscopic (TEM) observations, and an absolute qPCR analysis were conducted. Most starch synthesis-related genes exhibited temporarily reduced expression in the inner zone in accordance with the decrease in panicle water status. TEM observations provided evidence that vacuolar compartments remained among the loosely packed starch granules in the inner endosperm cells, where a chalky ring appeared after kernel dehydration. Taken together, we propose that vacuolar compartments sustained among the amyloplasts inadequately accumulated in rice endosperm cells and caused air space formation that leads to ring-shaped chalkiness under dry wind conditions.