Loss of the Golgi-localized v-ATPase subunit does not alter insulin granule formation or pancreatic islet ß-cell function.

Delayed Golgi export of proinsulin has recently been identified as an underlying mechanism leading to insulin granule loss and ß-cell secretory defects in type 2 diabetes (T2D). Because acidification of the Golgi lumen is critical for proinsulin sorting and delivery into the budding secretory granule, we reasoned that dysregulation of Golgi pH may contribute to proinsulin trafficking defects. In this report, we examined pH regulation of the Golgi and identified a partial alkalinization of the Golgi lumen in a diabetes model. To further explore this, we generated a ß-cell specific knockout (KO) of the v0a2 subunit of the v-ATPase pump, which anchors the v-ATPase to the Golgi membrane. Although loss of v0a2 partially neutralized Golgi pH and was accompanied by distension of the Golgi cisternae, proinsulin export from the Golgi and insulin granule formation were not affected. Furthermore, ß-cell function was well preserved. ß-cell v0a2 KO mice exhibited normal glucose tolerance in both sexes, no genotypic difference to diet-induced obesity, and normal insulin secretory responses. Collectively, our data demonstrate the v0a2 subunit contributes to ß-cell Golgi pH regulation but suggest that additional disturbances to Golgi structure and function contribute to proinsulin trafficking defects in diabetes.NEW & NOTEWORTHY Delayed proinsulin export from the Golgi in diabetic ß-cells contributes to decreased insulin granule formation, but the underlying mechanisms are not clear. Here, we explored if dysregulation of Golgi pH can alter Golgi function using ß-cell specific knockout (KO) of the Golgi-localized subunit of the v-ATPase, v0a2. We show that partial alkalinization of the Golgi dilates the cisternae, but does not affect proinsulin export, insulin granule formation, insulin secretion, or glucose homeostasis.

Respiration supports intraphagosomal filamentation and escape of Candida albicans from macrophages.

IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.

Mycobacterium tuberculosis Central Metabolism Is Key Regulator of Macrophage Pyroptosis and Host Immunity.

Metabolic dysregulation in Mycobacterium tuberculosis results in increased macrophage apoptosis or pyroptosis. However, mechanistic links between Mycobacterium virulence and bacterial metabolic plasticity remain ill defined. In this study, we screened random transposon insertions of M. bovis BCG to identify mutants that induce pyroptotic death of the infected macrophage. Analysis of the transposon insertion sites identified a panel of fdr (functioning death repressor) genes, which were shown in some cases to encode functions central to Mycobacterium metabolism. In-depth studies of one fdr gene, fdr8 (BCG3787/Rv3727), demonstrated its important role in the maintenance of M. tuberculosis and M. bovis BCG redox balance in reductive stress conditions in the host. Our studies expand the subset of known Mycobacterium genes linking bacterial metabolic plasticity to virulence and also reveal that the broad induction of pyroptosis by an intracellular bacterial pathogen is linked to enhanced cellular immunity in vivo.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK.

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Rab5 regulates macropinocytosis by recruiting the inositol 5-phosphatases OCRL and Inpp5b that hydrolyse PtdIns(4,5)P2.

Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

The Role of Membrane Surface Charge in Phagocytosis.

The formation and maturation of phagosomes are accompanied by acute changes in lipid metabolism. Phosphoinositides, in particular, undergo extensive modification as part of the signaling sequence that drives cytoskeletal and membrane remodeling. Because the phosphoinositides provide much of the anionic charge of the cytosolic leaflet of the plasmalemma and phagosomal membrane, the metabolic changes associated with signaling result in marked changes of the surface charge. Here we summarize the pathways involved in lipid remodeling during phagocytosis, the resultant alterations in the surface charge of the nascent and maturing phagosomes, and the consequent effects on the association of proteins attached to the membrane by electrostatic means.

Revisiting the role of calcium in phagosome formation and maturation.

Like other membrane receptor-mediated responses, execution of phagocytosis requires the transduction of signals to cytoplasmic effectors. Signaling in this case is particularly complex as the process involves not only the formation of phagosomes but also their subsequent maturation and resolution. Transient increases in cytosolic calcium, which mediate a variety of other transduction pathways, also feature prominently in phagocytosis. However, despite intensive study over the course of nearly 30 years, the occurrence, source, and functional relevance of such calcium bursts remain the subject of debate. Here, we have attempted to consolidate the information that was reviewed in the past with more recent studies in an effort to shed some light on the existing controversies.

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis.

Macrophages respond to several stimuli by forming florid membrane ruffles that lead to fluid uptake by macropinocytosis. This type of induced macropinocytosis, executed by a variety of non-malignant and malignant cells, is initiated by transmembrane receptors and is involved in nutrient acquisition and mTOR signalling. However, macrophages also perform a unique type of constitutive ruffling and macropinocytosis that is dependent on the presence of extracellular calcium. Calcium-sensing receptors are responsible for this activity. This distinct form of macropinocytosis enables macrophages to continuously sample their microenvironment for antigenic molecules and for pathogen- and danger-associated molecular patterns, as part of their immune surveillance functions. Interestingly, even within the monocyte lineage, there are differences in macropinocytic ability that reflect the polarized functional roles of distinct macrophage subsets. This review discusses the shared and distinct features of both induced and constitutive macropinocytosis displayed by the macrophage lineage and their roles in physiology, immunity and pathophysiology. In particular, we analyse the role of macropinocytosis in the uptake of modified low-density lipoprotein (LDL) and its contribution to foam cell and atherosclerotic plaque formation. We propose a combined role of scavenger receptors and constitutive macropinocytosis in oxidized LDL uptake, a process we have termed 'receptor-assisted macropinocytosis'. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

High-Throughput Screening Identifies Genes Required for Candida albicans Induction of Macrophage Pyroptosis.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes.

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae.

The vacuolar-type H⁺-ATPase at a glance - more than a proton pump.

The vacuolar H(+)-ATPase (V-ATPase) has long been appreciated to function as an electrogenic H(+) pump. By altering the pH of intracellular compartments, the V-ATPase dictates enzyme activity, governs the dissociation of ligands from receptors and promotes the coupled transport of substrates across membranes, a role often aided by the generation of a transmembrane electrical potential. In tissues where the V-ATPase is expressed at the plasma membrane, it can serve to acidify the extracellular microenvironment. More recently, however, the V-ATPase has been implicated in a bewildering variety of additional roles that seem independent of its ability to translocate H(+). These non-canonical functions, which include fusogenicity, cytoskeletal tethering and metabolic sensing, are described in this Cell Science at a Glance article and accompanying poster, together with a brief overview of the conventional functions of the V-ATPase.

The volume and hydration of the Cryptococcus neoformans polysaccharide capsule.

We present a new method to measure capsule size in the human fungal pathogen Cryptococcus neoformans that avoids the limitations and biases inherent in India ink measurements. The method is based on the use of gamma-radiation, which efficiently releases the capsule from the cell. By comparing the volume of irradiated and non-irradiated cells, one can accurately estimate the relative size of the capsule per cell. This method was also used to obtain an estimate of the capsule weight and water content. The C. neoformans capsule is a highly hydrated structure in all the conditions measured. However, after capsule enlargement, the amount of capsular polysaccharide significantly increases, suggesting a that capsule growth has a high energy cost for the cell.

Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule.

Exposure of Cryptococcus neoformans cells to gamma radiation results in a gradual release of capsular polysaccharide, in a dose-dependent manner. This method allows the systematic exploration of different capsular regions. Using this methodology, capsule density was determined to change according to the radial distribution of glucuronoxylomannan and total polysaccharide, becoming denser at the inner regions of the capsule. Scanning electron microscopy of cells following gamma radiation treatment confirmed this finding. The zeta potential of the capsule also increased as the capsule size decreased. However, neither charge nor density differences were correlated with any change in sugar composition (xylose, mannose, and glucuronic acid) in the different capsular regions, since the proportions of these sugars remained constant throughout the capsule. Analysis of the capsular antigenic properties by monoclonal antibody binding and Scatchard analysis revealed fluctuations in the binding affinity within the capsule but not in the number of antibody binding sites, suggesting that the spatial organization of high- and low-affinity epitopes within the capsule changed according to radial position. Finally, evidence is presented that the structure of the capsule changes with capsule age, since the capsule of older cells became more resistant to gamma radiation-induced ablation. In summary, the capsule of C. neoformans is heterogeneous in its spatial distribution and changes with age. Furthermore, our results suggest several mechanisms by which the capsule may protect the fungal cell against exogenous environmental factors.

Multiple promoters control expression of the Yersinia enterocolitica phage-shock-protein A (pspA) operon.

The widely conserved phage-shock-protein A (pspA) operon encodes an extracytoplasmic stress response system that is essential for virulence in Yersinia enterocolitica, and has been linked to other important phenotypes in Escherichia coli, Salmonella enterica and Shigella flexneri. Regulation of pspA operon expression is mediated through a promoter upstream of pspA that depends on sigma factor RpoN (sigma(54)) and the enhancer binding protein PspF. PspA, PspB and PspC, encoded within the pspA operon, also regulate expression by participating in a putative signal transduction pathway that probably serves to modulate PspF activity. All of this suggests that appropriate expression of the pspA operon is critical. Previous genetic analysis of the Y. enterocolitica pspA operon suggested that an additional level of complexity might be mediated by PspF/RpoN-independent expression of some psp genes. Here, an rpoN null mutation and interposon analysis were used to confirm that PspF/RpoN-independent gene expression does originate within the psp locus. Molecular genetic approaches were used to systematically analyse the two large non-coding regions within the psp locus. Primer extension, control region deletion and site-directed mutagenesis experiments led to the identification of RpoN-independent promoters both upstream and downstream of pspA. The precise location of the PspF/RpoN-dependent promoter upstream of pspA was also determined. The discovery of these RpoN-independent promoters reveals yet another level of transcriptional complexity for the Y. enterocolitica pspA operon that may function to allow low-level constitutive expression of psp genes and/or additional regulation under some conditions.

PspB and PspC of Yersinia enterocolitica are dual function proteins: regulators and effectors of the phage-shock-protein response.

The phage-shock-protein (Psp) stress-response system is conserved in many bacteria and has been linked to important phenotypes in Escherichia coli, Salmonella enterica and also Yersinia enterocolitica, where it is essential for virulence. It is activated by specific extracytoplasmic stress events such as the mislocalization of secretin proteins. From studies of the Psp system in E. coli, the cytoplasmic membrane proteins PspB and PspC have only been proposed to act as positive regulators of psp gene expression. However, in this study we show that PspB and PspC of Y. enterocolitica are dual function proteins, acting both as regulators and effectors of the Psp system. Consistent with the current model, they positively control psp gene expression in response to diverse inducing cues. PspB and PspC must work together to achieve this regulatory function, and bacterial two-hybrid (BACTH) analysis demonstrated a specific interaction between them, which was confirmed by in vivo cross-linking. We also show that PspB and PspC play a second role in supporting growth when a secretin protein is overexpressed. This function is independent from their role as regulators of psp gene expression. Furthermore, whereas PspB and PspC must work together for their regulatory function, they can apparently act independently to support growth during secretin production. This study expands the current understanding of the roles played by PspB and PspC, and demonstrates that they cannot be considered only as positive regulators of psp gene expression in Y. enterocolitica.

Improved system for construction and analysis of single-copy beta-galactosidase operon fusions in Yersinia enterocolitica.

We report a significantly improved system for studying single-copy lacZ operon fusions in Yersinia enterocolitica: a simple procedure for the stable integration of lacZ operon fusions into the ara locus and a strain with a deletion mutation that abolishes the low level of endogenous beta-galactosidase activity.

Identification of inducers of the Yersinia enterocolitica phage shock protein system and comparison to the regulation of the RpoE and Cpx extracytoplasmic stress responses.

Known inducers of the phage shock protein (Psp) system suggest that it is an extracytoplasmic stress response, as are the well-studied RpoE and Cpx systems. However, a random approach to identify conditions and proteins that induce the Psp system has not been attempted. It is also unknown whether the proteins or mutations that induce Psp are specific or if they also activate the RpoE and Cpx systems. This study addressed these issues for the Yersinia enterocolitica Psp system. Random transposon mutagenesis identified null mutations and overexpression mutations that increase Phi(pspA-lacZ) operon fusion expression. The results suggest that Psp may respond exclusively to extracytoplasmic stress. Null mutations affected glucosamine-6-phosphate synthetase (glmS), which plays a role in cell envelope biosynthesis, and the F0F1 ATPase (atp operon). The screen also revealed that in addition to several secretins, the overexpression of three novel putative inner membrane proteins (IMPs) induced the Psp response. We also compared induction of the Y. enterocolitica Psp, RpoE, and Cpx responses. Overexpression of secretins or the three IMPs or the presence of an atpB null mutation only induced the Psp response. Similarly, known inducers of the RpoE and Cpx responses did not significantly induce the Psp response. Only the glmS null mutation induced all three responses. Therefore, Psp is induced distinctly from the RpoE and Cpx systems. The specific IMP inducers may be valuable tools to probe specific signal transduction events of the Psp response in future studies.