Salmonella utilizes type 3 secreted effector proteins to induce plasma membrane (PM) perturbations during invasion of host cells1. The effectors drive mobilization of host membranes to generate cell surface ruffles, followed by invagination and scission of the PM to generate Salmonella-containing vacuoles (SCVs)2. Here, we show that LRRK2 kinase generates membrane reservoirs exploited by Salmonella during invasion. The reservoirs are tubular compartments associated with the PM under basal conditions and are formed through the phosphorylation of RAB10 GTPase by LRRK2. Mobilization of membrane reservoirs to generate invasion ruffles mediates delivery of phosphorylated RAB10 to invasion sites. Subsequently, RAB10 dephosphorylation is required for its inactivation by a bacterial GTPase activating protein and subsequent scission of the PM. RAB10 dephosphorylation is mediated by a TLR4/PIEZO1/TMEM16F-dependent pathway and is inhibited by hyperactive variants of LRRK2. Our findings reveal how Salmonella exploits LRRK2-dependent PM dynamics during invasion and provide new insight into how LRRK2 variants can protect against bacterial infection3,4.

Delayed proinsulin export from the Golgi in diabetic β-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 β-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.

Neuronal endosomal and lysosomal abnormalities are among the early changes observed in Alzheimer’s disease (AD) before plaques appear. However, it is unclear whether distinct endolysosomal defects are temporally organized and how altered γ-secretase function or amyloid precursor protein (APP) metabolism contribute to these changes. Inhibiting γ-secretase chronically, in mouse embryonic fibroblast and hippocampal neurons, led to a gradual endolysosomal collapse initiated by decreased lysosomal calcium and increased cholesterol, causing downstream defects in endosomal recycling and maturation. This endolysosomal demise is γ-secretase dependent, requires membrane-tethered APP cytoplasmic domains, and is rescued by APP depletion. APP C-terminal fragments (CTFs) localized to late endosome/lysosome-endoplasmic reticulum contacts; an excess of APP-CTFs herein reduced lysosomal Ca2+ refilling from the endoplasmic reticulum, promoting cholesterol accretion. Tonic regulation by APP-CTFs provides a mechanistic explanation for their cellular toxicity: failure to timely degrade APP-CTFs sustains downstream signaling, instigating lysosomal dyshomeostasis, as observed in prodromal AD. This is the opposite of substrates such as Notch, which require intramembrane proteolysis to initiate signaling.

ABSTRACTWhile it has been known for decades that luminal acidification is required for normal traffic along the endocytic pathway, the precise underlying mechanism(s) remain unknown. We found that dissipation of the endomembrane pH gradient resulted in acute formation of large Rab5- or Rab7-positive vacuoles. Vacuole formation was associated with and required hyperactivation of the Rabs, which was attributable to impaired GTPase activity, despite normal recruitment of cognate GAPs. Surprisingly, LRRK2 –a kinase linked to Parkinson’s disease–was recruited to endomembranes and markedly activated upon dissipation of luminal acidification. LRRK2 phosphorylated Rab GTPases, rendering them insensitive to deactivation. Importantly, genetic deletion of LRRK2 prevented the ΔpH-induced vacuolation, implying that the kinase is required to modulate vesicular traffic. We propose that by dictating the state of activation of LRRK2 and in turn that of Rab GTPases, the development of a progressive luminal acidification serves as a timing device to control endocytic maturation.

ABSTRACT For the human fungal pathogen Candida albicans , metabolic flexibility and the ability to transition between yeast and filamentous growth states are key virulence traits that enable disease in the host. These traits are particularly important during the interaction of C. albicans with macrophages, where the fungus must utilize multiple alternative carbon sources to survive after being phagocytosed, and filamentation is coupled to fungal escape and immune cell death. Here, we employed functional genomic screening of conditional-expression mutants covering >50% of the C. albicans genome to identify genes selectively required for filamentation inside macrophages. Through manual and machine learning-based image analyses, we uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex in governing filamentous growth within the phagosome, suggesting that C. albicans relies on respiration to evade the antifungal activities of macrophages. We demonstrate that downregulating the expression of these genes reduces ATP levels and impedes filamentation as well as growth under monoculture conditions in medium lacking glucose. In co-culture with physiological glucose concentration, downregulation of genes involved in mitochondrial function and respiration prevented C. albicans from expanding within the phagosome, escaping, and inducing immune cell death. Together, our work provides new insights into the impact of metabolism on the interaction between C. albicans and macrophages, highlighting respiration and the SNF1 AMP-activated kinase as key effectors of C. albicans metabolic flexibility and filamentation within phagocytes. 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.

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.

Letter to Blood| June 29, 2023 Novel immunodeficiency caused by homozygous mutation in solute carrier family 19 member 1, which encodes the reduced folate carrier Akira Shiraishi, Akira Shiraishi 1Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON, Canada2Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan https://orcid.org/0000-0003-2184-3587 Search for other works by this author on: This Site PubMed Google Scholar Vedat Uygun, Vedat Uygun 3Pediatric Bone Marrow Transplantation Unit, Istinye University Faculty of Medicine, Medical Park Antalya Hospital, Antalya, Turkey https://orcid.org/0000-0003-3257-7798 Search for other works by this author on: This Site PubMed Google Scholar Nigel Sharfe, Nigel Sharfe 1Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON, Canada4Canadian Centre for Primary Immunodeficiency and Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Hospital for Sick Children, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Serap Beldar, Serap Beldar 5Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Mark G. F. Sun, Mark G. F. Sun 6Oracle Therapeutics (Canada) Inc, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Harjit Dadi, Harjit Dadi 1Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON, Canada4Canadian Centre for Primary Immunodeficiency and Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Hospital for Sick Children, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Linda Vong, Linda Vong 1Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON, Canada4Canadian Centre for Primary Immunodeficiency and Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Hospital for Sick Children, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Michelle Maxson, Michelle Maxson 7Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, ON, Canada8Department of Biochemistry, University of Toronto, Toronto, ON, Canada https://orcid.org/0000-0002-1493-490X Search for other works by this author on: This Site PubMed Google Scholar Neslihan E. Karaca, Neslihan E. Karaca 9Department of Pediatrics, Faculty of Medicine, Ege University, Izmir, Turkey Search for other works by this author on: This Site PubMed Google Scholar Süleyman Mevlitoğlu, Süleyman Mevlitoğlu 10Dolunay Pediatric Clinic, Muratpasa, Antalya, Turkey Search for other works by this author on: This Site PubMed Google Scholar Sergio Grinstein, Sergio Grinstein 7Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, ON, Canada8Department of Biochemistry, University of Toronto, Toronto, ON, Canada https://orcid.org/0000-0002-0795-4160 Search for other works by this author on: This Site PubMed Google Scholar Reha Artan, Reha Artan 11Department of Pediatric Gastroenterology, Akdeniz University Faculty of Medicine, Antalya, Turkey https://orcid.org/0000-0001-6114-9210 Search for other works by this author on: This Site PubMed Google Scholar Daniele Merico, Daniele Merico 12Vevo Therapeutics, San Francisco, CA13The Centre for Applied Genomics, Hospital for Sick Children, Toronto, ON, Canada https://orcid.org/0000-0002-3728-4401 Search for other works by this author on: This Site PubMed Google Scholar Chaim M. Roifman Chaim M. Roifman 1Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON, Canada4Canadian Centre for Primary Immunodeficiency and Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Hospital for Sick Children, Toronto, ON, Canada Search for other works by this author on: This Site PubMed Google Scholar Blood (2023) 141 (26): 3226–3230. https://doi.org/10.1182/blood.2022017968 Article history Submitted: September 8, 2022 Accepted: January 29, 2023 First Edition: February 6, 2023 Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Request Permissions Cite Icon Cite Search Site Citation Akira Shiraishi, Vedat Uygun, Nigel Sharfe, Serap Beldar, Mark G. F. Sun, Harjit Dadi, Linda Vong, Michelle Maxson, Neslihan E. Karaca, Süleyman Mevlitoğlu, Sergio Grinstein, Reha Artan, Daniele Merico, Chaim M. Roifman; Novel immunodeficiency caused by homozygous mutation in solute carrier family 19 member 1, which encodes the reduced folate carrier. Blood 2023; 141 (26): 3226–3230. doi: https://doi.org/10.1182/blood.2022017968 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBlood Search Subjects: Immunobiology and Immunotherapy TO THE EDITOR: Primary immunodeficiency diseases, including severe combined immunodeficiency (SCID) and combined immunodeficiency (CID), are monogenic disorders with a wide spectrum of clinical phenotypes including immune dysregulation, autoimmunity, inflammation, and malignancy.1 Manifestations may vary depending on whether the defect is localized to the immune system or its expression overlaps with other organ systems. Genetic aberrations affecting metabolic pathways, such as the coenzyme folate, can lead to immunodeficiency.2,3 SCID has previously been reported in patients with MTHFD1 deficiency, encoding a key enzyme in the folate pathway.2,4 Folates are essential for the synthesis of purine and pyrimidines during DNA and RNA synthesis and are cofactors for tissue growth and development.5,6 Folate uptake in humans is dependent on 2 transporters: the ubiquitously expressed reduced folate carrier (RFC) encoded by solute carrier family 19... References 1.Roifman CM. Primary T-cell immunodeficiencies. In: Rich R, eds. Clinical Immunology: Principles and Practice. Fifth Edition. Elsevier Health Sciences; 2018:489-508.Google Scholar 2.Burda P, Kuster A, Hjalmarson O, et al. Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment. J Inherit Metab Dis. 2015;38(5):863-872.Google ScholarCrossrefSearch ADS 3.Tan J, Li X, Guo Y, et al. Hereditary folate malabsorption with a novel mutation on SLC46A1. Medicine. 2017;96(50):e8712.Google ScholarCrossrefSearch ADS PubMed 4.Watkins D, Schwartzentruber JA, Ganesh J, et al. Novel inborn error of folate metabolism: identification by exome capture and sequencing of mutations in the MTHFD1 gene in a single proband. J Med Genet. 2011;48(9):590-592.Google ScholarCrossrefSearch ADS 5.Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327(26):1832-1835.Google ScholarCrossrefSearch ADS 6.Crider KS, Yang TP, Berry RJ, Bailey LB. Folate and DNA methylation: a review of molecular mechanisms and the evidence for folate's role. Adv Nutr. 2012;3(1):21-38.Google ScholarCrossrefSearch ADS PubMed 7.Zhao R, Min SH, Qiu A, et al. The spectrum of mutations in the PCFT gene, coding for an intestinal folate transporter, that are the basis for hereditary folate malabsorption. Blood. 2007;110(4):1147-1152.Google ScholarCrossrefSearch ADS PubMed 8.Malatack JJ, Moran MM, Moughan B. Isolated congenital malabsorption of folic acid in a male infant: insights into treatment and mechanism of defect. Pediatrics. 1999;104(5 Pt 1):1133-1137.Google ScholarPubMed 9.Svaton M, Skvarova Kramarzova K, Kanderova V, et al. A homozygous deletion in the SLC19A1 gene as a cause of folate-dependent recurrent megaloblastic anemia. Blood. 2020;135(26):2427-2431.Google ScholarCrossrefSearch ADS PubMed 10.De Bruyn E, Gulbis B, Cotton F. Serum and red blood cell folate testing for folate deficiency: new features?. Eur J Haematol. 2014;92(4):354-359.Google ScholarCrossrefSearch ADS PubMed 11.Hou Z, Matherly LH. Biology of the major facilitative folate transporters SLC19A1 and SLC46A1. Curr Top Membr. 2014;73:175-204.Google ScholarCrossrefSearch ADS PubMed 12.Luteijn RD, Zaver SA, Gowen BG, et al. SLC19A1 transports immunoreactive cyclic dinucleotides. Nature. 2019;573(7774):434-438.Google ScholarCrossrefSearch ADS PubMed 13.Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations: application to cancer genomics. Nucleic Acids Res. 2011;39(17):e118.Google ScholarCrossrefSearch ADS PubMed 14.Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res. 2001;11(5):863-874.Google ScholarCrossrefSearch ADS PubMed 15.Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248-249.Google ScholarCrossrefSearch ADS PubMed 16.Yang J, Anishchenko I, Park H, Peng Z, Ovchinnikov S, Baker D. Improved protein structure prediction using predicted interresidue orientations. Proc Natl Acad Sci U S A. 2020;117(3):1496-1503.Google ScholarCrossrefSearch ADS PubMed 17.Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature. 2008;455(7213):674-678.Google ScholarCrossrefSearch ADS PubMed 18.Konno H, Konno K, Barber GN. Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell. 2013;155(3):688-698.Google ScholarCrossrefSearch ADS PubMed 19.Gutjahr A, Papagno L, Nicoli F, et al. The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells. JCI Insight. 2019;4(7):e125107.Google ScholarCrossrefSearch ADS PubMed 20.Evans JG, Chavez-Rueda KA, Eddaoudi A, et al. Novel suppressive function of transitional 2 B cells in experimental arthritis. J Immunol. 2007;178(12):7868-7878.Google ScholarCrossrefSearch ADS 21.Gelineau-van Waes J, Heller S, Bauer LK, et al. Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation. Birth Defects Res A Clin Mol Teratol. 2008;82(7):494-507.Google ScholarCrossrefSearch ADS PubMed © 2023 by The American Society of Hematology2023 © 2023 by The American Society of Hematology2023 You do not currently have access to this content. Sign in via your Institution

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.

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.

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)P-2 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.

Candida albicans is a common opportunistic fungal pathogen that causes a wide range of infections from superficial mucosal to hematogenously disseminated candidiasis. The hyphal form plays an important role in the pathogenic process by invading epithelial cells and causing tissue damage. Notably, the secretion of the hyphal toxin candidalysin is essential for both epithelial cell damage and activation of mucosal immune responses. However, the mechanism of candidalysin-induced cell death remains unclear. Here, we examined the induction of cell death by candidalysin in oral epithelial cells. Fluorescent imaging using healthy/apoptotic/necrotic cell markers revealed that candidalysin causes a rapid and marked increase in the population of necrotic rather than apoptotic cells in a concentration dependent manner. Activation of a necrosis-like pathway was confirmed since C. albicans and candidalysin failed to activate caspase-8 and -3, or the cleavage of poly (ADP-ribose) polymerase. Furthermore, oral epithelial cells treated with candidalysin showed rapid production of reactive oxygen species, disruption of mitochondria activity and mitochondrial membrane potential, ATP depletion and cytochrome c release. Collectively, these data demonstrate that oral epithelial cells respond to the secreted fungal toxin candidalysin by triggering numerous cellular stress responses that induce necrotic death. TAKE AWAYS: Candidalysin secreted from Candida albicans causes epithelial cell stress. Candidalysin induces calcium influx and oxidative stress in host cells. Candidalysin induces mitochondrial dysfunction, ATP depletion and epithelial necrosis. The toxicity of candidalysin is mediated from the epithelial cell surface.

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)P-2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P-2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P-2 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)P-2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

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.

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.

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'.

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’.

ABSTRACT 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. IMPORTANCE Candida 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.

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.

ABSTRACT 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.