scholarly journals Detection and quantification of the vacuolar H ATPase using the Legionella effector protein SidK

2021 ◽  
Author(s):  
Michelle Maxson ◽  
Yazan M. Abbas ◽  
Jing Ze Wu ◽  
Sergio Grinstein ◽  
John L Rubinstein
Keyword(s):  
Legionella Pneumophila ◽  
High Specificity ◽  
Proton Pumping ◽  
Eukaryotic Cells ◽  
Effector Protein ◽  
Receptor Recycling ◽  
Phagosome Maturation ◽  
A Subunit ◽  
Endocytic Vesicles ◽  
Vacuolar Atpases

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar 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 nor specific to test this hypothesis. We introduce a new probe to localize and quantify V ATPases in eukaryotic cells. 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 AlexaFluor-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 the subcellular localization of the lysosome.

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

2022 ◽  
Vol 221 (3) ◽  
Author(s):  
Michelle E. Maxson ◽  
Yazan M. Abbas ◽  
Jing Ze Wu ◽  
Jonathan D. Plumb ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
High Specificity ◽  
Proton Pumping ◽  
Effector Protein ◽  
Receptor Recycling ◽  
Phagosome Maturation ◽  
Alexa Fluor ◽  
A Subunit ◽  
Endocytic Vesicles ◽  
Detection And Quantification

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.

scholarly journals Loss of zebrafish atp6v1e1b, encoding a subunit of vacuolar ATPase, recapitulates human ARCL type 2C syndrome and identifies multiple pathobiological signatures

PLoS Genetics ◽  
2021 ◽  
Vol 17 (6) ◽  
pp. e1009603
Author(s):  
Lore Pottie ◽  
Wouter Van Gool ◽  
Michiel Vanhooydonck ◽  
Franz-Georg Hanisch ◽  
Geert Goeminne ◽  
...  
Keyword(s):  
Proton Pumping ◽  
Structural Defects ◽  
Protein Levels ◽  
Clinical Presentations ◽  
A Subunit ◽  
Systemic Manifestations ◽  
Skin Wrinkles ◽  
Vacuolar Atpases ◽  
Heritable Connective Tissue Disorders

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo.

scholarly journals Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

2020 ◽  
Author(s):  
Pottie Lore ◽  
Van Gool Wouter ◽  
Vanhooydonck Michiel ◽  
Hanisch Franz-Georg ◽  
Goeminne Geert ◽  
...  
Keyword(s):  
Biological Effects ◽  
Proton Pumping ◽  
Structural Defects ◽  
Connective Tissue Disorders ◽  
A Subunit ◽  
Cancer Multiple ◽  
Vacuolar Atpases ◽  
Heritable Connective Tissue Disorders

AbstractThe inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

scholarly journals Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aimin Yang ◽  
Supansa Pantoom ◽  
Yao-Wen Wu
Keyword(s):  
Legionella Pneumophila ◽  
Lipid Binding ◽  
Effector Protein ◽  
Intracellular Pathogen ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Acyl Chains ◽  
Pathogenic Microbes ◽  
Lir Motif ◽  
Lipid Binding Site

Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

scholarly journals Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E Minelli ◽  
Min Wan ◽  
Paul D Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Biochemical Analyses ◽  
Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

scholarly journals Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation

2009 ◽  
Vol 185 (5) ◽  
pp. 917-928 ◽  
Author(s):  
Tony Yeung ◽  
Bryan Heit ◽  
Jean-Francois Dubuisson ◽  
Gregory D. Fairn ◽  
Basil Chiu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chlamydia Trachomatis ◽  
Surface Charge ◽  
Legionella Pneumophila ◽  
Protein Targeting ◽  
Membrane Surface ◽  
Phagosome Maturation ◽  
Cationic Proteins ◽  
Fluorescent Biosensor ◽  
Membrane Surface Charge

During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.

scholarly journals MultipleLegionella pneumophilaeffector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

2017 ◽  
Vol 114 (48) ◽  
pp. E10446-E10454 ◽  
Author(s):  
Stephanie R. Shames ◽  
Luying Liu ◽  
James C. Havey ◽  
Whitman B. Schofield ◽  
Andrew L. Goodman ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Host Cells ◽  
Limiting Factor ◽  
Effector Protein ◽  
Host Immune System ◽  
Loss Of Function ◽  
High Throughput Analysis ◽  
Single Strain

Legionella pneumophilais the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain ofL. pneumophilaencodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number ofL. pneumophilaeffectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhancedL. pneumophilain the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute toL. pneumophilavirulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

scholarly journals V-ATPase expression in the mouse olfactory epithelium

2008 ◽  
Vol 295 (4) ◽  
pp. C923-C930 ◽  
Author(s):  
Teodor G. Păunescu ◽  
Abigail C. Jones ◽  
Robert Tyszkowski ◽  
Dennis Brown
Keyword(s):  
Ph Regulation ◽  
Proton Pumping ◽  
Sensory Cells ◽  
Apical Plasma Membrane ◽  
Ph Homeostasis ◽  
B Subunit ◽  
Sustentacular Cells ◽  
Type Iv ◽  
A Subunit ◽  
Intracellular Organelles

The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa “A” subunit) and ATP6V1E1 (the ubiquitous 31-kDa “E” subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 (“B1,” typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 (“B2”) in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection.

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