scholarly journals Phosphatidylinositol 3-kinase-independent synthesis of PtdIns(3,4)P2 by a phosphotransferase

2021 ◽  
Author(s):  
Glenn Walpole ◽  
Jonathan Pacheco ◽  
Neha Chauhan ◽  
Yazan Abbas ◽  
Fernando Montaño-Rendón ◽  
...  
Keyword(s):  
De Novo ◽  
Host Cells ◽  
Phosphoinositide Metabolism ◽  
Free System ◽  
Enteropathogenic Bacteria ◽  
Successful Infection ◽  
Host Signaling ◽  
A Cell ◽  
And Migration ◽  
Bacterial Effectors

Abstract Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival within host organisms. We demonstrate that PtdIns(3,4)P2 is generated in host cells by effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signaling to gain entry and even provoke oncogenic transformation.

scholarly journals Phosphatidylinositol 3-kinase-independent synthesis of PtdIns(3,4)P2 by a phosphotransferase

2021 ◽  
Author(s):  
Glenn Walpole ◽  
Jonathan Pacheco ◽  
Neha Chauhan ◽  
Yazan Abbas ◽  
Fernando Montano-Rendon ◽  
...  
Keyword(s):  
De Novo ◽  
Host Cells ◽  
Phosphoinositide Metabolism ◽  
Free System ◽  
Enteropathogenic Bacteria ◽  
Successful Infection ◽  
Host Signaling ◽  
A Cell ◽  
And Migration ◽  
Bacterial Effectors

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival within host organisms. We demonstrate that PtdIns(3,4)P2 is generated in host cells by effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signaling to gain entry and even provoke oncogenic transformation.

scholarly journals Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

2016 ◽  
Vol 113 (23) ◽  
pp. E3260-E3269 ◽  
Author(s):  
Eric Martinez ◽  
Julie Allombert ◽  
Franck Cantet ◽  
Anissa Lakhani ◽  
Naresh Yandrapalli ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Membrane Trafficking ◽  
Galleria Mellonella ◽  
Q Fever ◽  
Early Endosome ◽  
Host Cells ◽  
Phosphoinositide Metabolism ◽  
Vacuolar Protein ◽  
Bacterial Effectors

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

scholarly journals Hepatitis C Virus RNA Synthesis in a Cell-Free System Isolated from Replicon-Containing Hepatoma Cells

2003 ◽  
Vol 77 (3) ◽  
pp. 2029-2037 ◽  
Author(s):  
Richard W. Hardy ◽  
Joseph Marcotrigiano ◽  
Keril J. Blight ◽  
John E. Majors ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Hcv Rna ◽  
Free System ◽  
Cell Free System ◽  
Huh7 Cells ◽  
A Cell

ABSTRACT A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40°C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn2+ and Mg2+ cations could be used in the reaction; however, concentrations of Mn2+ greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.

scholarly journals Structural Profiling of Bacterial Effectors Reveals Enrichment of Host-Interacting Domains and Motifs

2021 ◽  
Vol 8 ◽  
Author(s):  
Yangchun Frank Chen ◽  
Yu Xia
Keyword(s):  
Protein Interactions ◽  
Effector Proteins ◽  
Host Cells ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Specific Domain ◽  
Bacterial Proteins ◽  
Domain Interactions ◽  
Host Signaling ◽  
Bacterial Effectors

Effector proteins are bacterial virulence factors secreted directly into host cells and, through extensive interactions with host proteins, rewire host signaling pathways to the advantage of the pathogen. Despite the crucial role of globular domains as mediators of protein-protein interactions (PPIs), previous structural studies of bacterial effectors are primarily focused on individual domains, rather than domain-mediated PPIs, which limits their ability to uncover systems-level molecular recognition principles governing host-bacteria interactions. Here, we took an interaction-centric approach and systematically examined the potential of structural components within bacterial proteins to engage in or target eukaryote-specific domain-domain interactions (DDIs). Our results indicate that: 1) effectors are about six times as likely as non-effectors to contain host-like domains that mediate DDIs exclusively in eukaryotes; 2) the average domain in effectors is about seven times as likely as that in non-effectors to co-occur with DDI partners in eukaryotes rather than in bacteria; and 3) effectors are about nine times as likely as non-effectors to contain bacteria-exclusive domains that target host domains mediating DDIs exclusively in eukaryotes. Moreover, in the absence of host-like domains or among pathogen proteins without domain assignment, effectors harbor a higher variety and density of short linear motifs targeting host domains that mediate DDIs exclusively in eukaryotes. Our study lends novel quantitative insight into the structural basis of effector-induced perturbation of host-endogenous PPIs and may aid in the design of selective inhibitors of host-pathogen interactions.

Pyruvate metabolism by Anaplasma marginale in cell-free culture

1999 ◽  
Vol 45 (2) ◽  
pp. 185-189 ◽  
Author(s):  
Linda E McHolland ◽  
Daniel R Caldwell
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Anaplasma Marginale ◽  
Acid Synthesis ◽  
Pyruvate Metabolism ◽  
Amino Acid Synthesis ◽  
Free System ◽  
Label Incorporation ◽  
A Cell ◽  
Body Components

Partially purified Anaplasma marginale initial bodies were cultivated in a cell-free system in the presence of [3-14C]pyruvate for 24 or 48 h. Experiments showed that a significant portion of the pyruvate supplied to the cultures was incorporated into initial body components. Label incorporation was reduced by 72% in the presence of oxytetracycline. Fractionation and chromatography of the organisms revealed radioactive incorporation as alanine. This is the first report of de novo amino acid synthesis by A. marginale demonstrating that the rickettsia is capable of using pyruvate, an erythrocyte glycolytic product, in its metabolism.Key words: Anaplasma marginale, pyruvate metabolism, amino acid synthesis.

scholarly journals A Multistep, ATP-dependent Pathway for Assembly of Human Immunodeficiency Virus Capsids in a Cell-free System

1997 ◽  
Vol 136 (3) ◽  
pp. 567-581 ◽  
Author(s):  
Jaisri R. Lingappa ◽  
Rebecca L. Hill ◽  
Mei Lie Wong ◽  
Ramanujan S. Hegde
Keyword(s):  
Human Immunodeficiency Virus ◽  
De Novo ◽  
Atp Depletion ◽  
Assembly Process ◽  
Free System ◽  
Cell Free System ◽  
Virus Capsids ◽  
Immunodeficiency Virus ◽  
A Cell

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.

A Bacterial Effector Acts as a Plant Transcription Factor and Induces a Cell Size Regulator

Science ◽  
2007 ◽  
Vol 318 (5850) ◽  
pp. 648-651 ◽  
Author(s):  
Sabine Kay ◽  
Simone Hahn ◽  
Eric Marois ◽  
Gerd Hause ◽  
Ulla Bonas
Keyword(s):  
Transcription Factor ◽  
Cell Size ◽  
Effector Proteins ◽  
Host Cells ◽  
Mesophyll Cells ◽  
Eukaryotic Transcription ◽  
Helix Loop Helix ◽  
Host Signaling ◽  
A Cell ◽  
Developmental Reprogramming

Pathogenicity of many Gram-negative bacteria relies on the injection of effector proteins by type III secretion into eukaryotic cells, where they modulate host signaling pathways to the pathogen's benefit. One such effector protein injected by Xanthomonas into plants is AvrBs3, which localizes to the plant cell nucleus and causes hypertrophy of plant mesophyll cells. We show that AvrBs3 induces the expression of a master regulator of cell size, upa20, which encodes a transcription factor containing a basic helix-loop-helix domain. AvrBs3 binds to a conserved element in the upa20 promoter via its central repeat region and induces gene expression through its activation domain. Thus, AvrBs3 and likely other members of this family provoke developmental reprogramming of host cells by mimicking eukaryotic transcription factors.

scholarly journals Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols

1999 ◽  
Vol 344 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Peter GEROLD ◽  
Nicole JUNG ◽  
Nahid AZZOUZ ◽  
Nicole FREIBERG ◽  
Sabine KOBE ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Mammalian Cells ◽  
Biosynthetic Pathway ◽  
Unique Feature ◽  
Host Cells ◽  
Free System ◽  
Incubation System ◽  
A Cell ◽  
Inositol Ring ◽  
Myristoyl Coa

The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy.

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