scholarly journals Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole inChlamydia trachomatisInfected Human Cells

2019 ◽  
Author(s):  
Macy G. Olson ◽  
Ray E. Widner ◽  
Lisa M. Jorgenson ◽  
Alyssa Lawrence ◽  
Dragana Lagundzin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Host Pathogen Interactions ◽  
Inclusion Membrane ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Membrane Bound ◽  
Labeling System ◽  
Chlamydial Inclusion

AbstractAs an obligate intracellular pathogenic bacterium,C. trachomatisdevelops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Anin vivounderstanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 inChlamydia trachomatisL2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complexin vivoprotein-protein interactions at the chlamydial inclusion.Author summaryMany intracellular bacteria, including the obligate intracellular pathogenChlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane sitein vivoin the context of infection.

scholarly journals Proximity Labeling To Map Host-Pathogen Interactions at the Membrane of a Bacterium-Containing Vacuole in Chlamydia trachomatis-Infected Human Cells

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Macy G. Olson ◽  
Ray E. Widner ◽  
Lisa M. Jorgenson ◽  
Alyssa Lawrence ◽  
Dragana Lagundzin ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Inclusion Membrane ◽  
Content Type ◽  
Host Interactions ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Chlamydial Inclusion

ABSTRACT Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.

scholarly journals Bacteria Use Structural Imperfect Mimicry To Hijack The Host Interactome

2020 ◽  
Author(s):  
Natalia Sanchez de Groot ◽  
Marc Torrent Burgas
Keyword(s):  
Protein Interactions ◽  
Rational Design ◽  
Protein Protein Interactions ◽  
Host Proteins ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Imperfect Mimicry ◽  
The Way

ABSTRACTBacteria use protein-protein interactions to infect their hosts and hijack fundamental pathways, which ensures their survival and proliferation. Hence, the infectious capacity of the pathogen is closely related to its ability to interact with host proteins. Here, we show that hubs in the host-pathogen interactome are isolated in the pathogen network by adapting the geometry of the interacting interfaces. An imperfect mimicry of the eukaryotic interfaces allows pathogen proteins to actively bind to the host’s target while preventing deleterious effects on the pathogen interactome. Understanding how bacteria recognize eukaryotic proteins may pave the way for the rational design of new antibiotic molecules.

PROGRESS IN COMPUTATIONAL STUDIES OF HOST–PATHOGEN INTERACTIONS

2013 ◽  
Vol 11 (02) ◽  
pp. 1230001 ◽  
Author(s):  
HUFENG ZHOU ◽  
JINGJING JIN ◽  
LIMSOON WONG
Keyword(s):  
Protein Interactions ◽  
Computational Studies ◽  
Interaction Data ◽  
Protein Protein Interactions ◽  
Host Pathogen Interactions ◽  
Basic Principles ◽  
Host Pathogen Interaction ◽  
Treatment And Prevention ◽  
Host Pathogen ◽  
Pathogen Protein

Host–pathogen interactions are important for understanding infection mechanism and developing better treatment and prevention of infectious diseases. Many computational studies on host–pathogen interactions have been published. Here, we review recent progress and results in this field and provide a systematic summary, comparison and discussion of computational studies on host–pathogen interactions, including prediction and analysis of host–pathogen protein–protein interactions; basic principles revealed from host–pathogen interactions; and database and software tools for host–pathogen interaction data collection, integration and analysis.

scholarly journals Bacteria use structural imperfect mimicry to hijack the host interactome

2020 ◽  
Vol 16 (12) ◽  
pp. e1008395
Author(s):  
Natalia Sanchez de Groot ◽  
Marc Torrent Burgas
Keyword(s):  
Protein Interactions ◽  
Rational Design ◽  
Protein Protein Interactions ◽  
Host Proteins ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Imperfect Mimicry ◽  
The Way

Bacteria use protein-protein interactions to infect their hosts and hijack fundamental pathways, which ensures their survival and proliferation. Hence, the infectious capacity of the pathogen is closely related to its ability to interact with host proteins. Here, we show that hubs in the host-pathogen interactome are isolated in the pathogen network by adapting the geometry of the interacting interfaces. An imperfect mimicry of the eukaryotic interfaces allows pathogen proteins to actively bind to the host’s target while preventing deleterious effects on the pathogen interactome. Understanding how bacteria recognize eukaryotic proteins may pave the way for the rational design of new antibiotic molecules.

scholarly journals Analysis of Virus-Host Interactomes through a network-centric approach

2020 ◽  
Author(s):  
Giuseppe Tradigo ◽  
Pierangelo Veltri ◽  
Pietro Hiram Guzzi
Keyword(s):  
Protein Interactions ◽  
Living Cells ◽  
Common Mechanism ◽  
Protein Protein Interactions ◽  
Host Pathogen Interactions ◽  
Comprehensive Knowledge ◽  
Host Pathogen ◽  
Supporting Tool ◽  
Human Infections

AbstractViruses are small microorganisms that invade living cells and use them to replicate themselves. Viruses cause many common human infections (such as cold flu) as well as many lethal diseases. Therefore the comprehensive knowledge of mechanisms used by viruses to infect living cells, also known as host-pathogen interactions, is crucial. Mechanisms of infections of viruses are mediated by Protein-protein interactions (PPIs). PPIs are often modelled using graphs, thus the use of such a model may also explain mechanisms of infection of viruses. In this work, we propose a methodology to model and analyse host-pathogen interactions and a supporting tool able to analyse such data. We also analyse host-pathogen interactions of some common viruses demonstrating common mechanism and differences.

scholarly journals Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

2006 ◽  
Vol 282 (7) ◽  
pp. 4821-4829 ◽  
Author(s):  
Theresa N. Operaña ◽  
Robert H. Tukey
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conclusive Evidence ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Dietary Nutrients ◽  
Membrane Bound

UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of β-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

scholarly journals Application of a Bioinformatics-Based Approach to Identify Novel Putative in vivo BACE1 Substrates

2013 ◽  
Vol 5 ◽  
pp. BECB.S8383 ◽  
Author(s):  
Joseph L. Johnson ◽  
Emily Chambers ◽  
Keerthi Jayasundera
Keyword(s):  
Protein Interactions ◽  
Senile Plaques ◽  
Amyloid Β ◽  
Surface Protein ◽  
Cleavage Sites ◽  
Cell Surface Protein ◽  
Protein Protein Interactions ◽  
Membrane Bound ◽  
Hallmark Feature

BACE1, a membrane-bound aspartyl protease that is implicated in Alzheimer's disease, is the first protease to cut the amyloid precursor protein resulting in the generation of amyloid-β and its aggregation to form senile plaques, a hallmark feature of the disease. Few other native BACE1 substrates have been identified despite its relatively loose substrate specificity. We report a bioinformatics approach identifying several putative BACE1 substrates. Using our algorithm, we successfully predicted the cleavage sites for 70% of known BACE1 substrates and further validated our algorithm output against substrates identified in a recent BACE1 proteomics study that also showed a 70% success rate. Having validated our approach with known substrates, we report putative cleavage recognition sequences within 962 proteins, which can be explored using in vivo methods. Approximately 900 of these proteins have not been identified or implicated as BACE1 substrates. Gene ontology cluster analysis of the putative substrates identified enrichment in proteins involved in immune system processes and in cell surface protein-protein interactions.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals Porcine small intestinal organoids as a model to explore ETEC–host interactions in the gut

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Bjarne Vermeire ◽  
Liara M. Gonzalez ◽  
Robert J. J. Jansens ◽  
Eric Cox ◽  
Bert Devriendt
Keyword(s):  
Intestinal Epithelial ◽  
Gut Health ◽  
Functional Responses ◽  
Host Pathogen Interactions ◽  
Epithelial Surface ◽  
Host Interactions ◽  
Intestinal Organoids ◽  
Host Pathogen ◽  
Small Intestinal

AbstractSmall intestinal organoids, or enteroids, represent a valuable model to study host–pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host–pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

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