In Vivo Methods to Study Protein–Protein Interactions as Key Players in Mycobacterium Tuberculosis Virulence

Veyron‐Churlet, Romain; Locht, Camille

doi:10.3390/pathogens8040173

Cited by 5 publications

(6 citation statements)

References 101 publications

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“…Prototypical examples of this are the MmpL-centered biosynthetic complexes described under Section and evidence of coupling between biosynthesis and export within some of these pathways. Accumulating evidence further shows that protein–protein interactions also serve to bridge multiple cell envelope-related processes, as illustrated by the apparent direct and indirect interaction of the mycolic acid transporter MmpL3 with a number of cell elongation and division proteins as well as PG, AG and lipoglycan biosynthetic enzymes (see Section and refs and ). The fact that many cell envelope and cell elongation/division-related proteins localize at the poles, subpoles, and septa of actively replicating bacilli and physical evidence for the existence of biosynthetic microdomains within the plasma membrane is direct proof of the existence of coordinated biosynthetic foci within the cells (reviewed in ref ).…”

Section: Coordination Of Cell Envelope Biogenesis With Cell Elongatio...mentioning

confidence: 99%

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope

et al. 2020

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The biology of mycobacteria is dominated by a complex cell envelope of unique composition and structure and of exceptionally low permeability. This cell envelope is the basis of many of the pathogenic features of mycobacteria and the site of susceptibility and resistance to many antibiotics and host defense mechanisms. This review is focused on the transporters that assemble and functionalize this complex structure. It highlights both the progress and the limits of our understanding of how (lipo)polysaccharides, (glyco)lipids, and other bacterial secretion products are translocated across the different layers of the cell envelope to their final extra-cytoplasmic location. It further describes some of the unique strategies evolved by mycobacteria to import nutrients and other products through this highly impermeable barrier.

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Section: Coordination Of Cell Envelope Biogenesis With Cell Elongatio...mentioning

confidence: 99%

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope

et al. 2020

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“…Considering the complex organization of the mycomembrane (for review, see (Angala et al ., 2014; Jackson et al ., 2020)), PPI taking place at the cell wall (CW) level in mycobacteria are difficult to identify. Although there are several methods to decipher PPI in mycobacteria (for review, see (Veyron‐Churlet and Locht, 2019)), they all have their limits when the protein of interest is membrane‐ or CW‐associated. BioID appears thus as an attractive tool to study PPI at the CW level.…”

Section: Introductionmentioning

confidence: 99%

Interconnection of the mycobacterial heparin‐binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity‐dependent biotin identification inMycobacterium smegmatis

Veyron‐Churlet

Saliou

Locht

2021

Environmental Microbiology

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Deciphering protein-protein interactions is a critical step in the identification and the understanding of biological mechanisms deployed by pathogenic bacteria. The development of in vivo technologies to characterize these interactions is still in its infancy, especially for bacteria whose subcellular organization is particularly complex, such as mycobacteria. In this work, we used the proximity-dependent biotin identification (BioID) to define the mycobacterial heparin-binding hemagglutinin (HbhA) interactome in the saprophytic bacterium Mycobacterium smegmatis. M. smegmatis is a commonly used model to study and characterize the physiology of pathogenic mycobacteria, such as Mycobacterium tuberculosis. Here, we adapted the BioID technology to in vivo protein-protein interactions studies in M. smegmatis, which presents several advantages, such as maintaining the complex organization of the mycomembrane, offering the possibility to study membrane or cell wall-associated proteins, including HbhA, in the presence of cofactors and post-translational modifications, such as the complex methylation pattern of HbhA. Using this technology, we found that HbhA is interconnected with cholesterol degradation and heme/iron pathways. These results are in line with previous studies showing the dual localization of HbhA, associated with the cell wall and intracytoplasmic lipid inclusions, and its induction under high iron growth conditions. AbbreviationsAPEX ascorbic acid peroxidase BioID proximity-dependent biotin identification BirA E. coli biotin ligase CW cell wall HbhA heparin-binding hemagglutinin HRP horseradish peroxidase ILI intracytoplasmic lipid inclusions Mtb Mycobacterium tuberculosis PPI protein-protein interactions

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“…Currently, a huge problem is the emergence of microbial strains resistant to a variety of antibiotics, such as tuberculosis pathogens. Protein-protein interactions also play a very important role in the physiology of Mycobacterium tuberculosis and the disease progress in the host [47]. Substances affecting these interactions could be the basis for a new generation of drugs.…”

Section: Differences In Two Approaches and Perspectives Of Bira-catal...mentioning

confidence: 99%

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Kulyyassov

Ramankulov

Ogryzko

2022

Life

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Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.

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In Vivo Methods to Study Protein–Protein Interactions as Key Players in Mycobacterium Tuberculosis Virulence

Cited by 5 publications

References 101 publications

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope

Interconnection of the mycobacterial heparin‐binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity‐dependent biotin identification inMycobacterium smegmatis

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

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