Duma, L. | bioRxiv Science

bioRxiv2024

Fluor NMR study of amino acid derived ligand to study TSPO

Translocator protein (TSPO, 18 kDa), previously known as peripheral-type benzodiazepine receptor, is an evolutionarily conserved membrane protein involved in various physiological processes and patho-physiological conditions. The endogeneous TSPO ligand is a polypeptide of 9 kDa, but dipeptides with biological activity have been previously synthesized and characterized. Herein, we synthesized a phenyl alanine derived ligand with a 19F labelling which opens prospective for 19F-MRI and potential 18F-PET applications. We characterized the coexistence of two conformers that are not equally sensitive to the media used for membrane protein studies. Interaction studies with the recombinant mouse TSPO (mTSPO) in different membrane-mimicking environments are presented using 19F NMR enabling structure/function characterizations. A change in the mTSPO environment from pure detergent to lipid/detergent mixture reveals different exchange rates between bound and free ligand forms. Competition experiments with the high-affinity drug ligand (R)-PK 11195 suggests that phenyl alanine derived ligand binds in the same protein cavity. HighlightsO_LIFluor labelling of ligands easily reveals the presence of conformers C_LIO_LIFluorinated phenyl alanine derived ligand interacts with TSPO C_LIO_LIFluor NMR enables characterization of TSPO ligand interactions C_LIO_LIFluor NMR facilitates exchange rate studies between free and bound ligand states C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/618300v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1913e31org.highwire.dtl.DTLVardef@8a3c30org.highwire.dtl.DTLVardef@175881borg.highwire.dtl.DTLVardef@13aaa7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

The impact of plasma membrane lipid composition on flagella-mediated adhesion of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC on host tissues is the first step enabling bacterial colonization. Adhesins like fimbriae and flagella mediate this mechanism. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using Giant Unilamellar Vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components including proteins and glycoproteins. In contrast, with GUVs we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild type EHEC but not of a non-flagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagella-mediated adhesion to plasma membranes has broad implications to host-pathogen interactions.Importance Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues and form biofilm. Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagella-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagella adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts are important in bacterial adhesion. These findings significantly strengthen plasma membrane adhesion via bacterial flagella in an important human pathogen. This mechanism represents a promising target for the development of novel anti-adhesion therapies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗