Hery, M. | Search bioRxiv

bioRxiv2021

Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on {beta}-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

cell biology↗

bioRxiv2021

Specificities and commonalities of carbapenemase producing Escherichia coli isolated in France from 2012 to 2015.

Carbapenemase-producing Escherichia coli (CP-Ec) represent a major public health threat with a risk of dissemination in the community as it has occurred for lineages producing extended spectrum {beta}-lactamases. To characterize the extend of CP-Ec spread in France, isolates from screening and infection samples received at the French National Reference Centre laboratory (F-NRC) for carbapenemase-producing Enterobacterales were investigated. Six hundred and ninety one CP-Ec isolates collected between 2012 and 2015 and 22 before were fully sequenced. Analysis of their genome sequences revealed some disseminating multidrug resistant (MDR) lineages frequently acquiring diverse carbapenemase genes mainly belonging to clonal complex (CC) 23 (ST 410) and CC10 (ST10, ST167) and sporadic isolates including rare ST131 isolates (n=17). However, the most represented ST was ST38 (n=92) with four disseminated lineages carrying blaOXA-48-like genes inserted in the chromosome. Globally, the most frequent carbapenemase gene (n=457) was blaOXA-48. It was also less frequently associated with MDR isolates being the only resistance gene in 119 isolates. Thus, outside the ST38 clades, its acquisition was frequently sporadic with no sign of dissemination, reflecting the circulation of the IncL plasmid pOXA-48 in France and its high frequency of conjugation. In contrast blaOXA-181 or blaNDM genes were often associated with the evolution of MDR E. coli lineages characterized by mutations in ftsI and ompC. IMPORTANCECarbapenemase-producing Escherichia coli (CP-Ec) might be difficult to detect, as minimal inhibitory concentrations can be very low. However, their absolute number and their proportion among carbapenem-resistant Enterobacterales have been increasing, as reported by WHO and national surveillance programs. This suggests a still largely uncharacterized community spread of these isolates. Here we have characterized the diversity and evolution of CP-Ec isolated in France before 2016. We show that carbapenemase genes are associated with a wide variety of E. coli genomic backgrounds and a small number of dominant phylogenetic lineages. In a significant proportion of CP-Ec, the most frequent carbapenemase gene blaOXA-48, was detected in isolates lacking any other resistance gene, reflecting the dissemination of pOXA-48 plasmids, likely in the absence of any antibiotic pressure. In contrast carbapenemase gene transfer may also occur in multi-drug resistant E. coli, ultimately giving rise to at-risk lineages encoding carbapenemases with a high potential of dissemination.

microbiology↗