The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.

In this work we demonstrate that time domain techniques can be used successfully to monitor realtively weak modulations of the fluorescence in sensing applications. The metal sensing complex Newport Green DCF™ can detect selected transition metals in vivo as well as in vitro. Incremental addition of Ni and/or Zn (in vitro) lead to a substantial reduction in the yield of the fast component in a bi-exponential fluorescence decay (τ1 = 150–250 ps) from 60% to 30–35%. This is rationalised as an inhibition of intra-molecular electron transfer in the NPG sensing complex due to metal complexation. In order to explore this effect in cellulo, NIH 3 T3 mouse skin fibroplast cells were pre-incubated with set levels of Ni and Zn, at a constant concentration of NPG. The fluorescence modulation in cellullo was subsequently studied employing both time-resolved fluorescence microscopy and confocal fluorescence microscopy. In correlation with the in vitro observations, similar effects were observed on the fluorescence decay in cellulo.Electronic supplementary materialThe online version of this article (10.1007/s10895-018-2335-z) contains supplementary material, which is available to authorized users.

Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein-protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast Saccharomyces cerevisiae, we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation promoting Las17-Vrp1 complex, and that the network of SH3 interactions coordinate both Las17-Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority uses the SH3 domain to recruit other proteins, and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a high number of acute and chronic hospital-acquired infections. As it develops more and more resistances against existing antibiotics, P. aeruginosa has been placed highest on the global priority list of antibiotic-resistant bacteria for which alternative treatments are urgently needed. Former studies have highlighted the crucial role of the bacterial lectin LecA and the host cell glycosphingolipid globotriaosylceramide (Gb3) for the cellular uptake of P. aeruginosa into epithelial cells via the lipid zipper mechanism. To further characterize the host cell plasma membrane domain for LecA-driven attachment and invasion, we analyzed the protein and lipid composition of pulled-down membrane domains for novel interaction partners of LecA by mass spectrometry. We unraveled a predilection of LecA for binding to saturated Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate clusters at the intracellular leaflet co-localizing with sites of LecA binding. Moreover, we identified the GPI-anchored protein CD59 and flotillins, known as cargo and eponymous component of flotillin-assisted endocytosis, as LecA interaction partners. Depletion of each of these host cell proteins resulted in more than 50% of reduction in invasiveness of the P. aeruginosa strain PAO1 highlighting the importance of this LecA-induced plasma membrane domain. Our strategy to reduce the complexity of host-pathogen interactions by first identifying interaction partners of a single virulence factor and subsequently transferring these findings to the bacterium has been proven to be a successful approach in elucidating the molecular mechanisms of bacterial infections.