The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

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.

Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi

SUMMARY There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets.

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

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.

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the Gβγ subunits dependent activation of the phospholipase C beta 3 (PLCβ3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism “ARTG” (AutoRegulation of TGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.

Role of MCC/Eisosome in Fungal Lipid Homeostasis

One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome’s proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.