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

Abstract

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.

Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

“…Other pathogenic bacteria are known to require ordered domains in order to establish their infection. Shigella flexneri and P. aeruginosa, for example, co-opt the lipid rafts of their host membranes for their entry (Grassmé et al, 2003;Goot et al, 2004;Brandel et al, 2021). In addition, it was also proposed that these pathogens use lipid rafts to damage the compartment in which they reside.…”

“…In addition, Anaplasma phagocytophilum inclusions are also enriched with flotillins, and it was proposed that flotillins participate in trafficking of free cholesterol to allow replication of A. phagocytophilum (Xiong et al, 2019). Recently, flotillins were found to be associated with the Pseudomonas aeruginosa lectin LecA, and to be required for bacteria entry into the host cell, together with saturated long-chain fatty acids and the GPI-anchored protein CD59 (Brandel et al, 2021). On the other hand, the Leishmania parasite actively depletes flotillin-1 from its compartment by preventing phagosome-lysosome fusion (Dermine et al, 2001).…”

The Protein Toxins Ricin and Shiga Toxin as Tools to Explore Cellular Mechanisms of Internalization and Intracellular Transport

“…Moreover, monounsaturated fatty acyl groups with 16-20 carbon atoms seem to be absent in glycosphingolipids [6,135]. However, minor amounts of Gb3 containing C18:1 were recently reported in H1299 cells [136]. Since the glycosphingolipids normally contain very little of species with N-amidated C18 and C20 fatty acyl groups, the glycosphingolipids have a bimodal distribution regarding the fatty acyl chain length [82], which is further discussed below.…”

Multiplicity of Glycosphingolipid-Enriched Microdomain-Driven Immune Signaling

“…LacCer-enriched microdomains are likely to interact in cis with the extracellular juxtamembrane region of CD18 [51]. In lung epithelial cells, the interaction of the Pseudomonas aeruginosa-derived virulence factor LecA with globotriaosylceramide (Gb3Cer) in the outer leaflet of plasma membranes was found to induce the formation of membrane domains enriched in saturated long fatty acyl chain-containing Gb3Cer species, the GPI-anchored protein CD59, phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and flotillin, thereby promoting the efficient uptake of P. aeruginosa [52]. Mechanisms were suggested by which Gb3Cer and its associated molecules mediate signal transduction from extracellular to intracellular sites through transbilayer coupling.…”