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Septin GTPases polymerize into higher-ordered structures as a part of the cytoskeleton and are involved in interactions of the host with a wide spectrum of pathogens. Many pathogens foster an ambiguous relationship with septins. They exploit septins for uptake, but septins also prevent their intracellular replication and target them for autophagy. We demonstrate that septins are involved in a defense mechanism against the pathogen Pseudomonas aeruginosa, which enters cells via a lipid zippering mechanism relying on interaction of the lectin LecA with the glycosphingolipid Gb3 on the host membrane. LecA-dependent invagination of the plasma membrane triggers septin recruitment to the site of bacterial attachment. We also find a septin-dependent reinforcement of cortical actin at attachment sites. Atomic force microscopy reveals formation of a septin-dependent rigid barrier below the membrane, preventing bacterial penetration. Our data suggest that septin barriers represent a cellular defense against bacteria inducing membrane curvature for invasion.

International audience; The human pathogen Pseudomonas aeruginosa induces phosphorylation of the adaptor protein CrkII by activating the non-receptor tyrosine kinase Abl to promote its uptake into host cells. So far, the bacterial as well as host cell factors which induce Abl/CrkII signalling are entirely unknown. In this research, we employed human lung epithelial cells H1299, Chinese hamster ovary cells and P. aeruginosa wild type strain PAO1 to study the invasion process of P. aeruginosa into host cells by using microbiological, biochemical and cell biological approaches such as Western Blot, immunofluorescence microscopy and flow cytometry. Here, we demonstrate that the host glycosphingolipid globotriaosylceramide, also termed Gb3, represents a signalling receptor for the P. aeruginosa lectin LecA to induce CrkII phosphorylation at tyrosine 221. Alterations in Gb3 expression and LecA function correlate with CrkII phosphorylation. Interestingly, phosphorylation of CrkII Y221 occurs independently of Abl kinase. We further show that Src family kinases transduce the signal induced by LecA binding to Gb3, leading to Crk Y221 phosphorylation. In summary, we identified LecA as a bacterial factor, which utilizes a so far unrecognized mechanism for phospho-CrkII Y221 induction by binding to the host glycosphingolipid receptor Gb3. The LecA/Gb3 interaction highlights the potential of glycolipids to mediate signalling processes across the plasma membrane and should be further elucidated to gain deeper insights into this non-canonical mechanism of activating host cell processes.

Pseudomonas aeruginosa is an opportunistic pathogen that induces severe lung infections such as ventilator-associated pneumonia and acute lung injury. Under these conditions, the bacterium diminishes epithelial integrity and inhibits tissue repair mechanisms, leading to persistent infections. Understanding the involved bacterial virulence factors and their mode of action is essential for the development of new therapeutic approaches. In our study we discovered a so far unknown effect of the P. aeruginosa lectin LecB on host cell physiology. LecB alone was sufficient to attenuate migration and proliferation of human lung epithelial cells and to induce transcriptional activity of NF-κB. These effects are characteristic of impaired tissue repair. Moreover, we found a strong degradation of β-catenin, which was partially recovered by the proteasome inhibitor lactacystin. In addition, LecB induced loss of cell–cell contacts and reduced expression of the β-catenin targets c-myc and cyclin D1. Blocking of LecB binding to host cell plasma membrane receptors by soluble l-fucose prevented these changes in host cell behavior and signaling, and thereby provides a powerful strategy to suppress LecB function. Our findings suggest that P. aeruginosa employs LecB as a virulence factor to induce β-catenin degradation, which then represses processes that are directly linked to tissue recovery., Highlights • The Pseudomonas aeruginosa lectin LecB reduces lung cell migration and proliferation • LecB also induces NF-kB-mediated inflammation in lung epithelial cells • ß-catenin degradation and reduction of c-myc and cyclin D1 protein levels inhibit cell proliferation and migration • Blocking of LecB binding to host cell receptors with L-fucose suppresses all effects caused by LecB

Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology.

Shiga toxin subunit B (STxB) binding to its cellular receptor Gb3 leads to the formation of protein-lipid clusters and bending of the membrane. A newly developed synthetic route allowed synthesizing the biologically most relevant Gb3-C24:1 2OH species with both, the natural (Gb3-R) as well as the unnatural (Gb3-S) configuration of the 2OH group. The derivatives bind STxB with identical nanomolar affinity, while the propensity to induce membrane tubules in giant unilamellar vesicles is more pronounced for Gb3-S. Fluorescence and atomic force microscopy images of phase-separated supported membranes revealed differences in the lateral organization of the protein on the membrane. Gb3-R favorably induces large and tightly packed protein clusters, while a lower protein density is found on Gb3-S doped membranes.

Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15 N-STxB) was synthesised in Escherichia coli using 15 NH 4 Cl as sole nitrogen source resulting in 15 N enrichment in the molecule. Using the NanOSIMS 50 ion microprobe (Cameca), different ion species ( 12 C 14 N - , 12 C 15 N 31 P - ) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12 C 15 N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15 N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15 N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

The current urge to understand the role of lipids in defense against pathogens is driven by two strategies: killing pathogens and protecting the cell from infection. Antimicrobial peptides and antimicrobial polymers offer a promising alternative to classical antibiotics through their action on membrane integrity. In principle, antimicrobial peptides and polymers are able to cluster lipids through lipid selection and recruitment from a mixed membrane. We show that antimicrobial polymers induce leakage in lipid vesicles by transient defects rather than defined pores.