Pathogen and Toxin Entry - How Pathogens and Toxins Induce and Harness Endocytotic Mechanisms

Humans have been exposed to a plethora of pathogens (bacteria, viruses) ever since. Infectious diseases are among the leading causes of death worldwide. For example, in 2011, 1.34 million people died of tuberculosis, which is caused by an infection with Mycobacterium tuberculosis. Even more died of an infection by the human immunodeficiency virus (HIV; 1.78 million) or lower respiratory tract infection (3.46 million) [1]. In addition, recurring pandemic outbreaks of the influenza A virus, as in 2009, or an epidemic outbreak of enterohemorrhagic E. coli (EHEC) in Germany in 2011, show quite plainly that pathogens in the 21th century still are a severe health problem, not only in developing countries

Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells

Glycans in autophagy, endocytosis and lysosomal functions

Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal

Synchronizing Protein Traffic to the Primary Cilium

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane

Pseudomonas aeruginosa lectin LecB inhibits tissue repair processes by triggering β-catenin degradation

AbstractPseudomonas 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

Lipid Reorganization Induced by Shiga Toxin Clustering on Planar Membranes

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb3 in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb3 lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb3 (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb3 (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb3, STxB-Gb3 complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

Impedance analysis and single ion channel recordings on pore-suspending lipid bilayers based on highly ordered pore arrays

Ion channels are one of the main drug targets and are in the focus of various membrane biosensor applications and drug screening assays. The aim of this thesis was to develop and characterize a novel membrane system suspending highly ordered porous substrates. This hybrid membrane system was supposed to combine the advantages of freestanding and solid supported lipid membranes. While part of the lipid bilayer anchored to the surface of the porous matrix resembles a solid supported membrane, the pore-suspending parts can be viewed as freestanding lipid membranes. Hexagonally ordered porous alumina and macroporous silicon substrates with pore diameters in the nano- and micrometer range were fabricated by different etching procedures and characterized in detail by impedance spectroscopy and scanning electron microscopy. These highly ordered sieve-like pore arrays of billions of pores per square centimeter were used as supports for lipid bilayer immobilization. Two different methods were successfully developed to obtain pore-suspending lipid bilayers based on these porous substrates: (1) a painting technique and (2) a technique based on vesicle spreading and fusion: (1) Painting technique: In order to ensure that the prepared bilayers suspend the pores and do not cover the inner pore walls, the top of the pore columns was selectively functionalized by coating with a thin gold layer followed by chemisorption of either 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol or 1-octadecanethiol. Lipid bilayers suspending the pores were obtained by painting 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane across the porous matrix. The membrane formation process was followed by means of electrical impedance spectroscopy, and membrane specific parameters were extracted from the impedance data by modeling the electrical behavior of these membrane systems by an adequate equivalent circuit consisting of a simple parallel RmCm-element. Specific membrane capacitances of CmA = 0.7 µF/cm2 were calculated indicating the formation of single lipid bilayers. Suspended lipid bilayers on porous alumina, which we termed nano-black lipid membranes (nano-BLMs) and those suspending macroporous silicon substrates, termed micro-BLMs, both exhibited membrane resistances in the gigaohm regime allowing for single ion channel recordings and an extraordinary high long-term stability. In contrast to classical BLMs, which rupture in one single step, the membrane resistance of nano- and micro-BLMs decreases continuously, which was attributed to the fact that each membrane suspending a single pore can rupture individually due to the separation of the freestanding bilayer parts by the chemisorbed hydrophobic submonolayer. To prove this hypothesis porous matrix-supported BLMs were formed by painting 1,2-diphytanoyl-sn-glycero-3-phosphocholine across macroporous silicon substrates without pre-functionalization. Indeed, without the chemisorbed submonolayer, these matrix-supported lipid bilayers resemble classical BLMs. Though they exhibit typical membrane specific parameters, they rupture in one single event. (2) Vesicle spreading and fusion: The formation of solvent-free pore-suspending lipid bilayers was achieved by spreading and fusion of thiolipid-containing large unilamellar vesicles on porous alumina substrates, which were covered on top of the pore columns with a thin gold layer. Impedance analysis revealed that these membranes were, however, not defect-free and thus, were as yet not suited for single channel recordings. Pore-suspending lipid bilayers formed by the painting technique were proven to be ideally suited as membrane biosensors with fully functional transmembrane ion channels. The peptide antibiotics gramicidin and alamethicin as well as the transmembrane domain of the HIV-1 accessory peptide Vpu were successfully inserted into these novel chip-based membrane systems and peptide-characteristic conductance states were recorded. For Vpu, different amiloride derivates were elucidated as potential drugs to inhibit its channel activity. These measurements confirm the potential of nano- and micro-BLMs as membrane biosensors. Pore-suspending lipid bilayers based on hexagonally ordered pore arrays will allow for automation and parallelization of ion channel recordings and will thus enable the development of high-throughput screening assays. Furthermore, the highly ordered porous structure serving as membrane support will allow adressing each substrate pore by space-resolved electrochemical techniques. This will enable one to perform several measurements on one support quasi-simultaneously

Impedance Analysis and Single-Channel Recordings on Nano-Black Lipid Membranes Based on Porous Alumina

Ordered porous alumina substrates with pore diameters of 55 and 280 nm, respectively, were produced and utilized as a support to prepare membranes suspending the pores of the material. Highly ordered porous alumina was prepared by an anodization process followed by dissolution of the remaining aluminum and alumina at the backside of the pores. The dissolution process of Al(2)O(3) at the backside of the pores was monitored by electrical impedance spectroscopy ensuring the desired sieve-like structure of the porous alumina. One side of the porous material with an area of 7 mm(2) was coated with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol. The hydrophobic monolayer on top of the upper surface was a prerequisite for the formation of suspending membranes, termed nano-black lipid membranes (nano-BLMs). The formation process, and long-term and mechanical stability of the nano-BLMs were followed by electrical impedance spectroscopy indicating the formation of lipid bilayers with typical specific membrane capacitances of (0.65 ± 0.2) μF/cm(2) and membrane resistances of up to 1.6 × 10(8) Ω cm(2). These high membrane resistances allowed for single-channel recordings. Gramicidin as well as alamethicin was successfully inserted into the nano-BLMs exhibiting characteristic conductance states