The transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli.

Enterohemorrhagic (EHEC) is a foodborne pathogen that colonizes the gastrointestinal tract and has evolved intricate mechanisms to sense and respond to the host environment. Upon the sensation of chemical and physical cues specific to the host's intestinal environment, locus of enterocyte effacement (LEE)-encoded virulence genes are activated and promote intestinal colonization. The LEE transcriptional activator GrlA mediates EHEC's response to mechanical cues characteristic of the intestinal niche, including adhesive force that results from bacterial adherence to epithelial cells and fluid shear that results from intestinal motility and transit.

Outer Membrane Vesicle-Host Cell Interactions.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes derived from the outer membrane of Gram-negative bacteria. They are ubiquitously produced both in culture and during infection and are now recognized to play crucial roles during host-microbe interactions. OMVs can transport a broad range of chemically diverse cargoes, including lipids and lipopolysaccharides, membrane-embedded and associated proteins and small molecules, peptidoglycan, and nucleic acids.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P.

Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies.

Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear.

Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells.

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe.

Cell-free reconstitution of multivesicular body (MVB) cargo sorting.

The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles.

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9.

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression.

UBE4B protein couples ubiquitination and sorting machineries to enable epidermal growth factor receptor (EGFR) degradation.

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown.

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown.

Cellular communication via nanoparticle-transporting biovesicles.

Aims: Endothelial cells are dynamic cells tasked with selective transport of cargo from blood vessels to tissues. Here we demonstrate the potential for nanoparticle transport across endothelial cells in membrane-bound vesicles.

Materials & Methods: Cell-free endothelial-derived biovesicles were characterized for cellular and nanoparticle content by electron microscopy.

UBE4B levels are correlated with clinical outcomes in neuroblastoma patients and with altered neuroblastoma cell proliferation and sensitivity to epidermal growth factor receptor inhibitors.

Background: The UBE4B gene, which is located on chromosome 1p36, encodes a ubiquitin ligase that interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a protein involved in epidermal growth factor receptor (EGFR) trafficking, suggesting a link between EGFR trafficking and neuroblastoma pathogenesis. The authors analyzed the roles of UBE4B in the outcomes of patients with neuroblastoma and in neuroblastoma tumor cell proliferation, EGFR trafficking, and response to EGFR inhibition.

Methods: The association between UBE4B expression and the survival of patients with neuroblastoma was examined using available microarray data sets.

Cell-free reconstitution of multivesicular body formation and receptor sorting.

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements.