Destruction of the brush border by sv. Typhimurium subverts resorption by polarized epithelial cells.

serovar Typhimurium is an invasive, facultative intracellular gastrointestinal pathogen that destroys the brush border of polarized epithelial cells (PEC). The brush border is critical for the functions of PEC because it resorbs nutrients from the intestinal lumen and builds a physical barrier to infecting pathogens. The manipuation of PEC during infection by was investigated by live-cell imaging and ultrastructural analysed of the brush border.

Quantitative Proteomics of the Intracellular Bacterial Pathogen Salmonella enterica Serovar Typhimurium.

Mass spectrometry-based proteomics provides a wealth of information about changes in protein production and abundance under diverse conditions, as well as mechanisms of regulation, signaling cascades, interaction partners, and communication patterns across biological systems. For profiling of intracellular pathogens, proteomic profiling can be performed in the absence of a host to singularly define the pathogenic proteome or during an infection-like setting to identify dual perspectives of infection. In this chapter, we present techniques to extract proteins from the human bacterial intracellular pathogen, Salmonella enterica serovar Typhimurium, in the presence of macrophages, an important innate immune cell in host defense.

Manipulation of microvillar proteins during invasion results in brush border effacement and actin remodeling.

Enterocyte invasion by the gastrointestinal pathogen is accompanied by loss of brush border and massive remodeling of the actin cytoskeleton, leading to microvilli effacement and formation of membrane ruffles. These manipulations are mediated by effector proteins translocated by the Pathogenicity Island 1-encoded type III secretion system (SPI1-T3SS). To unravel the mechanisms of microvilli effacement and contribution of SPI1-T3SS effector proteins, the dynamics of host-pathogen interactions was analyzed using live cell imaging (LCI) of polarized epithelial cells (PEC) expressing LifeAct-GFP.

Affinity Enrichment of Salmonella-Modified Membranes from Murine Macrophages for Proteomic Analyses.

Dissecting host-pathogen interaction requires the ability to specifically enrich distinct proteins along with their co-assembled constituents or complexes. Affinity technologies leverage specificity of reagents to desired targets and help to enrich proteins of interests along with specifically associated proteins. Coupled with mass-spectrometry-based proteomics, this technology has become a powerful tool to explore pathogen compartments of diverse facultative and obligate intracellular pathogens.

Protein Purification and Digestion Methods for Bacterial Proteomic Analyses.

Reproducible protein extraction is critical for the quantitative analysis of bacterial proteomes. While a wide range of techniques exist, there is no one-size-fits-all solution that will be suitable for all applications. In this report, we describe a set of standard extraction methods that have been adapted for a range of bacterial proteome analyses.

Evaluating the effect of spaceflight on the host-pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium.

Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host-pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight.

Proteomic Analysis of -modified Membranes Reveals Adaptations to Macrophage Hosts.

Systemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen commonly forms highly dynamic and extensive tubular membrane compartments built from -modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of is well researched, much less is known regarding specific adaptations to different host cell types.

Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction.

The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular serovar Typhimurium ( Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (Δ) elicited a unique metabolic profile.

Impact of ROS-Induced Damage of TCA Cycle Enzymes on Metabolism and Virulence of serovar Typhimurium.

serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the -containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron-sulfur (Fe-S) clusters are particularly sensitive and become non-functional upon oxidation.

Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense.

Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV.

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter.

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR.

Identification of Mature Atherosclerotic Plaque Proteome Signatures Using Data-Independent Acquisition Mass Spectrometry.

Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms.

Functional expression of the entire adhesiome of Salmonella enterica serotype Typhimurium.

Adhesins are crucial virulence factors of pathogenic bacteria involved in colonization, transmission and pathogenesis. Many bacterial genomes contain the information for a surprisingly large number of diverse adhesive structures. One prominent example is the invasive and facultative intracellular pathogen Salmonella enterica with an adhesiome of up to 20 adhesins.

Purification and proteomics of pathogen-modified vacuoles and membranes.

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.

The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties.

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes.

Elucidating the molecular basis of adverse health effects from exposure to anthropogenic polyfluorinated compounds using toxicoproteomic approaches.

Linear, short-chain polyfluorinated and perfluorinated alkyl compounds, often referred to as PFCs, have been in worldwide use as surfactants and polymer precursors for decades, and environmental dispersal of these highly persistent compounds represents a public health threat. Whereas ubiquitous low-level exposure to these compounds has been demonstrated in human populations from around the world, the exact mechanisms of toxicity and their toxic potency remain subject to investigation and scientific dispute. As with other environmental exposures, a major hurdle for gaining a better understanding of their human health impacts is the limited utility of cell culture and animal models serving as convenient, yet imperfect proxies to human physiology and disease.

Proteomes of host cell membranes modified by intracellular activities of Salmonella enterica.

Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies.

Salmonella enterica invasion of polarized epithelial cells is a highly cooperative effort.

The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system (T3SS) and the SPI4-encoded adhesin SiiE. The invasion of polarized cells is more efficient than that of nonpolarized cells, and we observed the formation of clusters of bacteria on infected cells. Here we demonstrate that the invasion of polarized cells is a highly cooperative activity.

Microfluidic devices for high-throughput proteome analyses.

Over the last decades, microfabricated bioanalytical platforms have gained enormous interest due to their potential to revolutionize biological analytics. Their popularity is based on several key properties, such as high flexibility of design, low sample consumption, rapid analysis time, and minimization of manual handling steps, which are of interest for proteomics analyses. An ideal totally integrated chip-based microfluidic device could allow rapid automated workflows starting from cell cultivation and ending with MS-based proteome analysis.

Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults.

Background: Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped.

The current state of microbial proteomics: where we are and where we want to go.

Proteomics allows the assessment of cellular processes in an unprecedented scale by providing a comprehensive quantitative and qualitative overview of the protein content of a cell. Consequently, proteomics has been employed to investigate a multitude of bacterial processes ranging from the analysis of environmental communities, identification of virulence factors to the proteome-guided optimization of production strains. Proteomics has, in short, become an indispensable tool for the global analysis of bacterial physiology.

Characterization and liquid chromatography-MS/MS based quantification of hydroxylated fullerenes.

Highly water-soluble hydroxylated fullerene derivatives are being investigated for a wide range of commercial products as well as for potential cytotoxicity. However, no analytical methods are currently available for their quantification at sub-ppm concentrations in environmental matrixes. Here, we report on the development and comparison of liquid chromatography-ultraviolet/visible spectroscopy (LC-UV/vis) and liquid chromatography-mass spectrometry (LC-MS) based detection and quantification methods for commercial fullerols.

Towards proteome standards: the use of absolute quantitation in high-throughput biomarker discovery.

The use of proteomics to profile biological fluids and identify therein biomarkers for cancer and other diseases was initially received with considerable excitement. However, results have fallen short of the expectations. Traditionally, protein biomarkers have been identified by measurement of relative expression changes between case and control samples from which differentially expressed proteins are then considered to represent biomarker candidates.

Comprehensive proteome profiling of the Fe(III)-reducing myxobacterium Anaeromyxobacter dehalogenans 2CP-C during growth with fumarate and ferric citrate.

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta-proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2-D reference map of the soluble A.