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Item Open Access Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults(National Institute of Environmental Health Sciences (NIEHS), 2012-05-01) Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R; Witter, Frank R; Halden, Rolf UEarly 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.Item Open Access Purification and proteomics of pathogen-modified vacuoles and membranes(Frontiers Media, 2015-06-02) Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C; Subbarayal, Prema; Prusty, Bhupesh K; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E; Rudel, Thomas; Hilbi, HubertCertain 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.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.Item Open Access Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica*(Elsevier, 2015-01) Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, NicoleItem Open Access Proteomic Analysis of Salmonella-modified Membranes Reveals Adaptations to Macrophage Hosts*(American Society for Biochemistry and Molecular Biology (ASBMB), 2020-05) Reuter, Tatjana; Vorwerk, Stephanie; Liss, Viktoria; Chao, Tzu-Chiao; Hensel, Michael; Hansmeier, NicoleSystemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen Salmonella enterica commonly forms highly dynamic and extensive tubular membrane compartments built from Salmonella-modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of S. enterica is well researched, much less is known regarding specific adaptations to different host cell types. Using an affinity-based proteome approach, we explored the composition of SMMs in murine macrophages. The systematic characterization provides a broader landscape of host players to the maturation of Salmonella-containing compartments and reveals core host elements targeted by Salmonella in macrophages as well as epithelial cells. However, we also identified subtle host specific adaptations. Some of these observations, such as the differential involvement of the COPII system, Rab GTPases 2A, 8B, 11 and ER transport proteins Sec61 and Sec22B may explain cell line-dependent variations in the pathophysiology of Salmonella infections. In summary, our system-wide approach demonstrates a hitherto underappreciated impact of the host cell type in the formation of intracellular compartments by Salmonella.Item Open Access Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction(American Society for Microbiology, 2019-12-11) Noster, Janina; Hansmeier, Nicole; Persicke, Marcus; Chao, Tzu-Chiao; Kurre, Rainer; Popp, Jasmin; Liss, Viktoria; Reuter, Tatjana; Hensel, MichaelThe tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S. 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 (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S. Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S. Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S. Typhimurium and cellular functions and virulence.Item Open Access Impact of ROS-Induced Damage of TCA Cycle Enzymes on Metabolism and Virulence of Salmonella enterica serovar Typhimurium(Frontiers Media, 2019-04-24) Noster, Janina; Persicke, Marcus; Chao, Tzu-Chiao; Krone, Lena; Heppner, Bianca; Hensel, Michael; Hansmeier, NicoleSalmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella-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. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (ΔsodAB). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. ΔsodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (ΔacnAB). Furthermore, we determined a decreased expression of acnA in STM ΔsodAB. While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM ΔacnAB, proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.Item Open Access Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense(Public Library of Science, 2019-04-22) Noster, Janina; Chao, Tzu-Chiao; Sander, Nathalie; Schulte, Marc; Reuter, Tatjana; Hansmeier, Nicole; Hensel, MichaelIntracellular 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 tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms.Item Open Access Evaluating the effect of spaceflight on the host–pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium(Nature Research, 2021-03-09) Barrila, Jennifer; Sarker, Shameema F.; Hansmeier, Nicole; Yang, Shanshan; Buss, Kristina; Briones, Natalia; Park, Jin; Davis, Richard R.; Forsyth, Rebecca J.; Ott, C. Mark; Sato, Kevin; Kosnik, Cristine; Yang, Anthony; Shimoda, Cheryl; Rayl, Nicole; Ly, Diana; Landenberger, Aaron; Wilson, Stephanie D.; Yamazaki, Naoko; Steel, Jason; Montano, Camila; Halden, Rolf U.; Cannon, Tom; Castro-Wallace, Sarah L.; Nickerson, Cheryl A.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. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.Item Open Access Manipulation of microvillar proteins during Salmonella enterica invasion results in brush border effacement and actin remodeling(Frontiers Media, 2023-03-02) Felipe-Lopez, Alfonso; Hansmeier, Nicole; Danzer, Claudia; Hensel, MichaelEnterocyte invasion by the gastrointestinal pathogen Salmonella enterica 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 Salmonella 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. PEC were infected with S. enterica wild-type and mutant strains with defined defects in SPI1-T3SS effector proteins, and pharmacological inhibition of actin assembly were applied. We identified that microvilli effacement involves two distinct mechanisms: i) F-actin depolymerization mediated by villin and ii), the consumption of cytoplasmic G-actin by formation of membrane ruffles. By analyzing the contribution of individual SPI1-T3SS effector proteins, we demonstrate that SopE dominantly triggers microvilli effacement and formation of membrane ruffles. Furthermore, SopE via Rac1 indirectly manipulates villin, which culminates in F-actin depolymerization. Collectively, these results indicate that SopE has dual functions during F-actin remodeling in PEC. While SopE-Rac1 triggers F-actin polymerization and ruffle formation, activation of PLCg and villin by SopE depolymerizes F-actin in PEC. These results demonstrate the key role of SopE in destruction of the intestinal barrier during intestinal infection by Salmonella.