Ulrich Schaible - Academia.edu (original) (raw)

Papers by Ulrich Schaible

Research paper thumbnail of Isoniazid@Fe 2 O 3 Nanocontainers and Their Antibacterial Effect on Tuberculosis Mycobacteria

Angewandte Chemie International Edition, 2015

Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter &lt... more Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.

Research paper thumbnail of Targeting the Lysosome: Fluorescent Iron(III) Chelators To Selectively Monitor Endosomal/Lysosomal Labile Iron Pools

Journal of Medicinal Chemistry, 2008

Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy hav... more Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy have led to the identification of a distinct subcellular compartmentation of intracellular redox-active "labile" iron. To investigate the distribution of labile iron, our research has been focused on the development of fluorescent iron sensors targeting the endosomal/lysosomal system. Following the recent introduction of a series of 3-hydroxypyridin-4-one (HPO) based fluorescent probes we present here two novel HPO sensors capable of accumulating and monitoring iron exclusively in endosomal/lysosomal compartments. Flow cytometric and confocal microscopy studies in murine macrophages revealed endosomal/lysosomal sequestration of the probes and high responsiveness toward alterations of vesicular labile iron concentrations. This allowed assessment of cellular iron status with high sensitivity in response to the clinically applied medications desferrioxamine, deferiprone, and deferasirox. The probes represent a powerful class of sensors for quantitative iron detection and clinical real-time monitoring of subcellular labile iron levels in health and disease. . Representative confocal microscopy images of the sensor distribution in BMMØ. Cells were cultured on µ-channel slides and treated with a 75 µM solution of 20 for 20 min. Fluorescence was excited at a 488 nm excitation wavelength using a 505 nm long-pass filter for emission. Fluorescence data are shown in comparison to the corresponding bright field images. Untreated 20-labeled cells were monitored after the initial 20 min incubation period (A). Following exchange of the incubation buffer with a 1 mM DFO solution, cells were monitored after an additional 20 min (B) and 40 min (C) incubation period. Subsequent application of a 500 µg/mL solution of iron dextran (FeDex) was measured after a further 20 min (D).

Research paper thumbnail of IL4 and T Cells Are Required for the Generation of IgG1 Isotype Antibodies Against Cardiolipin

Research paper thumbnail of Faculty of 1000 evaluation for Induction of colonic regulatory T cells by indigenous Clostridium species

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Infection with Helicobacter pylori is associated with protection against tuberculosis

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages

Journal of immunology (Baltimore, Md. : 1950), 1998

Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with... more Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases...

Research paper thumbnail of Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture

PLoS ONE, 2014

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect ... more Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specificpathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the OPEN ACCESS Citation: Yun Y, Srinivas G, Kuenzel S, Linnenbrink M, Alnahas S, et al. (2014) Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture. PLoS ONE 9(12): e113466.

Research paper thumbnail of A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry

Biochemical Journal, 2014

The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult... more The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult and generally time consuming. We have sought a simple and fast method for such a determination. We adopted a fluorescence assay and designed a fluorescent dye with a chelating agent attached to sense iron. To avoid autofluorescence from serum samples, the iron probes were linked to beads and the autofluorescence could be separated and excluded from the measurement by flow cytometry due to the size difference between beads and serum proteins. Fluorescent beads containing both fluorescent and chelating moieties have been synthesized. The nature of the chelating function has been systematically investigated using four different chelators: bidentate hydroxypyranone, bidentate hydroxypyridinone, hexadentate hydroxypyranone and hexadentate hydroxypyridinone, each with different iron affinity constants. Competition studies demonstrate that the hexadentate hydroxypyridinone-based beads are capable of scavenging most of low molecular mass and albumin-bound iron but negligible amounts of iron from transferrin and ferritin. Serum samples from 30 patients with different types of disease and normal volunteers were measured. The concentrations of NTBI fall in the range -0.41 to +6.5 μM. The data have been compared with those obtained from the traditional 'NTA' method.

Research paper thumbnail of Confrontation between Intracellular Bacteria and the Immune System

Advances in Immunology, 1998

AD\'AN (_; ES in IMMUNOLDCV. VOL. 71 Confronfation between Intracell... more AD\'AN (_; ES in IMMUNOLDCV. VOL. 71 Confronfation between Intracellular Bacteria and the Immune System ULRICH E. SCHAIBLE, HELEN L. COUINS, AND STEFAN HE KAUFAitANN Max-Plandc InsHtuie for Infection Biology, D-10117 Berlin, Germany I. ...

Research paper thumbnail of Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens: Proteomics of Mycobacterium tuberculosis

Molecular Microbiology, 2002

ABSTRACT

Research paper thumbnail of Maturation of Rhodococcus equi-Containing Vacuoles is Arrested After Completion of the Early Endosome Stage

Traffic, 2005

Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foal... more Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foals and AIDS patients. Here, we have analyzed R. equi-containing vacuoles (RCVs) in murine macrophages by confocal laser scanning microscopy, by transmission electron microscopy and by immunochemistry upon purification. We show that RCVs progress normally through the early stages of phagosome maturation acquiring PI3P, early endosome antigen-1, and Rab5, and loosing all or much of them within minutes. Although mature RCVs possess the normally late endocytic markers, lysosome-associated membrane proteins, lysobisphosphatidic acid and Rab7, they lack other hallmark features of late endocytic organelles such as possession of cathepsin D, acid beta-glucuronidase, proton-pumping ATPase and the ability to fuse with prelabeled lysosomes. Bacterial strains possessing a virulence-associated plasmid maintain a nonacidified compartment for 48 h, whereas isogenic strains lacking such plasmids acidify progressively. In summary, RCVs represent a novel phagosome maturation stage positioned after completion of the early endosome stage and before reaching a fully mature late endosome compartment. In addition, vacuole biogenesis can be influenced by bacterial plasmids.

Research paper thumbnail of An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria

PROTEOMICS, 2007

Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their... more Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein.

Research paper thumbnail of Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells

Proceedings of the National Academy of Sciences, 2004

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-rest... more A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Research paper thumbnail of Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters

PLoS ONE, 2010

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We con... more Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P mtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.

Research paper thumbnail of Malnutrition and Infection: Complex Mechanisms and Global Impacts

Research paper thumbnail of Why intracellular parasitism need not be a degrading experience for Mycobacterium

Philosophical Transactions of the Royal Society B: Biological Sciences, 1997

... R. Soc. Lond. B David G. Russell, Sheila Sturgill-Koszycki, Tambryn Vanheyningen, Helen Colli... more ... R. Soc. Lond. B David G. Russell, Sheila Sturgill-Koszycki, Tambryn Vanheyningen, Helen Collins and Ulrich E. Schaible Mycobacterium ... experience for Mycobacterium DAVID G. RUSSELL* , SHEILA STURGILL-KOSZYCKI, TAMBRYN VANHEYNINGEN, HELEN COLLINS{ ...

Research paper thumbnail of Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Research paper thumbnail of Saposin C is required for lipid presentation by human CD1b

Nature Immunology, 2004

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in hum... more Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.

Research paper thumbnail of Mycobacterium tuberculosis gene expression profiling within the context of protein networks

Microbes and Infection, 2006

As one of the world&a... more As one of the world's most successful intracellular pathogens, Mycobacterium tuberculosis, the causative agent of human tuberculosis, is responsible for two to three million deaths annually. The pathogenicity of M. tuberculosis relies on its ability to survive and persist within host macrophage cells during infection. It is of central importance, therefore, to identify genes and pathways that are involved in the survival and persistence of M. tuberculosis within these cells. Utilizing genome-wide DNA arrays we have identified M. tuberculosis genes that are specifically induced during macrophage infection. To better understand the cellular context of these differentially expressed genes, we have also combined our array analyses with computational methods of protein network identification. Our combined approach reveals certain signatures of M. tuberculosis residing within macrophage cells, including the induction of genes involved in DNA damage repair, fatty acid degradation, iron metabolism, and cell wall metabolism.

Research paper thumbnail of The IFN-Inducible Golgi- and Endoplasmic Reticulum- Associated 47-kDa GTPase IIGP Is Transiently Expressed During Listeriosis

The Journal of Immunology, 2002

Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mecha... more Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mechanism that confers innate resistance against infection with intracellular pathogens. Overt immunological parameters are apparently uncompromised in mice deficient for individual members and the prototype of this family, IGTP, localizes to the endoplasmic reticulum. This suggests that these GTPases are involved in intracellular defense. We analyzed the expression of the 47-kDa GTPase cognate, IIGP, in splenic sections from mice infected with the intracellular pathogen Listeria monocytogenes by immunohistochemistry. An early transient IIGP induction was observed revealing the IFN-gamma responsiveness of cellular subcompartments within the spleen in early listeriosis. Marginal metallophilic macrophages and endothelial cells within the red and white pulp strongly expressed IIGP, while other splenocytes remained negative. In vitro analyses show that both type I and type II IFNs are prime stimuli for IIGP induction in various cells, including L. monocytogenes-infected or LPS-stimulated macrophages, endothelial cells, and activated T cells. Contrary to the subcellular localization of IGTP, IIGP was predominantly associated with the Golgi apparatus and also localizes to the endoplasmic reticulum. We conclude that IIGP exerts a distinct role in IFN-induced intracellular membrane trafficking or processing.

Research paper thumbnail of Isoniazid@Fe 2 O 3 Nanocontainers and Their Antibacterial Effect on Tuberculosis Mycobacteria

Angewandte Chemie International Edition, 2015

Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter &lt... more Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.

Research paper thumbnail of Targeting the Lysosome: Fluorescent Iron(III) Chelators To Selectively Monitor Endosomal/Lysosomal Labile Iron Pools

Journal of Medicinal Chemistry, 2008

Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy hav... more Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy have led to the identification of a distinct subcellular compartmentation of intracellular redox-active "labile" iron. To investigate the distribution of labile iron, our research has been focused on the development of fluorescent iron sensors targeting the endosomal/lysosomal system. Following the recent introduction of a series of 3-hydroxypyridin-4-one (HPO) based fluorescent probes we present here two novel HPO sensors capable of accumulating and monitoring iron exclusively in endosomal/lysosomal compartments. Flow cytometric and confocal microscopy studies in murine macrophages revealed endosomal/lysosomal sequestration of the probes and high responsiveness toward alterations of vesicular labile iron concentrations. This allowed assessment of cellular iron status with high sensitivity in response to the clinically applied medications desferrioxamine, deferiprone, and deferasirox. The probes represent a powerful class of sensors for quantitative iron detection and clinical real-time monitoring of subcellular labile iron levels in health and disease. . Representative confocal microscopy images of the sensor distribution in BMMØ. Cells were cultured on µ-channel slides and treated with a 75 µM solution of 20 for 20 min. Fluorescence was excited at a 488 nm excitation wavelength using a 505 nm long-pass filter for emission. Fluorescence data are shown in comparison to the corresponding bright field images. Untreated 20-labeled cells were monitored after the initial 20 min incubation period (A). Following exchange of the incubation buffer with a 1 mM DFO solution, cells were monitored after an additional 20 min (B) and 40 min (C) incubation period. Subsequent application of a 500 µg/mL solution of iron dextran (FeDex) was measured after a further 20 min (D).

Research paper thumbnail of IL4 and T Cells Are Required for the Generation of IgG1 Isotype Antibodies Against Cardiolipin

Research paper thumbnail of Faculty of 1000 evaluation for Induction of colonic regulatory T cells by indigenous Clostridium species

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Infection with Helicobacter pylori is associated with protection against tuberculosis

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages

Journal of immunology (Baltimore, Md. : 1950), 1998

Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with... more Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases...

Research paper thumbnail of Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture

PLoS ONE, 2014

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect ... more Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specificpathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the OPEN ACCESS Citation: Yun Y, Srinivas G, Kuenzel S, Linnenbrink M, Alnahas S, et al. (2014) Environmentally Determined Differences in the Murine Lung Microbiota and Their Relation to Alveolar Architecture. PLoS ONE 9(12): e113466.

Research paper thumbnail of A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry

Biochemical Journal, 2014

The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult... more The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult and generally time consuming. We have sought a simple and fast method for such a determination. We adopted a fluorescence assay and designed a fluorescent dye with a chelating agent attached to sense iron. To avoid autofluorescence from serum samples, the iron probes were linked to beads and the autofluorescence could be separated and excluded from the measurement by flow cytometry due to the size difference between beads and serum proteins. Fluorescent beads containing both fluorescent and chelating moieties have been synthesized. The nature of the chelating function has been systematically investigated using four different chelators: bidentate hydroxypyranone, bidentate hydroxypyridinone, hexadentate hydroxypyranone and hexadentate hydroxypyridinone, each with different iron affinity constants. Competition studies demonstrate that the hexadentate hydroxypyridinone-based beads are capable of scavenging most of low molecular mass and albumin-bound iron but negligible amounts of iron from transferrin and ferritin. Serum samples from 30 patients with different types of disease and normal volunteers were measured. The concentrations of NTBI fall in the range -0.41 to +6.5 μM. The data have been compared with those obtained from the traditional 'NTA' method.

Research paper thumbnail of Confrontation between Intracellular Bacteria and the Immune System

Advances in Immunology, 1998

AD\'AN (_; ES in IMMUNOLDCV. VOL. 71 Confronfation between Intracell... more AD\'AN (_; ES in IMMUNOLDCV. VOL. 71 Confronfation between Intracellular Bacteria and the Immune System ULRICH E. SCHAIBLE, HELEN L. COUINS, AND STEFAN HE KAUFAitANN Max-Plandc InsHtuie for Infection Biology, D-10117 Berlin, Germany I. ...

Research paper thumbnail of Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens: Proteomics of Mycobacterium tuberculosis

Molecular Microbiology, 2002

ABSTRACT

Research paper thumbnail of Maturation of Rhodococcus equi-Containing Vacuoles is Arrested After Completion of the Early Endosome Stage

Traffic, 2005

Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foal... more Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foals and AIDS patients. Here, we have analyzed R. equi-containing vacuoles (RCVs) in murine macrophages by confocal laser scanning microscopy, by transmission electron microscopy and by immunochemistry upon purification. We show that RCVs progress normally through the early stages of phagosome maturation acquiring PI3P, early endosome antigen-1, and Rab5, and loosing all or much of them within minutes. Although mature RCVs possess the normally late endocytic markers, lysosome-associated membrane proteins, lysobisphosphatidic acid and Rab7, they lack other hallmark features of late endocytic organelles such as possession of cathepsin D, acid beta-glucuronidase, proton-pumping ATPase and the ability to fuse with prelabeled lysosomes. Bacterial strains possessing a virulence-associated plasmid maintain a nonacidified compartment for 48 h, whereas isogenic strains lacking such plasmids acidify progressively. In summary, RCVs represent a novel phagosome maturation stage positioned after completion of the early endosome stage and before reaching a fully mature late endosome compartment. In addition, vacuole biogenesis can be influenced by bacterial plasmids.

Research paper thumbnail of An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria

PROTEOMICS, 2007

Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their... more Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein.

Research paper thumbnail of Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells

Proceedings of the National Academy of Sciences, 2004

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-rest... more A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Research paper thumbnail of Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters

PLoS ONE, 2010

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We con... more Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P mtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.

Research paper thumbnail of Malnutrition and Infection: Complex Mechanisms and Global Impacts

Research paper thumbnail of Why intracellular parasitism need not be a degrading experience for Mycobacterium

Philosophical Transactions of the Royal Society B: Biological Sciences, 1997

... R. Soc. Lond. B David G. Russell, Sheila Sturgill-Koszycki, Tambryn Vanheyningen, Helen Colli... more ... R. Soc. Lond. B David G. Russell, Sheila Sturgill-Koszycki, Tambryn Vanheyningen, Helen Collins and Ulrich E. Schaible Mycobacterium ... experience for Mycobacterium DAVID G. RUSSELL* , SHEILA STURGILL-KOSZYCKI, TAMBRYN VANHEYNINGEN, HELEN COLLINS{ ...

Research paper thumbnail of Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Research paper thumbnail of Saposin C is required for lipid presentation by human CD1b

Nature Immunology, 2004

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in hum... more Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.

Research paper thumbnail of Mycobacterium tuberculosis gene expression profiling within the context of protein networks

Microbes and Infection, 2006

As one of the world&a... more As one of the world's most successful intracellular pathogens, Mycobacterium tuberculosis, the causative agent of human tuberculosis, is responsible for two to three million deaths annually. The pathogenicity of M. tuberculosis relies on its ability to survive and persist within host macrophage cells during infection. It is of central importance, therefore, to identify genes and pathways that are involved in the survival and persistence of M. tuberculosis within these cells. Utilizing genome-wide DNA arrays we have identified M. tuberculosis genes that are specifically induced during macrophage infection. To better understand the cellular context of these differentially expressed genes, we have also combined our array analyses with computational methods of protein network identification. Our combined approach reveals certain signatures of M. tuberculosis residing within macrophage cells, including the induction of genes involved in DNA damage repair, fatty acid degradation, iron metabolism, and cell wall metabolism.

Research paper thumbnail of The IFN-Inducible Golgi- and Endoplasmic Reticulum- Associated 47-kDa GTPase IIGP Is Transiently Expressed During Listeriosis

The Journal of Immunology, 2002

Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mecha... more Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mechanism that confers innate resistance against infection with intracellular pathogens. Overt immunological parameters are apparently uncompromised in mice deficient for individual members and the prototype of this family, IGTP, localizes to the endoplasmic reticulum. This suggests that these GTPases are involved in intracellular defense. We analyzed the expression of the 47-kDa GTPase cognate, IIGP, in splenic sections from mice infected with the intracellular pathogen Listeria monocytogenes by immunohistochemistry. An early transient IIGP induction was observed revealing the IFN-gamma responsiveness of cellular subcompartments within the spleen in early listeriosis. Marginal metallophilic macrophages and endothelial cells within the red and white pulp strongly expressed IIGP, while other splenocytes remained negative. In vitro analyses show that both type I and type II IFNs are prime stimuli for IIGP induction in various cells, including L. monocytogenes-infected or LPS-stimulated macrophages, endothelial cells, and activated T cells. Contrary to the subcellular localization of IGTP, IIGP was predominantly associated with the Golgi apparatus and also localizes to the endoplasmic reticulum. We conclude that IIGP exerts a distinct role in IFN-induced intracellular membrane trafficking or processing.