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Biomedical subjects

Thomas F Meyer

Publications and source records attributed to Thomas F Meyer.

At least 19 recordsLinked to original sources
PubMed2007

The orphan response regulator HP1021 of Helicobacter pylori regulates transcription of a gene cluster presumably involved in acetone metabolism.

Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with the tight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021-regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region.

Acetone↗
PubMed2007

The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin.

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.

Antigens, Bacterial↗
PubMed2006

Striptease on glass: validation of an improved stripping procedure for in situ microarrays.

Microarrays have rapidly become an indispensable tool for gene analysis. Microarray experiments can be cost prohibitive, however, largely due to the price of the arrays themselves. Whilst different methods for stripping filter arrays on membranes have been established, only very few protocols are published for thermal and chemical stripping of microarrays on glass. Most of these protocols for stripping microarrays on glass were developed in combination with specific surface chemistry and different coatings for covalently immobilizing presynthesized DNA in a deposition process. We have developed a method for stripping commercial in situ microarrays using a multi-step procedure. We present a method that uses mild chemical degradation complemented by enzymatic treatment. We took advantage of the differences in biochemical properties of covalently linked DNA oligonucleotides on in situ synthesized microarrays and the antisense cRNA hybridization probes. The success of stripping protocols for microarrays on glass was critically dependent on the type of arrays, the nature of sample used for hybridization, as well as hybridization and washing conditions. The protocol employs alkali hydrolysis of the cRNA, several enzymatic degradation steps using RNAses and Proteinase K, combined with appropriate washing steps. Stripped arrays were rehybridized using the same protocols as for new microarrays. The stripping method was validated with microarrays from different suppliers and rehybridization of stripped in situ arrays yielded comparable results to hybridizations done on unused, new arrays with no significant loss in precision or accuracy. We show that stripping of commercial in situ arrays is feasible and that reuse of stripped arrays gave similar results compared to unused ones. This was true even for biological samples that show only slight differences in their expression profiles. Our analyses indicate that the stripping procedure does not significantly influence data quality derived from post-primary hybridizations. The method is robust, easy to perform, inexpensive, and results after reuse are of comparable accuracy to new arrays.

Cost Control↗
PubMed2006

Cholesterol glucosylation promotes immune evasion by Helicobacter pylori.

Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell-dependent reduction of the H. pylori burden in the stomach. Intrinsic alpha-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-alpha-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host-pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.

Animals↗
PubMed2006

Cloning of a cholesterol-alpha-glucosyltransferase from Helicobacter pylori.

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

Amino Acid Sequence↗
PubMed2006

Robust Salmonella metabolism limits possibilities for new antimicrobials.

New antibiotics are urgently needed to control infectious diseases. Metabolic enzymes could represent attractive targets for such antibiotics, but in vivo target validation is largely lacking. Here we have obtained in vivo information about over 700 Salmonella enterica enzymes from network analysis of mutant phenotypes, genome comparisons and Salmonella proteomes from infected mice. Over 400 of these enzymes are non-essential for Salmonella virulence, reflecting extensive metabolic redundancies and access to surprisingly diverse host nutrients. The essential enzymes identified were almost exclusively associated with a small subgroup of pathways, enabling us to perform a nearly exhaustive screen. Sixty-four enzymes identified as essential in Salmonella are conserved in other important human pathogens, but almost all belong to metabolic pathways that are inhibited by current antibiotics or that have previously been considered for antimicrobial development. Our comprehensive in vivo analysis thus suggests a shortage of new metabolic targets for broad-spectrum antibiotics, and draws attention to some previously known but unexploited targets.

Animals↗
PubMed2006

Type IV secretion systems and their effectors in bacterial pathogenesis.

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.

Animals↗
PubMed2006

Naturally occurring amino acids differentially influence the development of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae.

The differential influence of individual amino acids on the growth of Chlamydia trachomatis versus Chlamydia (Chlamydophila) pneumoniae was investigated. Certain essential amino acids added in excess at the middle of the infection course resulted in varying degrees of abnormality in the development of the two species. If amino acids were added as early as 2 h post-infection, these effects were even more pronounced. The most effective amino acids in terms of C. trachomatis growth inhibition were leucine, isoleucine, methionine and phenylalanine. These amino acids elicited similar effects against C. pneumoniae, except methionine, which, surprisingly, showed a lower inhibitory activity. Tryptophan and valine marginally inhibited C. trachomatis growth and, paradoxically, led to a considerable enhancement of C. pneumoniae growth. On the other hand, some non-essential amino acids administered at the middle of or throughout the infection course differentially affected the development of the two species. For example, C. trachomatis growth was efficiently inhibited by glycine and serine, whereas C. pneumoniae was relatively less sensitive to these agents. Another difference was apparent for glutamate, glutamine and aspartate, which stimulated C. pneumoniae growth more than that of C. trachomatis. Overall, several distinctive patterns of susceptibility to excess amino acid levels were revealed for two representative C. trachomatis and C. pneumoniae isolates. Perturbation of amino acid levels, e.g. of leucine and isoleucine, might form a basis for the development of novel treatment or preventive regimens for chlamydial diseases.

Amino Acids↗
PubMed2006

Analysis of aerotactic band formation by Desulfovibrio desulfuricans in a stopped-flow diffusion chamber.

Aerotactic band formation by Desulfovibrio desulfuricans (DSM 9104) was studied in a stopped-flow diffusion chamber. This chamber allowed us to create reproducible, steep oxygen gradients in a flat capillary, time-lapse video recordings and spatio-temporal analysis of band formation. The cells formed two types of bands. Bands of the first type evolved quickly after starting the experiment and were located near the oxic-anoxic interface. Bands of the second type typically appeared several minutes later and a few millimeters inside the initially anoxic volume of the capillary. Band formation depended on metabolism and could be stimulated by lactate addition, and thus appears to be energy taxis. Mathematical modeling of oxygen diffusion and respiration within the chamber revealed that bands formed preferentially at oxygen concentrations close to 4% air saturation. The swimming speed of the cells was determined by digital single-cell tracking and found to be highest (up to 58 mum s(-1)) close to the oxic-anoxic interfaces. Motility patterns were influenced by surfaces, at which cells accumulated. Bioconvection sometimes occurred if very dense bands had formed. The ecological implications of these two phenomena are unknown.

Aerobiosis↗
PubMed2006

Low-phosphate-dependent invasion resembles a general way for Neisseria gonorrhoeae to enter host cells.

Obligate human-pathogenic Neisseria gonorrhoeae expresses numerous variant surface proteins mediating adherence to and invasion of target cells. The invariant major outer membrane porin PorB of serotype A (P.IA) gonococci triggers invasion into Chang cells only if the medium is devoid of phosphate. Since gonococci expressing PorB(IA) are frequently isolated from patients with severe disseminating infections, the interaction initiated by the porin may be of major relevance for the development of this serious disease. Here, we investigated the low-phosphate-dependent invasion and compared it to the well-known pathways of entry initiated by Opa proteins. P.IA-triggered invasion requires clathrin-coated pit formation and the action of actin and Rho GTPases. However, in contrast to Opa-initiated invasion via heparan sulfate proteoglycans, microtubules, acidic sphingomyelinase, phosphatidylinositol 3-kinase, and myosin light chain kinase are not involved in this entry pathway. Nor are Src kinases required, as they are in invasion, e.g., via the CEACAM3 receptor. Invasion by PorB(IA) occurs in a wide spectrum of cell types, such as primary human epithelial and endothelial cells and in cancer cells of human and animal origin. Low-phosphate-dependent invasion is thus a pathway of gonococcal entry distinct from Opa-mediated invasion.

Animals↗
PubMed2006

Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation.

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR approximately P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR approximately P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR approximately P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene.

Adaptation, Physiological↗
PubMed2005

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.

Amino Acid Sequence↗
PubMed2005

Gene expression and protein profiling of AGS gastric epithelial cells upon infection with Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens, colonizes the human stomach and causes a variety of gastric diseases. This pathogen elicits a range of phenotypic responses in infected cultured AGS gastric epithelial cells, including expression of proinflammatory genes and changes in the actin cytoskeleton. Some of these responses are mediated by the type IV secretion system (T4SS) encoded by the cag pathogenicity island. We have used two global approaches, namely 2-DE combined with PMF and cDNA expression array analyses, to study in both a comprehensive and quantitative manner the protein profile and the temporal patterns of mRNA accumulation in AGS cells upon infection with H. pylori and isogenic T4SS mutants. We identified 140 transcripts and detected 190 protein species that were differentially regulated upon infection. Infection with wild-type H. pylori induced expression of a variety of host genes and changes in protein pattern involved in transcriptional responses, cell shape regulation and signal transduction. Among them, some were differentially regulated in a cag PAI-dependent manner, as shown by both the proteomic and cDNA expression array approaches. While 2-DE and PMF allowed us to examine the protein profiles in the infected host, array analysis enabled us to demonstrate dynamic temporal changes in host gene expression profile. In conclusion, our combined application of the two global approaches provides further molecular details on how the host cell responds to infection by H. pylori and its isogenic T4SS mutants on both transcriptional and protein levels. The findings pinpoint host proteins such as serine/threonine and tyrosine kinases, transcription factors, cell cycle related components and actin cytoskeletal signaling molecules as potential targets of individual H. pylori virulence determinants. This study serves as a basis for future work on transcription and proteome analyses of the H. pylori infection model.

Adenocarcinoma↗
PubMed2005

Helicobacter pylori vaccine development: facing the challenge.

An effective vaccine would be a desirable way to control Helicobacter pylori-induced gastric disease. Initial studies in animal models demonstrated the feasibility of immunization and led to high hopes for a human vaccine. In the mouse model immunological approaches have to date not brought a satisfactory explanation for the mechanisms of protection against this largely luminal pathogen. Recently, transcriptome studies have identified new factors. It is now proposed that non-classical immune mediators may be the key to vaccine-induced protection. Human trials of H. pylori vaccines are going ahead but although at least some formulations are clearly immunogenic, their effectiveness remains untested. The recent development of a human challenge model has now opened up new prospects for testing candidate vaccines and this will undoubtedly have a great impact in the near future. Future priorities for H. pylori vaccine development must be a better understanding of the protective mechanisms and the identification of biomarkers which can be used as reliable predictors of efficacy in humans. Despite some important advances in recent years, important issues must be resolved before an H. pylori vaccine will become a reality.

Adjuvants, Immunologic↗
PubMed2005

Inhibition of Marburg virus protein expression and viral release by RNA interference.

High mortality rates and lack of an available vaccine against Marburg haemorrhagic fever (MHF) highlight the need for a defensive therapy against MHF and greater knowledge of the causative agent, the Marburg virus (MARV). Here, RNA interference (RNAi) is employed to destroy MARV transcripts, disrupting replication and allowing analysis of various roles of MARV proteins. Small interfering RNAs (siRNAs) homologous to three MARV transcripts (NP, VP35 and VP30) were co-transfected into cells with plasmids encoding the corresponding nucleocapsid proteins. The resulting decrease in MARV nucleocapsid-protein levels was shown to be specific, as siRNA that was not homologous to the MARV genome did not decrease the levels of viral nucleocapsid proteins. Additionally, transcript levels of double-stranded RNA (dsRNA)-sensor proteins, the dsRNA-activated protein kinase and 2',5'-oligoadenylate synthetase 1 remained unchanged, suggesting that the decrease in viral proteins was not a result of activation of the antiviral properties of the interferon system. Subsequently, siRNAs were shown to reduce intracellular viral proteins in MARV-infected cells and viral material released into the medium. Targeted reduction of VP30 downregulated the intracellular levels of all other viral proteins, suggesting that VP30 plays an essential role for transcription/replication. The efficient reduction of MARV replication also suggests that RNAi may provide an agent against MHF.

Animals↗
PubMed2005

The PilC adhesin of the Neisseria type IV pilus-binding specificities and new insights into the nature of the host cell receptor.

Type IV pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate the first contact to human mucosal epithelial cells, an interaction which is also critical for the interaction with vascular endothelial cells. The PilC proteins have been characterized as the principal pilus-associated adhesin. Here we show that PilC2 exhibits a defined cell and tissue tropism, as it binds to human epithelial and endothelial cell lines, but not to human T cells or fibroblasts. Piliated gonococci and PilC2 exhibit similar patterns of binding to human epithelial and endothelial cells, supporting the function of PilC as the key pilus adhesin. Although CD46 has previously been suggested to be a pilus receptor, several observations indicate that neisserial type IV pili and the pilus adhesin PilC2 interact with epithelial cells in a CD46 independent manner. Biochemical approaches were used to characterize the nature of host cell factors mediating binding of piliated gonococci and PilC2 protein. Our data indicate that the putative host cell receptor for gonococcal pili and the PilC2 pilus adhesin is a surface protein. Glycostructures were found to not be involved in binding. Moreover, we observed the uptake of purified PilC2 protein together with its receptor via receptor-mediated endocytosis and subsequent receptor re-exposure on the cell surface. Our data support the existence of a specific pilus receptor and provide intriguing information on the nature of the receptor.

Adhesins, Bacterial↗
PubMed2005

Detection of Chlamydophila pneumoniae in the bone marrow of two patients with unexplained chronic anaemia.

Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.

Adult↗