Armin Bosserhoff

Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies.

ABSTRACT Colonization of the gastric mucosa by Helicobacter pylori is the major cause of gastroduodenal pathologies in humans. Studying the outcome of the humoral immune response directed against this gastric pathogen may contribute substantially to vaccine development and to the improvement of diagnostic techniques based on serology. By using two-dimensional gel electrophoresis, 29 proteins from H. pylori G27 were identified which strongly react with sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. These antigens were characterized by mass spectrometry and proved to correspond to products of open reading frames predicted by the H. pylorigenome sequence. The comparison of the antigenic patterns recognized by these sera revealed no association of specific H. pyloriantigens with antibodies in patients with particular gastroduodenal pathologies.

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung Homology between the two subunits of the enzyme

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77 500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Gα4 and Gβ, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase

Although the annotation of the complete genome sequence of Mycoplasma pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we showed experimentally that such an activity must exist in this bacterium, by determining the N‐terminus of the N‐terminal gene product P40 of MPN142, formerly called ORF6 gene. Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino acids 25 and 26 of the P40 precursor protein. The experimental results were in agreement with the cleavage site predicted by computational methods providing experimental confirmation for these theoretical analyses.

A Point Mutation in the Sensor Histidine Kinase SaeS of Staphylococcus aureus Strain Newman Alters the Response to Biocide Exposure

Staphylococcus aureus reacts to changing environmental conditions such as heat, pH, and chemicals through global regulators such as the sae (S. aureus exoprotein expression) two-component signaling system. Subinhibitory concentrations of some antibiotics were shown to increase virulence factor expression. Here, we investigated the S. aureus stress response to sublethal concentrations of a commonly used biocide (Perform), by real-time quantitative PCR (qRT-PCR), promoter activity assay, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a flow cytometric invasion assay. Perform, acting through the production of reactive oxygen species, generally downregulated expression of extracellular proteins in strains 6850, COL, ISP479C but upregulated these proteins in strain Newman. Upregulated proteins were sae dependent. The Perform component SDS, but not paraquat (another oxygen donor), mimicked the biocide effect. Eap (extracellular adherence protein) was most prominently augmented. Upregulation of eap and sae was confirmed by qRT-PCR. Promoter activity of sae P1 was increased by Perform and SDS. Both substances enhanced cellular invasiveness, by 2.5-fold and 3.2-fold, respectively. Increased invasiveness was dependent on Eap and the sae system, whereas agr, sarA, sigB, and fibronectin-binding proteins had no major effect in strain Newman. This unique response pattern was due to a point mutation in SaeS (the sensor histidine kinase), as demonstrated by allele swapping. Newman saePQRS(ISP479C) behaved like ISP479C, whereas saePQRS(Newman) rendered ISP479C equally responsive as Newman. Taken together, the findings indicate that a point mutation in SaeS of strain Newman was responsible for increased expression of Eap upon exposure to sublethal Perform and SDS concentrations, leading to increased Eap-dependent cellular invasiveness. This may be important for understanding the regulation of virulence in S. aureus.

Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins

A systematic investigation of the influence of the detergent sodium dodecyl sulphate (SDS) on micropreparative peptide separations on microbore reversed-phase high-performance liquid chromatographic columns is reported. A tryptic digest of bovine serum albumin and a mixture of synthetic peptides were used to monitor the separation behaviour of a 1.6 mm I.D. Nucleosil C18 column in the presence of various amounts of SDS. The data demonstrate that even traces of SDS in the sample reduce the separation efficiency and peptide recovery. An extraction method is presented which reduces the SDS content in peptide mixtures below the critical concentration without affecting significantly the recovery of individual peptides. After acidification of the sample, the detergent is extracted into heptane-isoamyl alcohol (4:1, v/v). In combination with chemical or enzymatic fragmentation techniques, this extraction method facilitates the sequence analysis of minute amounts of SDS-solubilized hydrophobic proteins. The applicability of the method is demonstrated on the example of the integral membrane protein bacteriorhodopsin.

High-Speed Preparative Reversed-Phase High-Performance Liquid-Chromatography of Synthetic Oligonucleotides

A rapid method is described for the purification and analysis of synthetic oligonucleotides, based on reversed-phase high-performance liquid chromatography. Volatile buffers and a short column (40 mm X 4.6 mm) packed with Nucleosil 300-5 C4 were employed. Monitoring the column effluent with an UV detector provides an excellent means of controlling product quality. The method is suitable for the purification of crude synthesis products, as well as for desalting and removing gel contaminants from oligonucleotides eluted from polyacrylamide gels. The total time required per sample is less than 25 min.

The Influence of SDS on Micropreparative Peptide Separations on Reversed Phase Columns

Sequence analysis of proteins and peptides can routinely be performed at low picomole levels. However, the micropreparative isolation of peptides with suitable purity for micro sequence analysis appears to be more problematic. The limited solubility of hydrophobic proteins, e.g. integral membrane proteins and membrane associated proteins requires the presence of ionic detergents like SDS during the purification. Detergents are also required to solubilize Coomassie stained proteins before electroelution from the gel (Hunkapiller et al. 1983). The preparations of these proteins therefore contain various amounts of SDS which interferes (above 0.1%) with subsequent enzymatic fragmentation and peptide separation. The separation capacity of high performance liquid chromatograpy (HPLC) reversed phase columns is remarkably reduced or even destroyed in the presence of a few micrograms of SDS.