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M Frosch

Publications and source records attributed to M Frosch.

At least 127 records · Page 7Linked to original sources
PubMed1995

Use of a monoclonal antibody against the antigen B of Echinococcus granulosus for purification and detection of antigen B.

A monoclonal antibody directed against the 8 kDa subunit of antigen B of Echinococcus granulosus was raised. This antibody was used for purification of antigen B by affinity chromatography. SDS-PAGE and immunological analysis of the purified antigen B demonstrated that the 8 kDa antigen B subunit was purified to homogeneity. The purified antigen retained its strong immunoreactivity in ELISA using human hydatid sera. Furthermore, a sandwich ELISA was established for detection of antigen B from hydatid cysts. The usefulness of this test system was demonstrated by the detection of antigen B in human hydatid cyst fluids, thus confirming the diagnosis of an echinococcal disease.

Animals↗
PubMed1994

Isolation and characterization of the flagellar hook of Campylobacter jejuni.

A method for purification of the flagellar hook of Campylobacter jejuni is described. The hook was shown to be composed of a subunit protein, which has a molecular mass of 92,000 and an isoelectric point of pI 4.8. A monoclonal antibody and a polyvalent antiserum was raised against the purified flagellar hook of C. jejuni. Immuno-electronmicroscopy revealed that the epitope recognized by the monoclonal antibody is surface-located. However, this antibody reacted only with the hook of the immunization strain, but not with other strains or other flagellated bacteria. Thus, our data indicate that the immunodominant epitopes are located on the surface of the hook and that these epitopes are strain-specific.

Antibodies, Monoclonal↗
PubMed1994

Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis.

Within the capsule gene complex (cps) of Neisseria meningitidis B a 5.5 kb DNA fragment encodes proteins with strong homologies to enzymes of the lipopolysaccharide biosynthetic pathway of Salmonella typhimurium and Escherichia coli, GalE, RfbB, RfbC and RfbD. A meningococcal galE mutant expressed a truncated lipooligosaccharide (LOS), which terminated at the glucose residue between inner and outer core, and a second galE gene present outside the cps cluster was found to be transcriptionally and functionally inactive and, thus, unable to complement this defect. Because of the defect in the outer core, the LOS of the galE-defective meningococcal mutant was not sialylated. In contrast, carbohydrate analysis of the LOS of an rfb-defective meningococcal mutant revealed no difference from the LOS of the wild-type strain, suggesting that the rfb genes are inactive. This was supported by Northern blot analysis, which showed that expression of the rfb gene products was transcriptionally regulated. The inability of the meningococcal galE mutant, which cannot sialylate the LOS, allowed us to investigate the significance of LOS sialylation in relation to the presence of the polysialic acid capsule. Sialylated LOS, but not the polysialic acid capsule, is necessary to confer complete serum resistance on the meningococcus by inhibition of the alternative complement pathway.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of the biosynthesis pathway of the alpha-2,8 polysialic acid capsule by Neisseria meningitidis serogroup B.

The genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9 kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the sigma 70 consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that ORF2 encodes the CMP-NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the alpha-2,8 polysialyltransferase. ORF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2-4 were able to synthesize a high-molecular-weight alpha-2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP-NeuNAc hydrolase, which cleaves CMP-NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster.

Amino Acid Sequence↗
PubMed1994

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis.

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.

Antigens, Bacterial↗
PubMed1993

Paramyosin of Echinococcus granulosus: cDNA sequence and characterization of a tegumental antigen.

A lambda ZAPII cDNA library of Echinococcus granulosus larvae was expressed in Escherichia coli SURE cells. Screening of the library with a rabbit antiserum raised against total larval antigen yielded several immunoreactive clones. For analysis of the nucleotide sequence, in vivo excision into pBlueskript was carried out and the 3' end of the cloned insert was sequenced. Three of these clones exhibited identical nucleotide sequences, suggesting expression of identical genes. The complete nucleotide sequence of the largest clone, EG36, with a 3.4-kb insert was determined, presenting an open reading frame of 2.59 kb. The predicted amino acid sequence showed 71.4% identity to the Schistosoma mansoni paramyosin and a significant homology to a 17 amino-acid peptide sequence from antigen B of Taenia solium. From these data we conclude that EG36 is the paramyosin of E. granulosus. For protein purification, the coding sequence of the cDNA was amplified by polymerase chain reaction and ligated in frame into the expression vector pGEX-3X. Affinity-chromatography-purified GST fusion protein was used to induce a polyclonal rabbit antiserum. Immunoblot analysis revealed the expression of a 97-kDa protein by the E. coli clone and that of a protein with a similar molecular weight in protoscolices from E. granulosus and E. multilocularis as well as in E. granulosus cyst fluid. Immunofluorescence studies showed that EG36 was localized throughout the tegument of E. granulosus and E. multilocularis larvae. Sera from patients suffering from echinococcosis, schistosomiasis, and neurocysticercosis reacted with the purified fusion protein when tested in an enzyme-linked immunosorbent assay.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of an echinococcal microtrichal antigen immunoreactive in Echinococcus multilocularis disease.

A cDNA expression library of the larval stage of the cestode worm Echinococcus multilocularis has been established in the phage lambda ZAPII system. By immunoscreening with pooled sera from patients with alveolar echinococcosis an immunoreactive clone, termed pEM13, was isolated. EM13 was expressed using the expression vector pGEX-3X, resulting in the synthesis of a glutathione S-transferase fusion protein. 82% of the sera from 28 patients suffering from E. multilocularis disease had antibodies against EM13, whereas none of the 55 sera obtained from Echinococcus granulosus-infected patients and none of the 15 sera from patients with other helminthic infections reacted with recombinant EM13. By use of a polyclonal rabbit anti-EM13 hyperimmune serum native EM13 protein could be detected only in the protoscolices of E. multilocularis, but not in E. granulosus larvae or hydatid fluid. Immunoelectron microscopy suggested that EM13 is located in the microtriches on the surface of the larvae. In contrast, EM13 mRNA could be detected by Northern blot analysis in both E. multilocularis and E. granulosus larval RNA preparations. Nucleotide and amino acid sequence analysis of a cDNA clone coding for the corresponding antigen of E. granulosus larvae, termed EG13, revealed a 21-bp insertion, a 51-bp deletion and additional 22 nucleotide exchanges resulting in a 96.3% identity at the nucleotide sequence level and a 96.6% identity at the amino acid sequence level to the coding region of the cDNA pEM13. Cross-reactivity of the polyclonal anti-EM13 serum with the recombinant EG13 indicates a posttranscriptional regulation mechanism, resulting in an EG13 negative phenotype in E. granulosus.

Amino Acid Sequence↗
PubMed1993

Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis.

Within the capsule gene complex (cps) of Neisseria meningitidis two functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of alpha-2,8 polyneuraminic acid. The region-C gene products share characteristics with transporter proteins of the ABC (ATP-binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B- and region C-defective Escherichia coli clones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full-size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the polysaccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1 and 48.7 kDa.

Amino Acid Sequence↗
PubMed1993

The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide.

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.

Amino Acid Sequence↗
PubMed1993

Serological differentiation between cystic and alveolar echinococcosis by use of recombinant larval antigens.

Two recombinant antigens of the larval stages of Echinococcus granulosus and Echinococcus multilocularis, termed EG55 and EM10, respectively, were applied for serodiagnosis and serological differentiation between parasitic infections caused by the metacestode tissue of both tapeworms. Antigen EM10 is synthesized by E. multilocularis larvae. Antigen EG55 represents the recombinant form of the low-molecular-weight subunit of antigen B, which is an Echinococcus genus-specific antigen. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins. A sandwich enzyme-linked immunosorbent assay with monoclonal antibodies against EM10 and EG55 as capture reagents for the recombinant antigens was established and was evaluated with 74 serum samples from patients with histologically confirmed alveolar echinococcosis and 63 serum samples from patients with histologically confirmed cystic echinococcosis. A sensitivity of 93.2% and a specificity of 96.8% were achieved for the serodiagnosis of alveolar echinococcosis. Cystic echinococcosis could be detected with a sensitivity of 89.1% and a specificity of 98.6%.

Animals↗
PubMed1993

Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates.

In order to improve the diagnosis of a Mycoplasma pneumoniae infection, we developed a polymerase chain reaction (PCR)-based assay. The gene encoding elongation factor Tu (tuf) was selected as the target sequence. Oligonucleotides derived from variable stretches of the tuf gene were able to prime the amplification of a 950-bp fragment exclusively when M. pneumoniae DNA was used as the template. The sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal M. pneumoniae-specific oligonucleotide. The use of three to four genome copies for PCR was sufficient for obtaining a hybridization signal. In addition, we substituted radioactive filter hybridization with a microtiter plate assay. Via a biotin moiety of one PCR primer, the amplification products were immobilized on streptavidin-coated microtiter plates. Subsequent hybridization with a digoxigenin-labeled oligonucleotide resulted in the same sensitivity and specificity as those obtained by filter hybridization. Clinical application of the assay was performed on 102 throat swab specimens from patients with respiratory tract infections. Of 21 culture-positive samples, 19 were confirmed to be positive in the PCR-based assay (sensitivity, 90%). Furthermore, 14 of 19 seropositive but culture-negative samples gave a positive hybridization signal. Of 62 culture-negative and seronegative specimens, 60 gave a negative result in our assay (specificity, 97%). Of the 33 samples that were positive in our PCR-based assay, 5 samples initially gave false-negative results because of the presence of inhibitory substances in those specimens. Inhibition of Taq polymerase in these five cases was prevented by an additional step of phenol extraction and subsequent ethanol precipitation.

Bacteriological Techniques↗
PubMed1993

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha (2-8) polysialic acid. 2. Amino acid sequence of the heavy (H-) chain Fd' region.

The complete amino acid sequence of the Fd' region including the VH part, the CH1 domain, and the hinge segment of the biologically relevant monoclonal mouse anti-alpha (2-8) polysialic acid antibody mAb735 is presented. The reduced and carboxymethylated H-chain was digested with trypsin and cyanogen bromide. For subfragmentation selected peptides were cleaved with thermolysin and endoproteinase Asp-N. The generated peptides were isolated by RP-HPLC and characterized by sequence analysis, plasma desorption mass spectrometry (PDMS), and amino acid analysis. The N-terminal sequence was determined after enzymatic deprotection with pyroglutamate aminopeptidase. According to Kabat et al. the variable region of the H-chain belongs to the subgroup II. Sequence data from the constant region indicate that mAb735 represents the gamma 2a isotype.

Amino Acid Sequence↗
PubMed1992

Transformation-mediated exchange of virulence determinants by co-cultivation of pathogenic Neisseriae.

The horizontal flow of genetic material between microbes utilizes three principal routes: conjugation, transduction and transformation. While the significance in nature of the first two pathways is generally accepted, the in vivo role of transformation remains uncertain, despite the early observations by Griffith in 1928 on the transformation of streptococci from an avirulent to a virulent state [1]. Recently, circumstantial evidence was collected suggesting a role for transformation-mediated horizontal exchange in the modulation of virulence determinants of pathogenic Neisseriae and the variation of surface structures. In order to further assess the significance of transformation-mediated exchange we performed simple co-cultivation experiments of different Neisseria strains. We observed an efficient intra- and interspecies transfer of essential virulence determinants; the process was sensitive to the presence of DNaseI in the culture and was blocked in transformation-deficient recipients.

Genes, Bacterial↗
PubMed1992

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase.

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.

Amino Acid Sequence↗