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S Birkelund

Publications and source records attributed to S Birkelund.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity.

BACKGROUND: The association of Chlamydia pneumoniae with the development of atherosclerosis is based on serology and on detection of C pneumoniae-specific DNA by polymerase chain reaction in the atheromas. METHODS AND RESULTS: Because the humoral immune response frequently recognizes epitopes present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used these antibodies in immunofluorescence microscopy of experimentally infected mice. In lung sections, a massive infiltration with polymorph nuclear neutrophil cells was observed. In the bronchial epithelial cells, C pneumoniae inclusions were seen. Evidence was found of differential expression of the GGAI proteins. CONCLUSIONS: On the basis of surface localization, differential expression, and the fact that the proteins are recognized by the human humoral immune response, we speculate whether these proteins, in addition to the lipopolysaccharides, are of importance for the immunopathogenesis of C pneumoniae.

Animals↗
PubMed1999

Antibodies to Chlamydia trachomatis heat shock proteins in women with tubal factor infertility are associated with prior infection by C. trachomatis but not by C. pneumoniae.

The antibody response to heat shock proteins 60 and 10 were studied in 163 patients with tubal factor infertility and in 163 age-matched pregnant women. The associations of these antibodies with specific antibodies to Chlamydia trachomatis and to Chlamydia pneumoniae as well as with antibodies to the common chlamydial lipopolysaccharide antigen were studied. Patients with tubal factor infertility had significantly higher frequencies and titres of all antibodies except to C. pneumoniae. In a logistic regression model an association was found between the prevalence of antibodies to the heat shock proteins and to C. trachomatis but no independent influence of antibodies to C. pneumoniae. No interaction between C. trachomatis and C. pneumoniae suggesting a synergistic effect was found although the heat shock proteins from these two organisms are immunologically similar. Antibodies to the chlamydial lipopolysaccharide also seemed to be related to C. trachomatis and not to C. pneumoniae in these women.

Adult↗
PubMed1999

Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy showed that epitopes of Omp4 were exposed on the surface of C. pneumoniae elementary bodies, reticulate bodies, and outer membrane complex. Proteins encoded by the C. pneumoniae gene family seem to be dominant antigens in experimentally infected mice.

Amino Acid Sequence↗
PubMed1998

The Mycoplasma hominis vaa gene displays a mosaic gene structure.

Mycoplasma hominis contains a variable adherence-associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolated by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N-terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequences boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.

Adhesins, Bacterial↗
PubMed1998

Mycoplasma contamination of Chlamydia pneumoniae isolates.

We examined 6 C. pneumonia isolates from The American Type Culture Collection (ATCC) and 2 Finnish isolates for Mycoplasma contamination. Three of the ATCC isolates and both of the Finnish isolates were Mycoplasma-contaminated. The contaminants were characterized by means of growth in BEa and BEg media, immunoblotting, polymerase chain reaction and pulsed field gel electrophoresis. Two of the 6 ATCC isolates [ATCC VR1355 (TWAR strain 2043) and ATCC VR1356 (TWAR strain 2023)] were infected with Mycoplasma hominis and 1 isolate [ATCC VR2282 (TWAR strain TW183)] was contaminated with both Mycoplasma hominis and Mycoplasma orale, whereas 3 of the ATCC isolates [ATCC VR1310, ATCC VR1360 (TWAR strain CM-1) and ATCC 53592 (TWAR strain AR39)] were not contaminated. The Finnish C. pneumoniae isolates Kajaani 6 and Parola were found to be contaminated with M. hominis and M. orale, respectively. The contamination of C. pneumoniae stock cultures, frequently used in the microimmunofluorescence test, with human pathogens, could pose a serious problem in C. pneumoniae serology.

Bacteriological Techniques↗
PubMed1998

DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates.

To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified by PCR and the PCR products were sequenced. Three isolates had identical 16S rRNA sequences and two isolates had sequences that differed from the others by only one nucleotide.

Base Sequence↗
PubMed1998

Antigenic and genomic homogeneity of successive Mycoplasma hominis isolates.

Sixty Mycoplasma hominis isolates were obtained from the cervices of pregnant women and from the ears or pharynges of their newborn babies. The isolates were examined by SDS-PAGE and pulsed-field gel electrophoresis. Antigenic and genomic profiles were obtained for 16 series with two or more successive isolates. Both analyses led to the conclusion that isolates from the same woman were identical or nearly identical, while isolates from different women exhibited a high degree of variation with respect to both genomic and antigenic profiles.

Antigenic Variation↗
PubMed1998

Analysis of the humoral immune response to Chlamydia outer membrane protein 2.

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P < 0.0001). The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.

Antibodies, Bacterial↗
PubMed1998

Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2.

Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol, and protease digestions indicate that Omp2 has a possible two-domain structure.

Animals↗
PubMed1997

Molecular biology of Mycoplasma.

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.

Animals↗
PubMed1997

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Animals↗
PubMed1997

Molecular biology of the Chlamydia pneumoniae surface.

Chlamydia pneumoniaeis a fastidious microorganism with a characteristic biphasic lifecycle causing a variety of human respiratory tract infections. There is limited knowledge about the molecular biology of C. pneumoniae, and only a few genes have been sequenced. The structure of the chlamydial surface differs from that of Chlamydia trachomatis. In order to study the surface of C. pneumoniae we generated monoclonal antibodies (MAbs) against C. pneumoniae strain VR-1310 and selected 14 MAbs that reacted with the surface of C. pneumoniae. All MAbs reacted in immunoelectron microscopy with the surface of both whole C. pneumoniae VR-1310 elementary bodies and with purified sarcosyl extracted outer membrane complexes. However, only 2 of the MAbs reacted in immunoblotting with C. pneumoniae proteins and only with antigen that had not been heat treated in SDS-sample buffer. This indicates the dominance of conformational epitopes at the C. pneumoniae surface.

Antibodies, Monoclonal↗
PubMed1996

Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting.

Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein (OMP2), the DnaK-like, GroEL-like, and macrophage infectivity potentiator (MIP)-like proteins, the plasmid-encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein-elongation factor EF-Tu. Other proteins, for which gene assignment was not possible, have been identified by three parameters (Mr, pI and N-terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome-linked database of chlamydial proteins.

Acrylamides↗
PubMed1996

Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1.

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro. Our results imply that DNA condensation is not the only function of Hc2.

Antibodies, Bacterial↗
PubMed1996

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.

Adenosine Triphosphatases↗
PubMed1996

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped.

Amino Acid Sequence↗
PubMed1996

The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.

Amino Acid Sequence↗