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J G Kusters

Publications and source records attributed to J G Kusters.

At least 73 records · Page 4Linked to original sources
PubMed1998

Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes.

Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Antibodies↗
PubMed1998

At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice.

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.

Animals↗
PubMed1998

Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori.

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.

Conjugation, Genetic↗
PubMed1998

Helicobacter pylori ribBA-mediated riboflavin production is involved in iron acquisition.

In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of Helicobacter pylori NCTC 11638 which confers to a siderophore synthesis mutant of Escherichia coli (EB53 aroB hemA) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the ribA gene of Bacillus subtilis. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated ribBA) resulted in the formation of one translational product, which was able to complement both the ribA and the ribB mutation in E. coli. Expression of ribBA was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in H. pylori cells is iron regulated. E. coli EB53 containing the plasmid with H. pylori ribBA excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by E. coli EB53 transformed with the H. pylori ribBA gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of ribBA in H. pylori eliminates its ferric-iron-reduction activity, we conclude that ribBA has an important role in ferric-iron reduction and iron acquisition by H. pylori.

Aldehyde Oxidoreductases↗
PubMed1997

Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death.

Helicobacter pylori can transform from its normal helical bacillary morphology to a coccoid morphology. Since this coccoid form cannot be cultured in vitro, it has been speculated that it is a dormant form potentially involved in the transmission of H. pylori and in a patient's relapse after antibiotic therapy. In this study we determined the effects of aging, temperature, aerobiosis, starvation, and antibiotics on the morphologic conversion rate and culturability of H. pylori. Aerobiosis and the addition of a bactericidal antibiotic to the culture medium resulted in the highest conversion rate. During the conversion to coccoid forms, the cultures always lost culturability at the stage where 50% of the organisms were still in bacillary form; this result indicated that culturability and coccoid morphology are two separate but related entities. Independent of the conditions used to induce the conversion into coccoids, the morphological conversion was accompanied by several marked antigenic and ultrastructural changes. Also, both the total amounts and the integrity of RNA and DNA were significantly reduced in coccoid forms. With the potential-sensitive probe diOC(5)-3, a clear loss of membrane potential in coccoid forms was observed. Inhibition of protein or RNA synthesis by the addition of bacteriostatic antibiotics did not prevent the conversion to coccoid forms but resulted in an increased conversion rate. Hence, we conclude that conversion of H. pylori from the bacillary to the coccoid form is a passive process that does not require protein synthesis. Our data suggest that the coccoid form of H. pylori is the morphologic manifestation of bacterial cell death.

Anaerobiosis↗
PubMed1996

Human serum antibody response against iron-repressible outer membrane proteins of Helicobacter pylori.

In Helicobacter pylori, in vitro iron limitation induces the expression of several iron repressible outer membrane proteins (IROMPs), which are not expressed under normal growth conditions. To substantiate their proposed role in virulence of H. pylori, we determined whether these IROMPs are also expressed in vivo. Therefore, we tested whether sera of patients with H. pylori infection contained antibodies against IROMPs. All sera from 20 H. pylori positive patients showed a clear immune response against a 77 kDa heme-binding IROMP in an immunoblot assay. Antibody responses against the other IROMPs were also found, but with lower frequencies. Serum samples from 18 patients negative for H. pylori infection did not show any immunoreactivity with IROMPs. These results indicate that the IROMPs of H. pylori are immunogenic and are expressed in vivo.

Antibodies, Bacterial↗
PubMed1996

Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori.

Seventy-three Helicobacter pylori-positive patients were treated with a combination of clarithromycin and ranitidine in order to eradicate the bacterium. Eradication was successful in 79.5%. In 15 patients eradication failed, and in 11 cases this was due to clarithromycin resistance. In one patient the infecting strain was resistant at the onset of treatment, while in the remaining 10 patients resistance developed during therapy. These isolates had also become resistant to various other antibiotics. Random amplified polymorphic DNA and restriction fragment end-labeling analysis of the isolates showed close genetic relatedness between pre- and post-treatment isolates, indicating that resistance was the result of selection of variants of the infecting strain rather then infection with an exogenous resistant strain. Nucleotide sequence comparisons revealed that all resistant isolates had a single base pair mutation in the 23S rRNA. Since this single point mutation results in co-resistance to various antibiotics at high frequencies, caution should be taken when using clarithromycin as a single antibiotic.

Anti-Bacterial Agents↗
PubMed1996

The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse.

We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine. In an organ culture model, a mutant of S. typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild-type. Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model. Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E. coli strain expressing PE fimbriae to thin sections of mouse small intestine. The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice. Intragastric injection of S. typhimurium 14028 and SR-11 caused fluid accumulation in infant mice. In contrast, pefC mutants of S. typhimurium 14028 and SR-11 were negative in the infant mouse assay. Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay. However, heterologous expression of PE fimbriae in E. coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation.

Animals↗
PubMed1996

Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.

Amino Acid Sequence↗
PubMed1995

Induction of the phoE promoter upon invasion of Salmonella typhimurium into eukaryotic cells.

Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes. Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo. In vivo-inducible promoters may be a good alternative for constitutive promoters. The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants. Here we studied whether its expression from a plasmid is induced in S. typhimurium upon invasion of eukaryotic cells. This appeared to be the case. Furthermore, a S. typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied. Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected. Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.

Cell Line↗
PubMed1995

Identification of the domain which determines the g,m serotype of the flagellin of Salmonella enteritidis.

Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed and screened with a g,m-specific monoclonal antibody. Results showed that the g,m epitope is localized between amino acids 258 and 348 of the flagellin. The fliC gene, encoding the flagellin of S. enteritidis, was proven to be the only flagellin gene present in S. enteritidis.

Amino Acid Sequence↗
PubMed1995

Identification of the hopG gene, a component of Escherichia coli K-12 type II export system, and its conservation among different pathogenic Escherichia coli and Shigella isolates.

The Escherichia coli K-12 gene coding for a component of a type II export system was identified and characterized. The HopG protein contains a typical prepilin peptidase cleavage site and has a high degree of homology with proteins PulG, OutG, and ExeG, which are components of type II secretion systems from Klebsiella pneumoniae, Erwinia carotovora, and Aeromonas hydrophila.

Amino Acid Sequence↗
PubMed1994

CDR1 T-cell receptor beta-chain peptide induces major histocompatibility complex class II-restricted T-T cell interactions.

T-T cell interactions have been proposed in postulated network theories of immunoregulation and autoimmunity. Despite previous reports of protection induced by T-cell receptor (TcR)-derived peptides in experimental autoimmunity, no evidence for T-T cell interactions by direct recognition of processed TcRs on native T cells was obtained. Here we report that immunization of rats with overlapping sets of peptides of the TcR alpha or beta chain allowed us to detect immunogenic TcR peptides. Remarkably enough, these TcR peptides appeared to cluster within the hypervariable complementarity-determining regions of the TcR. Immunization of rats with these TcR peptides induced CD4+ TcR peptide-specific T cells, which recognized both rDNA TcR proteins and the original, arthritogenic T cell in a major histocompatibility complex class II-restricted way. These findings indicate that activated T cells can process and present their own TcR in the context of major histocompatibility complex class II molecules and, furthermore, that such peptides can be recognized by TcR variable gene-specific T cells.

Amino Acid Sequence↗
PubMed1994

Characterization of three putative Serpulina hyodysenteriae hemolysins.

Serpulina hyodysenteriae hemolysin is though to be an important virulence factor in swine dysentery. One gene, tlyA, previously called tly, encoding a hemolysin in S. hyodysenteriae strain B204 has been characterized (Muir et al. Infect Immun 1992; 60: 529-35). Two other genes of S. hyodysenteria strain B204, designated tlyB and tlyC, encoding hemolytic proteins in Escherichia coli strain DH5 alpha were cloned and sequenced. The tlyB and tlyC genes, when expressed in E. coli, encode heat-labile, protease-sensitive proteins which exhibit both hemolytic and cytotoxic activity in vitro. The calculated molecular weights of the tlyB and tlyC gene products are 93.3 kDa and 30.8 kDa, respectively. The tlyB gene product has sequence homology with the Clp proteins, whereas for the tlyC-encoded protein no homology with other protein sequences was observed. Southern hybridization showed that the tlyB and tlyC genes are present in a single copy on the chromosome of S. hyodysenteriae.

Amino Acid Sequence↗