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J G Kusters

Publications and source records attributed to J G Kusters.

At least 91 records · Page 5Linked to original sources
PubMed1994

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid.

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV+, its plasmidless isogeneic derivative W22703 pYV- and four recent field isolates was examined. The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV- strains grew consistently faster than pYV+ strains. This difference in growth rate was high at 30-35 degrees C, moderate at 1-10 degrees C and 25 degrees C, but hardly significant at 15-20 degrees C. Increasing the growth temperature above 25 degrees C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1-20 degrees C range no obvious temperature- or plasmid-related differences in protein patterns could be detected. The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1 degrees C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16: 1 and cyclic fatty acid contents in the 5-25 degrees C and < or = 15 degrees C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.

Adaptation, Biological↗
PubMed1994

Salmonella typhimurium loci involved in survival within macrophages.

A set of Tn10 mutants of Salmonella typhimurium which have a diminished capacity to survive in murine macrophages and decreased virulence in mice has been described previously. In this study, we characterized 30 of these mutants and determined map locations of Tn10 insertions for 23 of these strains. In addition, short fragments of transposon-flanking DNA were cloned, and the nucleotide sequence was determined for 23 mutants. Seven mutants carried transposon insertions in known genes, representing six loci: htrA, prc, purD, fliD, nagA, and smpB. The possible roles of these genes in Salmonella virulence are discussed. One insertion was found to be in an unknown gene which shared homology with the open reading frames Bv' and Bv located in the pin inversion system of Shigella boydii. In one mutant, Tn10 was found to be inserted in a gene with significant homology to adhE of Escherichia coli and Clostridium acetobutylicum. The map location and degree of homology indicate that the Salmonella gene encodes a related, but different, dehydrogenase. In 14 of the mutants analyzed, Tn10 was inserted into genes which had no significant homologies to entries in the DNA and protein data bases. In conclusion, 16 insertions define loci, termed ims for impaired macrophage survival, which have not yet been described in S. typhimurium but have been shown previously to be necessary for full virulence in mice. Although most ims loci are distributed randomly throughout the genome, a cluster was found between 75 and 78 min on the Salmonella chromosome.

Amino Acid Sequence↗
PubMed1994

Entrance and survival of Salmonella typhimurium and Yersinia enterocolitica within human B- and T-cell lines.

Lymphocytes, located within the Peyer's patches, might be involved in the dissemination of enteropathogenic Salmonella typhimurium and Yersinia enterocolitica bacteria. To test this hypothesis, we have investigated the susceptibility of human B- and T-cell lines to bacterial adhesion and invasion. The two S. typhimurium strains analyzed were highly invasive, while the two Y. enterocolitica (O:8) strains adhered to the B- and T-cell lines but did not enter the cell lines in significant amounts. We hypothesize that the incapability of the Y. enterocolitica (O:8) strains to enter the human B- and T-cell lines is most probably due to the bacterial inability to induce the internalization process upon adhesion to both cell lines. Although immortalized B- and T-cell lines were used in this study, the results presented suggest the possibility that both cell types could play a role in the dissemination of intracellularly residing S. typhimurium in vivo.

B-Lymphocytes↗
PubMed1993

Invasion of HeLa cells by Bordetella bronchiseptica.

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis, is able to invade HeLa cells. Comparison of the invasiveness of Bvg+ and Bvg- B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg- (flagellated) phase was ten-fold lower than when Bvg+ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica, which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.

Bacterial Adhesion↗
PubMed1993

The role of hemolysin(s) in the pathogenesis of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae, an anaerobic beta hemolytic spirochaete, is the etiologic agent of swine dysentery. Not much is known at present about the virulence factors of S. hyodysenteriae. However, the hemolysin production of this bacterium is generally accepted to be a virulence factor. To study the exact role of hemolysin production in the pathogenesis of swine dysentery, the gene encoding a hemolysin, tly, was cloned and its nucleotide sequence determined. After inactivation of this gene, the virulence of a tly-minus mutant in mice was tested. The mutant had reduced hemolysis indicating that the tly-encoded hemolysin was not the only hemolysin produced by S. hyodysenteriae. Mice infected with the tly-minus mutant had fewer cecal lesions than mice infected with the wild-type S. hyodysenteriae. It was concluded that the tly-encoded hemolysin might be an important virulence factor, but not the only one. Since it was demonstrated that spirochaetes can be transformed through electroporation, this has made a genetic approach to elucidate the pathogenesis of spirochaetal infections possible.

Animals↗
PubMed1993

Molecular analysis of a flagellar core protein gene of Serpulina (Treponema) hyodysenteriae.

The flaB2 gene encoding a protein located in the core of the periplasmic flagella of Serpulina hyodysenteriae was cloned and sequenced. The FlaB2 protein consists of 285 amino acids and has a calculated molecular mass of 31.1 kDa. Southern blot analysis indicated that at least one, and possibly two genes related to flaB2 are present in the genome of S. hyodysenteriae. Comparison of the amino acid sequence of FlaB2 to sequences present in data banks showed significant similarity with the core flagellins of other spirochaetes, in particular with a FlaB2 protein from Treponema phagedenis.

Amino Acid Sequence↗
PubMed1993

Effects of multiplicity of infection, bacterial protein synthesis, and growth phase on adhesion to and invasion of human cell lines by Salmonella typhimurium.

Monolayers of intestine 407 (Int-407) cells were infected with the virulent Salmonella typhimurium strain C52, and the adhesion to and invasion of these cells were studied. The effects of the multiplicity of infection and growth phase of the bacteria (logarithmic versus stationary) on the interaction with eukaryotic cells were investigated. In contrast to other reports, we found no differences in the adhesive and invasive capacities of bacteria derived from logarithmic- or stationary-phase cultures. Invasion by S. typhimurium required bacterial protein synthesis and live Int-407 cells. Bacteria adhered equally well to dead or live Int-407 cells, which indicates that adhesion does not require metabolically active cells. Adhesion of S. typhimurium followed saturation kinetics, with a maximum of 10 adhesive bacteria per cell. This indicates that there is a limited number of bacterial adhesion sites (receptors) available on the surface of the host cell. Killed and live bacteria adhered equally well and competed with each other for cellular adhesion sites. This and adhesion experiments performed in the presence of chloramphenicol showed that bacterial protein synthesis is not required for adhesion. The general validity of the results obtained with S. typhimurium C52 was confirmed by comparing the invasion and adhesion data with those of the frequently used SL1344 and SR11 strains. In addition, we assayed the adhesion and invasion of S. typhimurium C52, SL1344, and SR11 and 27 S. typhimurium field isolates with Int-407, HeLa, and HEp-2 cells.

Bacterial Adhesion↗
PubMed1993

Genetic similarity of intestinal spirochetes from humans and various animal species.

The chromosomal DNA of spirochetes isolated from human, swine, dog, mouse, rat, and chicken intestine or feces was subjected to restriction enzyme analysis and hybridization with three different DNA probes, derived from a flagellin gene, a hemolysin gene, and the 16S rDNA sequence of the pathogenic swine intestinal spirochete Serpulina hyodysenteriae. This genetic analysis showed that intestinal spirochetes represent a heterogeneous but related population of bacteria. In general, unique genotypes were distinguished among isolates from the same host species; they were not present among isolates from other host species. This suggests the host specificity of some strains. An exception to this are isolates from humans and dogs suffering from gastrointestinal disorders; these isolates showed highly similar or even identical genotypes. None of them resembled any of the genotypes of isolates found in other host species without apparent disease.

Animals↗
PubMed1992

Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide.

Neutralizing monoclonal antibodies directed against five antigenic sites on the spike (S) S1 glycopolypeptide of avian infectious bronchitis virus (IBV) were used to select neutralization-resistant variants of the virus. By comparing the nucleotide sequence of such variants with the sequence of the IBV parent strain, we located five antigenic sites on the amino acid sequence of the S1 glycopolypeptide. The variants had mutations within three regions corresponding to amino acid residues 24 to 61, 132 to 149 and 291 to 398 of the S1 glycopolypeptide. The location of three overlapping antigenic sites on the IBV spike protein was similar to the location of antigenic sites on the spike protein of other coronaviruses.

Antibodies, Monoclonal↗
PubMed1992

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene.

Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.

Amino Acid Sequence↗
PubMed1992

Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein.

A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.

Amino Acid Sequence↗
PubMed1992

Rapid detection and identification of avian infectious bronchitis virus.

A rapid and sensitive method for the detection and unambiguous typing of infectious bronchitis virus (IBV) is described. RNA was isolated from IBV-infected allantoic fluid and was transcribed into cDNA. This cDNA was amplified by the polymerase chain reaction. The polymerase chain reaction products were subsequently analyzed on an agarose gel. The presence of IBV-specific RNA in the allantoic fluid then allowed the amplification of a 438-bp DNA fragment from the nucleocapsid (N) gene. For the typing of IBV isolates, we used amplified double-stranded DNA as a template in a sequencing reaction. We report 360 bases of the N gene of 18 IBV isolates. The sequence of the N gene was different between serologically indistinguishable IBV strains and may be a valuable tool in epidemiologic studies. A phylogenetic tree that was based on the sequences obtained did not agree with trees that were based on other parts of the sequence, illustrating the high frequency of recombination between IBV strains.

Animals↗
PubMed1991

Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification.

Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR alpha/beta expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Z1a T cell line was shown to use the same TcR V gene segments (V alpha 2 and V beta 8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the V beta D beta J beta junction was found. Alignment of a large number of human V alpha and V beta gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.

Amino Acid Sequence↗
PubMed1991

Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus.

In a previous study, two murine T-cell hybridomas generated after immunization with infectious bronchitis virus (IBV) were shown to be responsive to the internally localized viral nucleocapsid protein. In the present study, the antigenic determinants were mapped using recombinant expression products and synthetic peptides. Both hybridomas recognized the region spanning amino acid residues 71 to 78 of the nucleocapsid protein. The experimentally determined epitope corresponded with predicted motifs. Both an I-Ed binding motif and a predicted cleavage site for the aspartyl protease cathepsin D were contained within the sequence. The epitope was shown to prime cellular immune responses to IBV in the chicken.

Amino Acid Sequence↗
PubMed1991

MHC class II-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV.

Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.

Animals↗