scholarly journals Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers

1998 ◽  
Vol 66 (3) ◽  
pp. 1113-1120 ◽  
Author(s):  
Travis Grant ◽  
Vicki Bennett-Wood ◽  
Roy M. Robins-Browne
Keyword(s):  
Yersinia Enterocolitica ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Electron Microscopic Examination ◽  
Clinical Isolates ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Electron Microscopic ◽  
Virulence Markers ◽  
Culture Cells

ABSTRACT Yersinia enterocolitica is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of Y. enterocoliticabiotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb Yersinia virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes: ail (7.2%), myfA(11.7%), ystA (0.9%), and ystB (85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (P ≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (P ≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of Y. enterocolitica may invade cells by a novel mechanism. These results indicate that some clinical isolates of Y. enterocolitica which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic Yersiniaspecies.

scholarly journals Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly.

1983 ◽  
Vol 97 (1) ◽  
pp. 30-39 ◽  
Author(s):  
F Cabral ◽  
L Wible ◽  
S Brenner ◽  
B R Brinkley
Keyword(s):  
Cell Cycle ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Electron Microscopic Examination ◽  
Microtubule Assembly ◽  
Electron Microscopic ◽  
Cytoplasmic Microtubule ◽  
Ovary Cells ◽  
Essentially Normal

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

Scanning Electron Microscopic Examination of Yersinia enterocolitica Attached to Stainless Steel at Selected Temperatures and pH values1

1988 ◽  
Vol 51 (6) ◽  
pp. 445-448 ◽  
Author(s):  
PAULA J. HERALD ◽  
EDMUND A. ZOTTOLA
Keyword(s):  
Stainless Steel ◽  
Yersinia Enterocolitica ◽  
Steel Surface ◽  
Electron Microscopic Examination ◽  
Growth Conditions ◽  
Stainless Steel Surface ◽  
Electron Microscopic ◽  
Scanning Electron Microscopic ◽  
Steel Surfaces ◽  
Scanning Electron

Attachment of Yersinia enterocolitica to stainless steel surfaces at 35, 21, and 10°C was investigated using scanning electron microscopy (SEM). Cells adhered at all three temperatures, but, in general, the greatest number of adhered cells were observed at pH 8 and 21°C. Multi-flagellated cells were noted under these growth conditions. When grown at pH 9.5 and 21°C, fibrils were observed between cells and extending to the stainless steel surface. Fewer cells with flagella were seen at this pH. Adherence may be related to the flagella and any exopolymer surrounding the cells.

scholarly journals Identification of a Cytolethal Distending Toxin Gene Locus and Features of a Virulence-Associated Region inActinobacillus actinomycetemcomitans

1999 ◽  
Vol 67 (3) ◽  
pp. 1227-1237 ◽  
Author(s):  
Marcia P. A. Mayer ◽  
Lina C. Bueno ◽  
Eric J. Hansen ◽  
Joseph M. DiRienzo
Keyword(s):  
Dna Sequences ◽  
Recombinant Strain ◽  
Integration Site ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequence Similarity ◽  
Toxin Gene ◽  
Virulence Plasmid ◽  
Cytolethal Distending Toxin ◽  
Oral Pathogen

ABSTRACT A genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome ofActinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to the cdtABC genes of some pathogenicEscherichia coli strains and Haemophilus ducreyi, respectively. Sonic extracts from recombinant E. coli, containing the A. actinomycetemcomitans ORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins of H. ducreyi recognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an ΩKan-2 interposon was inserted into the cdtA andcdtB genes. Expression of the CDT-like activities inA. actinomycetemcomitans was strain specific. Naturally occurring expression-negative strains had large deletions within the region of the cdt locus. The cdtABC genes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen.

Electron Microscopic Examination of DNA in an Alkaline Sucrose Gradient

1975 ◽  
Vol 33 ◽  
pp. 612-613
Author(s):  
W. D. Garner ◽  
R. E. Nordquist ◽  
R. H. Bottomley ◽  
J. M. Hawrylko
Keyword(s):  
Mammalian Cells ◽  
Sucrose Gradient ◽  
Electron Microscopic Examination ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Ultrastructural Characteristics ◽  
Rat Hepatoma ◽  
Hepatoma Tissue

The technique of lysing cells on top of an alkaline sucrose gradient prior to sedimentation was first designed to examine single stranded DNA from protoplasts. The lysis of mammalian cells and sedimentation of the released DNA has not been examined thoroughly by EM. The present work describes the ultrastructural characteristics of DNA species found after whole cell lysing and sedimentation of the DNA through an alkaline sucrose gradient.The DNA of H4-II-E rat hepatoma tissue culture cells was prelabelled with H3TdR (luci/ml) for 18 to 24 hours prior to lysis. Cells were layered into a lysing layer (0.1 M EDTA and 0.5 N NaOH, pH 13.25) on top of a 5-202or 5-25% alkaline sucrose gradient (0.01 M EDTA, 0.1 N NaOH and 0.9 M NaCl). After centrifugation the gradients were fractionated from the bottom. The fractions containing the majority of H3TdR were applied directly on grids at pH 8.5 or 12. Samples were stained with uranyl acetate.

scholarly journals Role of Decay-Accelerating Factor Domains and Anchorage in Internalization of Dr-Fimbriated Escherichia coli

2000 ◽  
Vol 68 (3) ◽  
pp. 1391-1399 ◽  
Author(s):  
R. Selvarangan ◽  
P. Goluszko ◽  
V. Popov ◽  
J. Singhal ◽  
T. Pham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Interaction Model ◽  
Electron Microscopic ◽  
Decay Accelerating Factor ◽  
Glycosylphosphatidylinositol Anchor ◽  
E Coli ◽  
Short Consensus Repeat

ABSTRACT Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr+ E. coli was characterized in a cell-cell interaction model. Binding of Dr+ E. coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser165 by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr+ E. coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr+ E. coli were morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr+ E. coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.

scholarly journals Chinese hamster ovary cell mutants defective in the internalization of ricin.

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Enhanced internalization of ricin in nigericin-pretreated Chinese hamster ovary cells.

1981 ◽  
Vol 1 (6) ◽  
pp. 560-567 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Electron Microscopic ◽  
Ovary Cells

Biochemical and electron microscopic autoradiographic studies with [125I] ricin have revealed that nigericin-pretreated Chinese hamster ovary cells are more efficient than untreated cells in the internalization of the toxin into the cells. These results suggest that the enhanced rate of internalization of ricin in nigericin-pretreated cells may account for the enhancement of cytotoxicity of ricin in Chinese hamster ovary cells by nigericin.

scholarly journals Evidence of Extensive Circulation of Yersinia enterocolitica in Rodents and Shrews in Natural Habitats from Retrospective and Perspective Studies in South Caucasus

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 939
Author(s):  
Tata Imnadze ◽  
Lile Malania ◽  
Neli Chakvetadze ◽  
Irma Burjanadze ◽  
Natalia Abazashvili ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Flagellar Apparatus ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
South Caucasus ◽  
Natural Habitats ◽  
Single Nucleotide ◽  
E Coli ◽  
A Chain ◽  
Culture Positive

Yersinia enterocolitica culture-positive rodents and shrews were reported in different territories across Georgia during 14 of 17 years of investigations conducted for the period of 1981–1997. In total, Y. enterocolitica was isolated from 2052 rodents (15 species) and 33 shrews. Most isolates were obtained from Microtus arvalis, Rattus norvegicus, Mus musculus, and Apodemus spp. During the prospective study (2017−2019), isolates of Yersinia-like bacteria were cultured from 53 rodents collected in four parts of Georgia. All the Yersinia-like isolates were confirmed as Y. enterocolitica based on the API 20E and the BD Phenix50 tests. Whole-genome (WG) sequencing of five rodents and one shrew strain of Y. enterocolitica revealed that they possessed a set of virulence genes characteristic of the potentially pathogenic strains of biogroup 1A. All isolates lacked distinguished virulence determinants for YstA, Ail, TccC, VirF, and virulence plasmid pYV but carried the genes for YstB, YmoA, HemPR-HmuVSTU, YaxAB, PhlA, PldA, ArsCBR, and a flagellar apparatus. One strain contained a gene highly homologous to heat-labile enterotoxin, a chain of E. coli, a function not previously described for Y. enterocolitica. The WG single-nucleotide polymorphism-based typing placed the isolates in four distinct phylogenetic clusters.

scholarly journals Homologues of Insecticidal Toxin Complex Genes in Yersinia enterocolitica Biotype 1A and Their Contribution to Virulence

2005 ◽  
Vol 73 (10) ◽  
pp. 6860-6867 ◽  
Author(s):  
Sharon M. Tennant ◽  
Narelle A. Skinner ◽  
Angela Joe ◽  
Roy M. Robins-Browne
Keyword(s):  
Yersinia Enterocolitica ◽  
Virulence Genes ◽  
Gastrointestinal Disease ◽  
Subtractive Hybridization ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Photorhabdus Luminescens ◽  
Insecticidal Toxin ◽  
Toxin Complex

ABSTRACT Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo.

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