Biomedical subjects

J E Samuel

Publications and source records attributed to J E Samuel.

At least 37 records · Page 2Linked to original sources

PubMed1993

Cloning and sequencing of Coxiella burnetii outer membrane protein gene com1.

The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii. The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of the gene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence. The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Com1 protein expressed in E. coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane. While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases.

Amino Acid Sequence↗

PubMed1993

Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice.

In earlier studies using a streptomycin-treated mouse model of infection caused by enterohemorrhagic Escherichia coli (EHEC), animals fed Shiga-like toxin type II (SLT-II)-producing strains developed acute renal cortical necrosis and died, while mice fed Shiga-like toxin type I (SLT-I)-producing clones did not die (E. A. Wadolkowski, L. M. Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien, Infect. Immun. 58:3959-3965, 1990). To examine the bases for the differences we noted between the two toxins in the murine infection model, we injected mice with purified toxins and carried out histopathological examinations. Despite the genetic and structural similarities between the two toxins, SLT-II had a 50% lethal dose (LD50) which was approximately 400 times lower than that of SLT-I when injected intravenously or intraperitoneally into mice. Histopathologic examination of toxin-injected mice revealed that detectable damage was limited to renal cortical tubule epithelial cells. Passive administration of anti-SLT-II antibodies protected mice from SLT-II-mediated kidney damage and death. Immunofluorescence staining of normal murine kidney sections incubated with purified SLT-I or SLT-II demonstrated that both toxins bound to cortical tubule and medullary duct epithelial cells. Compared with SLT-I, SLT-II was more heat and pH stable, suggesting that SLT-II is a relatively more stable macromolecule. Although both toxins bound to globotriaosylceramide, SLT-I bound with a higher affinity in a solid-phase binding assay. Differences in enzymatic activity between the two toxins were not detected. These data suggest that structural/functional differences between the two toxins, possibly involving holotoxin stability and/or receptor affinity, may contribute to the differential LD50s in mice.

Animals↗

PubMed1992

An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine.

Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.

Animals↗

PubMed1992

An experimental model for subclinical edema disease (Escherichia coli enterotoxemia) manifest as vascular necrosis in pigs.

An experimental model for subclinical edema disease was developed in weanling pigs. In multiple experiments, 3-week-old pigs were weaned, then inoculated intragastrically with 10(10) colony-forming units of an SLT-IIv-positive strain of Escherichia coli originally isolated from a pig with edema disease (principals). Control pigs were inoculated with a nonpathogenic E coli strain. Of 39 principals, 8 developed clinical edema disease within 14 days after inoculation. However, 20 of 21 principals that did not develop clinical signs of edema disease, but were submitted for necropsy examination at 14 days after inoculation, had characteristic vascular lesions of edema disease. Vascular lesions, found principally in ileum and brain, consisted of segmental necrosis of myocytes in the tunica media of small arteries and arterioles. None of the pigs inoculated with a nonpathogenic strain of E coli developed edema disease or vascular lesions. None of the principals necropsied at 2 days after inoculation had vascular lesions. Development of vascular lesions by 14 days after inoculation was used as the end point for detecting subclinical edema disease in the model.

Animals↗

PubMed1991

Evaluation of the role of Shiga and Shiga-like toxins in mediating direct damage to human vascular endothelial cells.

Infection with Shiga toxin- and Shiga-like toxin-producing strains of Shigella dysenteriae and Escherichia coli, respectively, can progress to the hemolytic-uremic syndrome. It has been hypothesized that circulating Shiga toxin, Shiga-like toxins, and endotoxins may contribute to the disease by directly damaging glomerular endothelial cells. The effects of these toxins on HeLa, Vero, and human vascular endothelial cells (EC) were examined. Confluent EC were sensitive to Shiga toxin but were at least 10(6)-fold less sensitive to the toxins than were Vero cells. Shiga toxin was the predominant cytotoxic factor. Lipopolysaccharides were not cytotoxic and did not augment Shiga toxin-mediated toxicity. Lower doses of Shiga toxin caused cytotoxicity when coincubated with tumor necrosis factor. The relative resistance of EC to Shiga toxin and Shiga-like toxins may be due to reduced toxin binding, as low levels of globotriaosylceramide (Gb3), the toxin-specific receptor, were found in EC membranes.

Animals↗

PubMed1991

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Animals↗

PubMed1991

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

Amino Acid Sequence↗

PubMed1991

Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity.

Shiga toxin of Shigella dysenteriae type I and Shiga-like toxins I and II (SLT-I and SLT-II, respectively) of enterohemorrhagic Escherichia coli are functionally similar protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriaosylceramide glycolipid receptors on eukaryotic cells. Regionally directed chemical mutagenesis of the B subunit of SLT-II was used to identify amino acids in the B subunit that are critical for SLT-II holotoxin cytotoxic activity. Three noncytotoxic mutants were isolated, and their mutations were mapped. The substitutions of arginine with cysteine at codon 32, alanine with threonine at codon 42, and glycine with aspartic acid at codon 59 in the 70-amino-acid mature SLT-II B polypeptide resulted in the complete abolition of cytotoxicity. The analogous arginine, alanine, and glycine residues were conserved at codons 33, 43, and 60 in the 69-amino-acid mature B polypeptide of Shiga toxin. Comparable mutations induced in the B-subunit gene of Shiga toxin by oligonucleotide-directed, site-specific mutagenesis resulted in drastically decreased cytotoxicity (10(3)- to 10(6)-fold) as compared with that of wild-type Shiga toxin. The mutant SLT-II and Shiga toxin B subunits were characterized for stability, receptor binding, immunoreactivity, and ability to be assembled into holotoxin.

Amino Acids↗

PubMed1990

DNA probes for the identification of Coxiella burnetti strains.

Isolation of Coxiella Burnetii in the standard laboratory setting is hazardous; therefore most diagnoses are based on retrospective detection of a rising antibody titer to C. burnetti. As a result, this disease is usually undiagnosed or misdiagnosed. Methods for the rapid detection of C. burnetti have now been developed that utilize specific hybridization of labeled DNA probes to nucleic acid in clinical samples. One method detects the presence of C. burnetii 16S ribosomal RNA (rRNA); another uses plasmid sequences. We have developed a probe that detects C. burnetii and one that differentiates between Coxiella strains capable of causing chronic disease and those that cause the acute form. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. A third method differentiates between chronic (endocarditis-causing) strains and those that cause acute Q fever. This method uses the polymerase chain reaction (PCR), in which the target regions of DNA are amplified by iterative cycles of Taq I DNA polymerase chain extension to produce up to a 10(6) amplification of the target sequences. When Southern blotting is used in conjunction with PCR, the test detects as few as 2-9 C. burnetti cells.

Animals↗

PubMed1990

Identification and cloning of a 27-kDa Coxiella burnetii immunoreactive protein.

Patients with acute Q fever develop Coxiella burnetii-specific antibody, and chronic disease patients often develop extremely high levels of C. burnetii-specific antibody. Antibody-reactive LPS has been well characterized, but only a few immunoreactive proteins have been identified. An immunoreactive ca. 62-kDa protein has been cloned, sequenced, and shown to be related to an Escherichia coli heat-shock protein. A ca. 27-kDa immunoreactive outer membrane protein(s) has also been identified. We have begun characterizing C. burnetii immunoreactive proteins by gene-cloning methods. A gene bank of total C. burnetii Nine Mile phase 1 (acute strain) DNA was created using the lambda vector EMBL3. This bank was screened for plaques which reacted with E. coli-pre-absorbed rabbit antisera specific for purified, Nine Mile C. burnetii whole cells. Twenty-three immunoreactive plaques were identified from a screening of 3000 plaques. Twenty-two plaques produced a ca. 60-65-kDa immunoreactive protein. One plaque produced a ca. 27-kDa immunoreactive protein, as well as a ca. 60-65-kDa immunoreactive protein. Phage extracts of this plaque were used to enrich the antisera to produce a ca. 27-kDa-specific antisera. This enriched sera reacted with a ca. 27-kDa protein in all C. burnetii isolates tested, comprising isolates from both chronic and acute strains. The DNA contained in the immunoreactive clone was C. burnetii-specific, as shown by DNA hybridization. We are currently subcloning the ca. 27-kDa protein-coding region for sequencing to determine if this gene encodes the previously identified ca. 27-kDa immunodominant protein. This protein may ultimately have both diagnostic and vaccinogenic potential.

Antigens, Bacterial↗

PubMed1990

Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type II.

Escherichia coli O157:H7 strains have been implicated as etiologic agents in food-borne outbreaks of hemorrhagic colitis and the hemolytic-uremic syndrome. A prototype E. coli O157:H7 strain, designated 933, produces Shiga-like toxin I (SLT-I) and SLT-II and harbors a 60-MDa plasmid. In a previous study, streptomycin-treated mice were fed 933 together with a derivative cured of the 60-MDa plasmid (designated 933cu). Strain 933cu colonized poorly, but in approximately one-third of the animals, an isolate of 933cu was obtained from the feces that had regained the ability to colonize well. This isolate, designated 933cu-rev, killed all of the animals when fed alone to mice. In this investigation, two types of experiments were done to assess whether SLT-I, SLT-II, or both contributed to the death of mice fed 933cu-rev. (i) Mice were pretreated with monoclonal antibodies to SLT-I, SLT-II, SLT-I and SLT-II, or cholera toxin (as a control) before infection with 933cu-rev. (ii) Mice were fed either an E. coli K-12 strain carrying cloned SLT-I genes or the same K-12 strain carrying cloned SLT-II genes. The results of both types of experiments indicated that the deaths of the orally infected mice were due solely to SLT-II. Extensive histological and selected electron microscopic examinations of various tissues from the infected animals suggested that death was due to acute renal cortical tubular necrosis consistent with toxic renal damage. These data indicate a critical role for SLT-II, but not SLT-I, in renal damage associated with E. coli O157:H7 infection of streptomycin-treated mice.

Animals↗

PubMed1990

Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants.

The antigenically distinct Shiga-like toxins (SLTs) SLT-1 and SLT-II are cytotoxic for both Vero and HeLa cells and use Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb3) molecules as functional receptors. SLT-II-related variants SLT-IIvp and SLT-IIvh, produced by a porcine isolate and a human isolate, respectively, are cytotoxic for Vero but not HeLa cells. To investigate the basis for these differences in cytotoxic specificity among SLTs, the nature of the receptor for the SLT-II variants was examined. First, the patterns of binding of SLT-II and the SLT-II variants to Gb3 receptor analogs Gal alpha 1-4Gal-bovine serum albumin and Gal alpha 1-4Gal beta 1-4Glc-bovine serum albumin were compared. SLT-IIvp bound the trisaccharide neoglycoprotein preferentially, while SLT-IIvh bound both analogs equally but with less affinity than did SLT-II. Next, the glycolipids to which the SLT-II variants bound in Vero and HeLa cells were identified by thin-layer chromatography. SLT-IIvp bound to Gb3, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb4), and Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb5) in Vero cells but only Gb3 in HeLa cells. However, SLT-IIvh bound to Gal alpha 1-4Gal beta 1-1Cer (Gb2) and Gb3 in HeLa cells but only Gb3 in Vero cells. In addition, hybrid toxins (SLT-IIvp subunit A with SLT-II subunit B or SLT-II subunit A with SLT-IIvp subunit B) were used to show that the receptor specificities of the SLTs was B subunit specific. These differences in receptor specificities are important in vivo, as evidenced by a 400-fold difference in the 50% lethal doses of purified SLT-IIvp and SLT-II (200 versus 0.5 ng, respectively) for mice. These data indicate that SLT-II-cytotoxic variants can occur as a consequence of differences in receptor specificity and affinity.

Animals↗

PubMed1989

Phylogenetic diversity of the Rickettsiae.

Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the alpha subdivision of the purple bacteria. E. risticii also belongs to the alpha subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the gamma subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.

Bacteria↗

PubMed1988

Stability of plasmid sequences in an acute Q-fever strain of Coxiella burnetii.

The rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host. Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors. Genetically, the regions of DNA responsible for phase variation have not been identified. We have sought to determine whether the plasmid identified in acute disease isolates, QpH1, which represents approximately 5% of the coding capacity of this organism is involved in phase variation. Plasmids from phase 1 and phase 2 variants (designated QpH1 and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells. Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage. The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type. Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E. coli extract was also identical. Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical. These data indicate that within the limits of our analysis, the plasmid DNA from C. burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation.

Base Sequence↗

PubMed1988

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.

Amino Acid Sequence↗

PubMed1985

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases.

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.

Coxiella↗

PubMed1985

Correlation of plasmid type and disease caused by Coxiella burnetii.

The obligate intracellular bacterium Coxiella burnetii is the etiological agent of acute Q-fever and chronic endocarditis in humans and of several zoonotic infections. The DNA from a variety of these disease isolates was compared for homology to the plasmid QpH1, found in the Nine Mile strain. Three patterns of homology were found in these isolates, i.e., one pattern identical to that of QpH1, one common to several endocarditis isolates and goat abortion isolates, and one common to the remaining group of endocarditis isolates. Plasmid DNA from the endocarditis-abortion isolate group, designated QpRS, was mapped by restriction enzyme analysis and compared with QpH1. These data show that QpRS was 2 to 3 kilobase pairs larger, contained DNA not found in QpH1, but was not generated from QpH1 by a single insertional event. Isolation of plasmid DNA from the second endocarditis group of isolates was not successful and may indicate that the plasmid has integrated into the chromosome. This analysis provides the first clear evidence that differences exist between C. burnetii isolates which cause various diseases, indicating that different C. burnetii strains may have unique virulence characteristics.

Animals↗

PubMed1984

System for the exposure of cell suspensions to power-frequency electric fields.

A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line. In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated. Electric fields in the range 0.02-3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5%. The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 microT . The temperatures of the experimental and control cell suspensions are held fixed within +/- 0.1 degrees C by a water bath. Special chambers were developed to hold cell cultures during exposure and sham exposure. Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16-17 h, approximately the same as for CHO cells grown under standard cell-culture conditions.

Animals↗