PubMed Health⌕ Search

Biomedical subjects

D J Kopecko

Publications and source records attributed to D J Kopecko.

At least 55 records · Page 3Linked to original sources
PubMed1987

Introduction of Shigella flexneri 2a type and group antigen genes into oral typhoid vaccine strain Salmonella typhi Ty21a.

For protection against dysentery caused by Shigella flexneri 2a, an in vivo-constructed recombinant plasmid with genes specifying the S. flexneri type and group antigens located near the pro (min 6) and his (min 44) chromosomal markers, respectively, was made and transferred to the galE Salmonella typhi strain Ty21a. Strain Ty21a carrying this recombinant plasmid was shown by immunological and biochemical analyses to express the S. flexneri 2a type and group antigens. Mice immunized with this vaccine strain were found to be protected against challenge with virulent S. flexneri 2a, but not significantly against S. typhi challenge, presumably because synthesis of the Shigella antigens interfered with expression of the typhoid antigens. Elimination of the recombinant plasmid from Ty21a allowed this strain to again express typical S. typhi O antigens. Mouse protection against both S. typhi and S. flexneri 2a challenges was achieved with a whole-cell vaccine mixture composed of equal parts of Ty21a and the Ty21a-S. flexneri 2a hybrid strain.

Administration, Oral↗
PubMed1987

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.

Antigens, Bacterial↗
PubMed1985

Development of a DNA probe to detect Salmonella typhi.

This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection.

Antigens, Bacterial↗
PubMed1984

Unusual lipopolysaccharide antigens of a Salmonella typhi oral vaccine strain expressing the Shigella sonnei form I antigen.

Salmonella typhi 5076-1C, a potential live, oral vaccine for protection against typhoid fever and Shigella sonnei shigellosis, expresses the S. sonnei form I antigen and normal S. typhi somatic antigens. Polysaccharide antigens of this galactose epimeraseless genetic derivative strain were hot phenol-water extracted from cells grown with (+gal) and without (-gal) galactose. Ultracentrifugation of the aqueous layer from (+gal) cells resulted in a lipopolysaccharide (LPS) pellet having core-linked S. typhi O-antigen but no core-linked form I antigen; the LPS from (-gal) cells lacked O-antigen. The form I antigen, obtained from the supernatant, was purified by alcohol precipitation and ion exchange chromatography. Unlinked form I and S. typhi O-polysaccharide antigens, both present in the (+gal) supernatant, were further separated by gel filtration. Chemical analyses revealed the 5076-1C form I antigen to be a polymer (Mr = 14,000-20,000) having O-disaccharide repeating units comprised of 2-acetamido-4-amino-2, 4,6-trideoxy-D-galactose and 2-acetamido-2-deoxy-L-altruronic acid. Unlike parental S. sonnei form I LPS, the 5076-1C form I antigen lacked core lipid A, had low phosphorus content, and migrated in polyacrylamide gels with lower relative mobility. In contrast to current concepts of LPS assembly, these data indicate that 5076-1C form I antigen is transported to the cell surface without covalent linkage to core lipid A, and exists as a polymerized, antigenic surface entity.

Administration, Oral↗
PubMed1983

Progressive increase in antibiotic resistance of gram-negative bacterial isolates. Walter Reed Hospital, 1976 to 1980: specific analysis of gentamicin, tobramycin, and amikacin resistance.

During a five-year period at Walter Reed Army Hospital, Washington, DC, the frequency of aminoglycoside resistance among clinical bacterial isolates increased from less than 1% in 1976 to 13% of all isolates in later years. This resistance was seen among frequently isolated species of gram-negative bacilli isolated from patients throughout the hospital and from all anatomical sites, including blood. The incidence of gentamicin and amikacin resistance rose with increased administration of these antibiotics; however, the incidence of tobramycin resistance increased despite its minimal usage. From 1977 to 1978 and from 1979 to 1980, there was a decrease in the conjugal transmissibility of resistance to gentamicin and tobramycin. There was no detectable transmissible amikacin resistance. These findings suggest that high priority must be given to strategies that limit the emergence and dissemination of organisms resistant to these important antibiotics.

Amikacin↗
PubMed1982

Genetic and molecular studies of the regulation of atypical citrate utilization and variable Vi antigen expression in enteric bacteria.

1. The atypical citrate-utilizing ability to two strains of E. coli has been shown to be plasmid-encoded. Strain V414 carries a 130 Mdal conjugative Cit+ plasmid that also specifies Tcr and Cmr. Strain V517 carries 9 different plasmid species but only the 36 Mdal species is correlated with Cit+ ability. These plasmids are different from previously reported Cit+ plasmids of E. coli and Salmonella, which express thermosensitive conjugal transfer systems. 2. A 9 kb Pstl fragment, carrying the Cit+ genes of pWR60, has been cloned into the pBR325 plasmid. 3. Metabolic studies indicate that intact citrate is not incorporated directly into whole cells. Rather, atypical citrate utilization by these E. coli strains appears to involve partial metabolism of citrate at the cell surface before or during uptake. 4. The expression of atypical Cit+ ability by the parental pWR60 plasmid or by the recombinant pWR61 plasmid appears reversible and may involve an expression switch mechanism (i.e., insertion sequence element). 5. Two widely separated genetic loci, viaA and viaB, are necessary for Vi antigen synthesis in Salmonella and Citrobacter. In some strains of C. freundii, Vi antigen expression is reversible, a phenomenon which can be visualized by a colonial morphology transition between Vi-expressing and -nonexpressing forms. 6. The C. freundii viaB locus appears to encode the Vi antigen as well as the genetic "switch" mechanism controlling reversible Vi antigen expression. The viaA locus, which is found in several different bacterial species, may encode some common property (e.g., cell surface structure or enzymatic activity) that is needed for Vi antigen expression. 7. S. typhi and E. coli K12 hybrid strains which carry the C. freundii viaB locus have been constructed. These hybrid strains express reversible Vi antigen expression, even in the absence of general recombination (i.e., functional recA gene product). 8. The C. freundii viaB locus was transposed via Mu-mediated events to an F'lac plasmid in the E. coli K12 hybrid strain WR2376. F' plasmids carrying the viaB locus should serve as a highly enriched source of viaB DNA for physical examination of the switch mechanism. 9. Genetic manipulations such as those described herein can be used to study virtually any plasmid, viral, or chromosomally-encoded property. The resultant better understanding of biochemical pathways and of genetic regulatory control systems, and the isolation of desired gene sequences should provide ample information and materials for improving chemical processes and constructing vaccines against various organisms.

Antigens, Bacterial↗
PubMed1982

Involvement of a plasmid in the invasive ability of Shigella flexneri.

Representative Shigella flexneri strains were studied to determine whether plasmids are involved in their virulence. All invasive S. flexneri strains, irrespective of serotype, were found to harbor a large plasmid of approximately 140 megadaltons in size, although some strains carried additional plasmid species. Spontaneous variants of strains of serotypes 1, 2, and 5 had lost this 140-megadalton plasmid and had concomitantly become avirulent, i.e., could neither invade HeLa cell monolayers nor produced keratoconjunctivitis in guinea pigs. To monitor plasmid transfer, the 140-megadalton plasmid of strain M90T (serotype 5) was tagged with the kanamycin resistance transposon Tn5. This tagged plasmid, pWR110, was not self-transmissible, but was mobilized by one of several different conjugative plasmids into avirulent derivatives of the heterologous serotypes 1 and 2 which had lost the comparable large plasmid. Transconjugants of both serotypes which had received pWR110 regained virulence. These data directly demonstrate that this large S. flexneri plasmid encodes or regulates some function(s) required for epithelial cell penetration.

Animals↗
PubMed1982

Intestinal immunoglobulin A responses in rabbits to a Salmonella typhi strain harboring a Shigella sonnei plasmid.

Salmonella typhi 5076-IC, which contains a plasmid that encodes the form I antigen of Shigella sonnei and which expresses S. typhi 9 and 12 and S. sonnei form I antigens, was used to immunize rabbits via chronically isolated ileal loops. Intestinal immunoglobulin A activity was detected against S. typhi, S. sonnei form I, and S. typhi strain 5076-IC. Thus S. typhi 5076-IC can effectively elicit mucosal immunoglobulin A to both S. typhi and S. sonnei.

Animals↗
PubMed1981

Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence.

Virulent form I Shigella sonnei strains contain a 120-megadalton plasmid that is absent in their form II derivatives, which are always avirulent and devoid of O side chains. In the present study, 165 biochemical and antibiotic traits were assessed, but no experimentally useful phenotype could be associated with this large form I plasmid. Therefore, the form I plasmids of several S. sonnei strains were tagged with the antibiotic resistance transposons Tn3, Tn5, or Tn10. Transposon-tagged form I plasmids were not self-transmissible, but could be mobilized by the plasmid R386. Form II S. sonnei transconjugants for the form I plasmid acquired both virulence and the ability to synthesize form I antigen, establishing that these properties are plasmid mediated. Further studies indicate that this 120-megadalton form I plasmid is physically unstable in any of several host bacteria and suggest that it is a member of the FI incompatibility group. Also, two commonly observed, small plasmids of S. sonnei, of 3.2 and 3.9 megadaltons, were shown to encode either colicin E1 production or resistance to streptomycin and sulfonamide, respectively.

Anti-Bacterial Agents↗
PubMed1981

Construction of a potential bivalent vaccine strain: introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain.

Shigella sonnei, an intestinal pathogen, produces a characteristic form I cell surface antigen now known to be plasmid encoded. We considered that the GalE Salmonella typhi Ty21a oral vaccine strain, highly effective against typhoid, might be modified so as to be protective also against shigellosis due to S. sonnei. The plasmid responsible for form I antigen synthesis was therefore conjugally transferred to the galE S. typhi strain. Serological studies revealed that the derivative strain produces the form I antigen in addition to the normal S. typhi somatic antigens. Testing in mice demonstrated that the derivative form I galE S. typhi strain is protective against both S. sonnei and S. typhi challenges. These data suggest that the galE S. Ty21a oral vaccine strain, which presumably stimulates the local immune system in the intestine, may also serve as a useful carrier for other antigenic determinants to protect against different intestinal infections.

Animals↗
PubMed1981

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.

Antigens, Bacterial↗
PubMed1980

Genetic and physical evidence for plasmid control of Shigella sonnei form I cell surface antigen.

Virulent Shigella sonnei synthesize a surface antigen (form I) which appears to be one of several requirements needed for this host to invade epithelial cells. Upon restreaking on agar media, form I cells readily and irreversibly generate form II cells that lack the form I antigen. All form II cells are avirulent. Plasmid deoxyribonucleic acid of form I and II cells of four different S. sonnei isolates, obtained from different areas of the world, was analyzed by agarose gel electrophoresis. A large plasmid (approximately 120 megadaltons in three of the strains) that is present in form I cells was always absent from form II derivatives. Attempts to transfer conjugally only this large plasmid from form I to genetically marked form II cells were unsuccessful. However, a composite molecule, apparently formed by recombination between the large form I plasmid and a self-transmissible plasmid, was found to transfer the form I trait. Transconjugant S. sonnei strains acquiring the form I antigen could retransfer this trait to S. sonnei, Shigella flexneri, or Salmonella typhi. These preliminary findings demonstrate that S. sonnei form I antigen synthesis is mediated by a large plasmid which is lost spontaneously at a relatively high frequency from S. sonnei strains.

Antigens, Bacterial↗