Computerization of data bases for genetic stock collections.
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Animals↗
Biomedical subjects
R Curtiss
Publications and source records attributed to R Curtiss.
Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium.
Plasmid DNA of 68 strains of Salmonella that belonged to 18 serovars and exhibited 48 different plasmid profiles was examined for hybridization with a 32P-labelled DNA probe which consisted of a 3750 base pairs (bp) HindIII-HindIII fragment derived from the virulence region of the 60 megadalton (Mdal) plasmid of Salmonella typhimurium. The 32 Mdal plasmid of S. cholerae-suis, the 50 Mdal plasmid of S. dublin, the 36 Mdal plasmid of S. enteritidis, the 60 Mdal plasmid of S. gallinarum, the 60 Mdal plasmid of S. pullorum, and the 60 Mdal plasmid of S. typhimurium, plasmids that have been associated with virulence, all hybridized with the probe. Digestion of plasmid DNA of these strains with PvuII and hybridization with the probe revealed that the plasmids of strains of all six serovars contained fragments of approximately 2520 and 1520 bp that hybridized with the probe. Similarly, hybridization with BglI digests of DNA of the virulence-associated plasmids of strains of these six serovars showed that all six plasmids contained a fragment of approximately 3690 bp that hybridized with the probe. No other plasmids of these strains nor any plasmids of 12 other Salmonella serovars hybridized with the probe. Chromosomal DNA did not hybridize with the probe. The 60 Mdal plasmids of S. gallinarum and S. pullorum showed similar digestion patterns with restriction endonucleases BglI, BglII and PvuII.
Animals↗
Nucleotide sequence of the gtfA gene from S. mutans GS-5.
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Amino Acid Sequence↗
Avirulent Salmonella typhimurium delta cya delta crp oral vaccine strains expressing a streptococcal colonization and virulence antigen.
Salmonella typhimurium SR-11 strains lacking adenylate cyclase and the cyclic AMP receptor protein (CRP) due to deletion (delta) mutations in the cya and crp genes, respectively, are avirulent for mice and induce high level protective immunity against subsequent challenge with wild-type virulent S. typhimurium SR-11 cells. The avirulence of these delta cya delta crp mutants has been enhanced by elimination of the 100 kb virulence plasmid pStSR100 without impairing immunogenicity. The present report confirms the avirulence and immunogenicity of these mutant strains, demonstrates that immunization of both four- and eight-week-old mice has no adverse effect on weight gain, and that immunity lasts at least ninety days following initial immunization. Avirulent S. typhimurium strains have been endowed with the ability to produce several streptococcal colonization and virulence antigens for the purpose of constructing recombinant bivalent oral vaccine strains. Important antigenic determinants of the Streptococcus sobrinus surface protein antigen A (SpaA), presumed to be a critical colonization antigen of S. sobrinus, are expressed at high level by the delta cya delta crp S. typhimurium strains. The recombinant vaccine strains are stable in vitro and in animals (for a period of at least eight days) where they localize to the gut-associated lymphoid tissue (GALT).
Administration, Oral↗
Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.
We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.
Animals↗
Analysis of the virulence of Streptococcus mutans serotype c gtfA mutants in the rat model system.
The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton protein which catalyzes the synthesis of a small glucan (1.5 kilodaltons) from sucrose (J.P. Robeson, R.G. Barletta, and R. Curtiss III, J. Bacteriol. 153:211-221, 1983). To investigate the role of the GtfA enzyme in virulence, we constructed S. mutans gtfA mutants from three cariogenic serotype c strains. A plasmid that carried an erythromycin resistance determinant and an internal fragment of the gtfA gene but that was unable to replicate in streptococci was used to transform S. mutans. The erythromycin-resistant transformants carried a partial duplication of the internal gtfA fragment, because of the integration of plasmid sequences within the S. mutans gtfA gene, which also resulted in the inactivation of the gtfA gene. This was verified by Southern DNA hybridization analysis and Western blot studies of cellular protein extracts of the mutant strains with GtfA antiserum. Mutants were fully virulent in both germfree and conventional rats. These results do not rule out the involvement of the GtfA protein in virulence. Pucci and Macrina (M.J. Pucci and F.L. Macrina, Infect. Immun. 54:77-84, 1986) have suggested that the GtfA enzyme synthesizes a primer for water-insoluble glucans. Another S. mutans protein, presumably a glucosyltransferase, may have a similar function and, thus, may obscure the relevance of the GtfA enzyme in pathogenesis.
Animals↗
Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella avium.
We examined Bordetella avium for virulence factors common to Bordetella pertussis, including pertussis toxin, filamentous hemagglutinin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin. B. avium produced a dermonecrotic toxin and a tracheal cytotoxin. The dermonecrotic toxin of B. avium is a 155,000-molecular-weight, heat-labile protein which was lethal for mice, guinea pigs, young chickens, and turkey poults and produced dermonecrosis when injected intradermally into guinea pigs, chickens, and turkey poults. High-pressure liquid chromatography of B. avium culture supernatant fluid revealed the presence of a tracheal cytotoxin chemically identical to that produced by B. pertussis. B. avium isolates were negative for B. pertussis-like filamentous hemagglutinin and pertussis toxin when assayed with antibody against B. pertussis filamentous hemagglutinin and pertussis toxin. Furthermore, B. avium failed to induce the clustered CHO cell morphology characteristic of pertussis toxin. Adenylate cyclase assays indicated that B. avium does not produce an extracytoplasmic adenylate cyclase, even after passage through embryonated turkey eggs. Since production of virulence proteins by B. pertussis is regulated by growth in media containing nicotinamide or MgSO4 or by growth at reduced temperatures, we determined the effect of these supplements and growth conditions on production of dermonecrotic toxin by B. avium. Production of dermonecrotic toxin in B. avium was not altered by growth in media containing 100 microM FeSO4 or 500 micrograms of nicotinamide per ml or by growth at 25 or 42 degrees C, but production was significantly decreased by growth in media containing 20 mM MgSO4 and slightly reduced by growth in media containing 500 micrograms of nicotinic acid per ml. These studies revealed that B. avium is similar to B. pertussis in that both species produce a dermonecrotic toxin and a tracheal cytotoxin and production of dermonecrotic toxin is regulated by nicotinamide and MgSO4. The presence of dermonecrotic toxin and tracheal cytotoxin in all Bordetella species indicates that these products may be important virulence factors in bordetellosis.
Adenylate Cyclase Toxin↗
Nucleotide sequence of the asd gene of Streptococcus mutans. Identification of the promoter region and evidence for attenuator-like sequences preceding the structural gene.
The complete nucleotide sequence of the asd gene of Streptococcus mutans encoding aspartate beta-semialdehyde dehydrogenase (EC 1.2.1.11), an enzyme comprised of 357 amino acids, having an Mr of 38,897 and active in the biosynthetic pathway of lysine, threonine, methionine, diaminopimelic acid, and isoleucine, has been determined. In addition we report the 276 nucleotides upstream of the structural gene which contain a highly efficient promoter identified by both RNA polymerase binding and in vitro transcription analysis. A leader transcript which terminates at a fixed point immediately preceding the asd promoter region was identified in the DNA sequence and confirmed by in vitro transcription analysis as well. The close proximity of this transcript and its p-independent transcriptional terminator to the asd coding sequence suggests involvement in a mechanism of regulation. Message stability experiments indicate the half-life of asd specific messages to be comparable to that of Escherichia coli messages. Conditions of varying concentrations of lysine, threonine, and methionine exert no apparent control over expression of the S. mutans asd gene in Escherichia coli suggesting the requirement of an accessory regulatory element specific for the S. mutans asd gene.
Aspartate-Semialdehyde Dehydrogenase↗
Plasmid-associated virulence of Salmonella typhimurium.
We investigated the role of the 100-kilobase (kb) plasmid of Salmonella typhimurium in the virulence of this organism for mice. Three strains, LT2-Z, SR-11, and SL1344, which possessed 100-kb plasmids with identical restriction enzyme digestion profiles, were cured of their respective 100-kb plasmids after Tnmini-tet was used to label plasmids. Curing wild-type virulent strains SR-11 and SL1344 raised peroral 50% lethal doses from 3 x 10(5) and 6 x 10(4) CFU, respectively, to greater than 10(8) CFU. Both wild-type strains had intraperitoneal 50% lethal doses of less than 50 CFU, whereas the intraperitoneal 50% lethal doses for cured SR-11 and SL1344 were less than 50 and 400 CFU, respectively. Reintroduction of the Tnmini-tet-labeled, 100-kb plasmid restored wild-type virulence. Invasion from Peyer's patches to mesenteric lymph nodes and spleens after peroral inoculation was the stage of pathogenesis most affected by curing S. typhimurium of the 100-kb plasmid. Wild-type S. typhimurium replicated in spleens of mice inoculated intravenously to a greater extent than did plasmid-cured derivatives. Wild-type and cured strains equally adhered to and invaded Henle-407, HEp-2, and CHO cells; furthermore, the presence of the 100-kb plasmid was not necessary for replication of S. typhimurium within CHO cells. The 100-kb plasmid had no effect on phagocytosis and killing of S. typhimurium by murine peritoneal macrophages in vitro and in vivo. Similarly, wild-type and plasmid-cured strains were resistant to killing by 90% normal human, rabbit, and guinea pig sera. All wild-type and plasmid-cured S. typhimurium strains possessed complete lipopolysaccharide, as determined by silver staining solubilized cells in sodium dodecyl sulfate-polyacrylamide gels. We have confirmed the role of the 100-kb plasmid of S. typhimurium in virulence, primarily in invasion to mesenteric lymph nodes and spleens after peroral inoculation of mice. Involvement of the 100-kb plasmid in infection of mesenteric lymph nodes and spleens suggests a role for the plasmid in the complex interaction of S. typhimurium with cells of the reticuloendothelial system.
Animals↗
Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic.
Salmonella typhimurium SR-11 mutants with cya::Tn10 or crp::Tn10 mutations were found to be avirulent and immunogenic for BALB/c mice. Fusaric acid-resistant derivatives with deletions of the Tn10 and adjacent DNA sequences were constructed in S. typhimurium SR-11 strains with or without the virulence plasmid pStSR100. These delta cya delta crp strains grew more slowly than wild-type strains. They possessed wild-type ability to attach to, invade, and persist in gut-associated lymphoid tissue for up to a week but exhibited a diminished ability to reach mesenteric lymph nodes and the spleen. Mice 4 to 8 weeks old were resistant to oral infection with 10(9) cells of several different delta cya and delta cya delta crp strains (the equivalent to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11) and 30 days after immunization became resistant to oral challenge with 10(3) to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11.
Adenylyl Cyclases↗
Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene.
The presence of proteases in culture supernatant fluids and on the cell surface of Streptococcus sobrinus and the aggregation of multicomponent enzyme complexes make the isolation and characterization of cell surface proteins difficult. We report a simple purification procedure for dextranase and the cloning of the dextranase structural gene. S. sobrinus culture supernatant fluids were precipitated with 70% ammonium sulfate, and the precipitate was dialyzed against sodium acetate buffer and loaded onto a hemoglobin-Sepharose 4B column connected to a blue dextran-agarose column at 4 degrees C. After being washed with low concentrations of salt, the dextranase and the dextran-binding proteins were eluted with 5 M KI and further purified by gel filtration. Two dextranases (molecular weights, 175,000 and 160,000) were purified and partially characterized. The structural gene for the dextranase of S. sobrinus 6715 strain UAB66, serotype g, was cloned into the cosmid vector, pHC79. Clones were selected for expression of dextranase activity by detection of zones of enzyme-mediated hydrolysis of a blue dextran substrate incorporated into minimal medium agar plates. Release of dextranase was achieved by induction of thermoinducible, excision-defective Escherichia coli K-12 lysogens containing recombinant cosmid molecules of S. sobrinus DNA. Recombinant cosmid molecules were repackaged simultaneously into infectious lambdoid particles. Recombinant clones expressing dextranase activity which varied in size from the high-molecular-weight protein produced by S. sobrinus (i.e., 175,000) to lower-molecular-weight forms expressed by S. sobrinus have been identified and partially characterized.
Bacterial Proteins↗
Binding of laminin to oral and endocarditis strains of viridans streptococci.
Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent studies by us and others have shown that matrix proteins which serve as adhesion proteins for eucaryotic cells may also be recognized by some bacteria. In the present communication, we report that several strains of viridans streptococci are able to bind to laminin. Most strains isolated from blood and heart valves of patients with endocarditis expressed laminin receptors, whereas only a few of the strains isolated from the oral cavity recognized this protein. This observation indicates that laminin binding might be an important factor in the pathogenesis of viridans endocarditis. Laminin binding to two strains (Streptococcus mitis UAB594 and UAB597) isolated from patients with endocarditis was characterized further. The bacterial cells expressed a limited number of laminin receptors (4 X 10(2) to 1 X 10(3) per cell) which bound the protein in a high-affinity interaction (Kd, 40 to 80 nM). This receptor of S. mitis UAB594 was heat labile and could be solubilized from bacteria by brief digestion with trypsin. Solubilized receptors which competed with cell-bound receptors for 125I-laminin could be adsorbed on laminin-Sepharose but not on Sepharose substituted with fibrinogen or fibronectin. Comparison of laminin receptors from S. mitis with those previously described for Streptococcus pyogenes suggest that different sites in the laminin molecule are recognized by the two bacteria and hence that the corresponding receptor molecules are not identical.
Animals↗
Isolation and characterization of mutants of Streptococcus mutans using selective removal of wild-type cells by agglutination with an agglutinin from Persea americana.
Persea americana agglutinin (PAA), a substance known to bind basic proteins and inhibit the sucrose-independent adherence of Streptococcus mutants to saliva = coated hydroxyapatite (Staat et al., 1980) was used to selectively enrich for mutants defective in a variety of cell surface associated virulence characteristics from cultures UAB62 (PS14 Riff, serotype c), UAB66 (6715 Strr Spcr, serotype g) and UAB77 (GS5, serotype c). Following mutagenesis and growth for segregation and phenotypic expression, washed cells of each strain were exposed to PAA overnight at 37 degrees C. Aggregated cells were removed by low-speed centrifugation and cells remaining in the supernatant fluids were concentrated, grown to stationary phase and the enrichment with PAA repeated. Mutants isolated following enrichment were phenotypically diverse and included strains defective in one or more of the following characteristics: adherence to glass in a sucrose-containing medium, aggregation with sucrose, dextran or PAA. dextranase production, colony morphology, cell or chain morphology, fermentation of sorbitol, lactose, galactose, raffinose, melibiose, or fructose, and production of surface protein antigen A (SpaA). The diversity of mutant phenotypes identified along with the observation that PAA could still cause aggregation (with a lower efficiency) of all mutants leads us to infer that the interaction of this agglutinin with proteins on the S. mutans cell surface is relatively nonspecific and that the observed inhibition of S. mutants attachment to saliva-coated hydroxyapatite caused by PAA is not due to a highly specific unique interaction of PAA with the protein(s) responsible for sucrose-independent adherence.
Agglutination↗
Novel oral vaccines: the effectiveness of cloned gene products on inducing secretory immune responses.
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Administration, Oral↗
Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecylsulfate polyacrylamide gel electrophoresis.
A rapid and reproducible method for the assay of individual fractions of the multicomponent dextranase activity of Streptococcus sobrinus after reduction/denaturation and electrophoresis in acrylamide gels is described. Multiple forms of dextranase, possible virulence factors in the formation of dental caries by oral Streptococci (S. mutans, S. sobrinus, S. sanguis, S. cricetus, and S. rattus), have been separated in sodium dodecylsulfate-polyacrylamide gels into which an indicator substrate, blue dextran, has been incorporated, and identified after renaturation to remove the reducing/denaturing agents of the Laemmli buffer system.
Animals↗
Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12.
Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate beta-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the beta-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amount than when expressed from the S. mutans asd promoters. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.
Aspartate-Semialdehyde Dehydrogenase↗
In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.
Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.
ATP-Dependent Proteases↗
Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs.
Transformation (i.e., DNase-sensitive genetic transfer) of strains of Streptococcus mutans representing serotypes c and e was accomplished by using chromosomal DNA from a Rifr Strr Spcr isolate of strain GS5 (UAB525) and a chimeric plasmid, pYA629. Shuttle plasmid pYA629 comprises the S. mutans plasmid pVA318, an inducible erythromycin resistance determinant originally isolated from a group A streptococcal strain, the tetracycline resistance gene and replication region of the Escherichia coli plasmid pBR322, and the promoter region of the S. mutans gene for aspartate beta-semialdehyde dehydrogenase. The strains examined for recipient ability included those known to lack a cryptic plasmid (GS5, UA130, UA159, and MT8148) and those known to contain a widely disseminated 5.8-kilobase cryptic plasmid (LM7, V318, UA101, UA174, and 3098791). The transformation frequencies in GS5 for GS5 chromosomal antibiotic resistance markers were comparable to those reported by others, but UA101, UA130, UA159 and UA174 were transformed with both chromosomal and plasmid markers at much higher efficiencies. In a larger strain survey, strains containing the 5.8-kilobase cryptic plasmid were more frequently transformable with both chromosomal and pYA629 DNAs than were strains lacking this cryptic plasmid. All plasmid-containing strains except LM7 lost their resident cryptic plasmids when transformed with pYA629. LM7 transformed with pYA629 retained pLM7. There are therefore at least two incompatibility groups among S. mutans cryptic plasmids. yPA629 DNA isolated from either E. coli or S. mutans transformed S. mutans with equal efficiency. pYA629 DNA isolated from S. mutans transformed both restriction-deficient and restriction-proficient E. coli recipients. Therefore, the strains of S. mutans used lack a restriction-modification system for pYA629 DNA sequences. S. mutans strains that are readily transformable, display maximal cariogenicity in gnotobiotic rats, and give high scores for in vitro measures of important virulence attributes have been identified to facilitate studies on the genetic basis and control of virulence.
Animals↗