Search PubMed⌕ Search

Biomedical subjects

R Curtiss

Publications and source records attributed to R Curtiss.

At least 91 records · Page 5Linked to original sources
PubMed1991

Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella typhi are deficient for entry into mammalian cells.

Invasion of intestinal epithelial cells is an essential virulence factor of salmonellae. A group of genes, invABC and invD, that allow Salmonella typhimurium to penetrate cultured epithelial cells have previously been characterized (J. E. Galán and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). The distribution of these genes among Salmonella isolates belonging to 37 different species or serovars was investigated by Southern and colony blot hybridization analyses. Regions of high sequence similarity to the invABC genes were present in all Salonella isolates examined, while regions of sequence similarity to the invD gene were present in all but one (S. arizonae) of the isolates tested, with little restriction fragment length polymorphism. Sequences similar to these genes were not detected in strains of Escherichia coli, Yersinia spp., or Shigella spp. invA mutants (unable to express the invABC genes) of several Salmonella species or serovars, including S. typhi, were constructed and examined for their ability to penetrate Henle-407 cells. All mutants were deficient for entry into cultured epithelial cells, indicating that the invABC genes were not only present in these strains but also functional.

Blotting, Southern↗
PubMed1991

Hormonotoxins: abrogation of ribosome inactivating property of gelonin in the disulfide linked ovine luteinizing hormone-gelonin conjugates.

In order to synthesize a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain ribosome-inactivating protein obtained from an Indian plant called Gelonium multiflorum was covalently linked to ovine luteinizing hormone (oLH) by a disulfide bond. Ovine LH-S-S-gelonin conjugates of different molar ratios were subjected to determine the ribosome-inactivating property in a cell-free translation assay using rabbit reticulocyte lysate system. A single amino group modification with N-succinimidyl-3-(2-pyridyldithio)propionate resulted in a loss of 90% protein synthesis inhibition activity. Upon conjugation of gelonin to oLH, the activity was further inhibited ranging from 2.5-6.4%. A 1:1 to 1:1.5 molar ratio (oLH-S-S-gelonin) conjugates showed 2.5-4.6% activity while 1:2.8 to 1:2.2 molar ratio exhibited 5.5-6.4% inhibition ability.

Animals↗
PubMed1990

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains.

The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP. This has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutant Salmonella hosts [Nakayama et al., Biotechnology 6 (1988) 693-697]. We have cloned the asd gene from S. typhimurium, characterized the gene product and used this gene to construct Asd+ expression cloning vectors. In addition we have constructed an asd cassette and a transposon derived from Tn5 that allow the rapid modification of other vectors for use with delta asd vaccine strains of S. typhimurium adding versatility to the Asd+ vector/delta asd host system of plasmid maintenance.

Amino Acid Sequence↗
PubMed1990

Antigen delivery systems for analysing host immune responses and for vaccine development.

Overall, the meeting was timely and worthwhile. It is evident that there is a need for comparative studies to establish a firm foundation for future work. This is particularly important since it appears that the responses observed are influenced by the particular strain used for attenuation, the means of attenuation, the antigen-presentation system employed and the animal most immunized. In this last regard, one must recognize that most work to date has been with inbred strains of mice which differ from the genetic diversity of most hosts that are the intended beneficiaries of the immunization strategies being developed.

Animals↗
PubMed1990

Occurrence of secondary attenuating mutations in avirulent Salmonella typhimurium vaccine strains.

The attenuating delta aroA554 mutation in Salmonella typhimurium strain SL3261 was complemented in vitro by selecting for AroA+ recombinant DNA clones. SL3261 containing cloned aroA+ genes did not require exogenous phenylalanine, tryptophan, tryosine, p-aminobenzoic acid, or dihydroxybenzoic acid for growth in defined media. Cloned aroA+ genes did not restore wild-type virulence to SL3261, however, in a murine typhoid model. The delta aroA554 mutation was transduced into S. typhimurium strain SR-11, a mouse-virulent strain recently passaged in mice. The SR-11 delta aroA554 mutant was highly attenuated for mice challenged parenterally. The same cloned aroA+ genes isolated in SL3261 restored the virulence of the SR-11 delta aroA554 mutant to that of wild-type SR-11. These results suggest that while the delta aroA554 allele remains effective in reducing S. typhimurium virulence, laboratory passage of attenuated vaccine strains may lead to the accumulation of additional attenuating defects.

Animals↗
PubMed1990

Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice.

Nonmotile flagellated (mot) and nonflagellated (fla) mutants of Salmonella typhimurium LT-2 were isolated from a collection of mutants with random Tn10-insertion mutations. Both classes of mutants were resistant to infection by the flagellotropic bacteriophage chi. The nonflagellated (fla::Tn10) mutants did not react with H antigen-specific antisera and did not possess flagella when examined by electron microscopy, and sheared-cell extracts were devoid of flagellin. The nonmotile (mot::Tn10) mutants reacted with H-specific antisera and expressed paralyzed flagella that were indistinguishable from wild-type flagella. The Tn10 insertions in strain LT-2 were mapped to loci in regions II (flh and mot) and III (fli) of the flagellar genes, and the mutations were transduced into the mouse-virulent S. typhimurium strains SR-11 and SL1344. Lack of motility reduced the ability of S. typhimurium to invade Henle cells in vitro, yet the virulence in mice of the nonmotile mutants of SR-11 and SL1344 was unaffected by the inactivity or loss of flagella. Wild-type SR-11 had a 50% lethal dose (LD50) in BALB/c mice following oral (p.o.) challenge of 2.4 x 10(4) CFU. The p.o. LD50 of the SR-11 fli-8007::Tn10 mutant was 4.5 x 10(4) CFU. The mot-8008::Tn10 mutation in SR-11 conferred paralyzed flagella and increased the p.o. LD50 in mice to 2.2 x 10(5) CFU, but this was not statistically significant. A similar increase in the p.o. LD50 was observed when the SL1344 mot-8008::Tn10 mutant was tested in mice. Wild-type SR-11 and the isogenic nonflagellated and nonmotile mutants were equally virulent in mice challenged via intraperitoneal injection.

Animals↗
PubMed1990

Regulation of expression of Streptococcus mutans genes important to virulence.

Studies were initiated to investigate the regulation of Streptococcus mutans genes which are believed to be important to virulence. Operon fusions were constructed between S. mutans gene regulatory regions and a promoterless chloramphenicol acetyltransferase gene (cat) found on the plasmid pMH109. Specifically, fusions were generated between cat and the S. mutans genes encoding fructosyltransferase (ftf) and the glucosyltransferase B/C (gtfB/C) operon. Constructs were confirmed by restriction enzyme analysis, and the fusions were subcloned into the integration vehicle pVA891. Following generation of multimeric DNA, recombinant plasmids were introduced into the s. mutans genome by Campbell-type insertion, resulting in single-copy operon fusions. Chloramphenicol acetyltransferase specific activities were used to monitor the expression of the S. mutans gtfB/C operon and ftf determinants. The expression of these genes is increased by the presence of sucrose and is followed by a rapid decline in expression over time. Additionally, expression of the gtfB/C operon is increased in S. mutans cells bound to artificial tooth pellicles.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling.

The ability to enter intestinal epithelial cells is an essential virulence factor of salmonellae. We have previously cloned a group of genes (invA, B, C, and D) that allow S. typhimurium to penetrate tissue culture cells (J. E. Galán and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). Transcriptional and translational cat and phoA fusions to invA (the proximal gene in the invABC operon) were constructed, and their expression was studied by measuring the levels of alkaline phosphatase or chloramphenicol acetyltransferase activity in mutants grown under different conditions. It was found that when strains containing the fusions were grown on media with high osmolarity, a condition known to increase DNA superhelicity, the level of invA transcription was approximately eightfold higher than that in strains grown on media with low osmolarity. The osmoinducibility of invA was independent of ompR, which controls the osmoinducibility of other genes. Strains grown in high-osmolarity media in the presence of subinhibitory concentrations of gyrase inhibitors (novobiocin or coumermycin A1), which reduce the level of DNA supercoiling, showed reduced expression of invA. Nevertheless, invA was poorly expressed in topA mutants of S. typhimurium, which have increased DNA superhelicity. In all cases, the differential expression of the invasion genes was correlated with the ability of S. typhimurium to penetrate tissue culture cells. These results taken together indicate that expression of S. typhimurium invasion genes is affected by changes in DNA supercoiling and suggest that this may represent a way in which this organism regulates the expression of these genes.

Animals↗
PubMed1990

Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group.

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

Animals↗
PubMed1990

Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing.

The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence plasmids specifically. Colony blots revealed that the par and repB regions of the S. typhimurium virulence plasmid hybridized with 80% of the isolates of S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum, S. pullorum, and S. typhimurium, while the repC region was not detected in any of the isolates of S. dublin, S. gallinarum, or S. pullorum. None of these maintenance regions was found in any of the 30 additional serovars tested. The large plasmids of those serovars that hybridized with par were labeled with a Kmr insert within parA via P22HTint or P1L4 transduction, which destabilized the plasmids and allowed the rapid isolation of plasmid-free derivatives for all of the serovars, except for S. dublin, which exhibited weak homology with par.

Base Sequence↗
PubMed1990

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.

Amino Acid Sequence↗
PubMed1990

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.

Cloning, Molecular↗
PubMed1989

Selective delivery of antigens by recombinant bacteria.

The means to attenuate Salmonella and to endow such avirulent strains with the ability to express colonization and virulence antigens from other pathogens has achieved considerable progress during the past several years. One can therefore begin to design and construct strains with specificity to a given animal host and to express in a defined way specific colonization and virulence antigens in a manner to stimulate long-lasting immunity to the Salmonella and to the pathogen supplying the genetic information for the colonization and virulence antigens. Since most pathogens colonize on or invade through mucosal surfaces, the use of recombinant bivalent Salmonella vaccine strains to stimulate a mucosal immune response would induce the development of a first line of defense against a diversity of pathogens. Mucosal immunity should therefore reduce contagious spread of many pathogens since the dose to overcome the mucosal immune barrier would be increased to result in a diminished likelihood of infection. The fact that the recombinant Salmonella vaccine strains also induce humoral and cellular immune responses justifies their use for induction of long-lasting immunity. Although considerable progress has been made in targeting antigens to the GALT by use of avirulent Salmonella, a similar strategy for delivery of antigens to the BALT has yet to be discovered and developed. In addition to constituting a system for induction of immunity against a diversity of pathogens, the recombinant avirulent Salmonella system should provide a means to explore parameters of the mucosal immune response. This would include investigation of the location and duration of memory, the age dependence of induction of mucosal immunity, and the means for the possible induction of oral tolerance with regard to either the mucosal or humoral response to an antigen expressed by the recombinant Salmonella. It is also possible to contemplate using the avirulent Salmonella to target expression of various modulators of the immune system such as interleukin-2 and interferon-gamma to the GALT and thus further enhance the immune response. Lastly, one can introduce into avirulent Salmonella strains genes for putative colonization antigens in order to investigate whether induction of an immune response against the putative colonization antigen does or does not interfere with infection. This system, therefore, permits another means to analyze the relative importance of various bacterial surface attributes in conferring pathogenicity to the microbe.

Animals↗
PubMed1989

Liposomes as oral adjuvants.

In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for mucosal responses when used as oral vaccines. Evidence was provided that oral administration of antigen in liposomes resulted in an augmented mucosal response, compared to the response obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were further enhanced by the use of lipophilic MDP in the antigen/liposome vaccines. In order to better understand the properties of liposomes important for their functional activities, a rapid and reproducible method employing flow cytometry was described which can be conveniently used for the characterization of liposome preparations. Finally, evidence was presented which further supports the potential of recombinant DNA techniques in developing effective and safe oral vaccines against a variety of infectious diseases.

Adjuvants, Immunologic↗
PubMed1989

Virulence and vaccine potential of phoP mutants of Salmonella typhimurium.

We have constructed Salmonella typhimurium phoP mutants and found them to be avirulent and able to induce a protective immune response. BALB/c mice survived challenge with phoP derivatives of the highly virulent S. typhimurium strains SR-11 and SL1344 when inoculated intraperitoneally and per oral with doses equivalent to 10(4) 50% lethal doses (LD50) of the parent virulent strains. The avirulent mutants were able to establish an infection of the Peyer's patches of orally infected animals for up to 10 days after inoculation but were very inefficient at reaching the spleens. Despite the low level of infectivity of these mutants, immunized animals developed a delayed-type hypersensitivity (DTH) response to Salmonella antigens and resisted challenge with up to 10(4) LD50 of the virulent parent strain 30 days after immunization.

Animals↗
PubMed1989

Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells.

Invasion of the intestinal epithelium is thought to be an important step in the pathogenesis of Salmonella infections. Using an in vitro system, we have isolated a genetic locus, inv, that confers to a noninvasive strain of Salmonella typhimurium the ability to penetrate tissue culture cells. Highly virulent S. typhimurium strains carrying inv mutations were defective for entry into Henle-407 cells while remaining unaffected in their ability to attach to cultured cells. When administered perorally to BALB/c mice, inv mutants of S. typhimurium had higher 50% lethal doses (LD50) than their wild-type parent strains. To the contrary, there were no differences in the observed LD50 when strains were administered intraperitoneally. In addition, inv mutants presented decreased ability to colonize the Peyer's patches, the small intestinal wall, and the spleen when administered perorally, although when administered intraperitoneally, they showed no difference in their ability to colonize the spleen compared to the wild-type parent strain.

Animals↗
PubMed1989

Impairment of melibiose utilization in Streptococcus mutans serotype c gtfA mutants.

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gtfA mutant strains synthesized less alpha-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of alpha-galactosidase in the mutant strains. We hypothesize that melibiose use by S. mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved in melibiose transport or hydrolysis.

Disaccharides↗