PubMed Health⌕ Search

Biomedical subjects

R M Roop

Publications and source records attributed to R M Roop.

At least 19 recordsLinked to original sources
PubMed1999

The siderophore 2,3-dihydroxybenzoic acid is not required for virulence of Brucella abortus in BALB/c mice.

2,3-Dihydroxybenzoic acid (DHBA) is the only siderophore described for Brucella, and previous studies suggested that DHBA might contribute to the capacity of these organisms to persist in host macrophages. Employing an isogenic siderophore mutant (DeltaentC) constructed from virulent Brucella abortus 2308, however, we found that production of DHBA is not required for replication in cultured murine macrophages or for the establishment and maintenance of chronic infection in the BALB/c mouse model.

Animals↗
PubMed1998

Identification and characterization of a 14-kilodalton Brucella abortus protein reactive with antibodies from naturally and experimentally infected hosts and T lymphocytes from experimentally infected BALB/c mice.

A low-molecular-weight recombinant Brucella abortus protein reactive with antibodies from a variety of naturally and experimentally infected hosts and T lymphocytes from experimentally infected mice was identified and given the designation BA14K. The gene encoding BA14K was cloned and characterized, and the predicted amino acid sequence of this immunoreactive protein showed no significant homology with previously described proteins. Sequences homologous to the cloned fragment encoding BA14K were identified by Southern blot analysis of genomic DNAs from representatives of all of the currently recognized Brucella species. Studies employing BA14K should contribute to our efforts to better understand the antigenic specificity of protective immunity to brucellosis.

Amino Acid Sequence↗
PubMed1997

Cloning and nucleotide sequence analysis of a Brucella abortus gene encoding an 18 kDa immunoreactive protein.

A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli. This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans. Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment. DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529. Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera. These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B. abortus strain 544 designated Omp 19.

Amino Acid Sequence↗
PubMed1996

In vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A (htrA) gene from the bovine vaccine strain Brucella abortus S19.

An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H2O2 (P < 0.001) and O(2)- generated by the redox cycling agent paraquat (P < 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 htrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P < 0.01) than the parental strain. Enhanced clearance (P < 0.05) was also observed at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the capacity of this strain to elicit Brucella cellular or humoral immune responses in this experimental host.

Animals↗
PubMed1996

Behaviour of a high-temperature-requirement A (HtrA) deletion mutant of Brucella abortus in goats.

Previous studies have shown that high-temperature-requirement A (HtrA) mutants of Brucella abortus are more sensitive to oxidative killing in vitro, are less able to survive in cultured murine macrophages and are attenuated in BALB/c mice. To measure the effect of an HtrA mutation on the virulence of B abortus in ruminants, pregnant goats in late gestation were exposed to infection by the conjunctival route with B abortus 2308 or an isogenic htrA mutant, PHE1. Infection with either 2308 or PHE1 resulted in abortion, but the serological responses to infection were consistent with 2308 but variable with PHE1. Strain 2308 was recovered post mortem both from aborted fetuses and infected dams, whereas PHE1 was recovered from neither. Nevertheless, short term studies revealed that PHE1 could be recovered from infected goats for up to two weeks after infection, suggesting that although the HtrA mutation may change the colonising ability of B abortus, the virulence of the mutant in pregnant goats is not reduced.

Abortion, Veterinary↗
PubMed1996

The HtrA stress response protease contributes to resistance of Brucella abortus to killing by murine phagocytes.

Compared with virulent Brucella abortus 2308, the isogenic htrA mutant PHE1 shows decreased resistance to killing by cultured murine neutrophils and macrophages and significant attenuation during the early stages of infection in the BALB/c mouse model. These findings further define the contributions of the htrA gene product to the pathogenesis of B. abortus infections.

Animals↗
PubMed1995

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

Bacteria↗
PubMed1995

In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species.

Previous studies have shown that the broad-host-range plasmid pBBR1MCS can be used for genetic complementation in Brucella abortus. To extend these observations, the in vivo and in vitro stability of pBBR1MCS was evaluated in the six currently recognized species of the genus Brucella. pBBR1MCS was readily introduced into all of the strains tested by electroporation and was stably maintained in broth cultures without antibiotic selection during five serial passages over a 10-day period. Furthermore, isolates of all six Brucella strains containing pBBR1MCS obtained from the spleens of BALB/c mice 1 week postinfection maintained the plasmid. Although pBBR1MCS maintains the mobilization locus present in the parental plasmid pBBR1CM, attempts to detect transfer of pBBR1MCS between Brucella strains by conjugation were unsuccessful. These results demonstrate the in vitro and in vivo stability of pBBR1MCS in Brucella spp. and reinforce the usefulness of this cloning vector for the genetic analysis of these organisms.

Animals↗
PubMed1995

Selective humoral immune response of Balb/C mice to Brucella abortus proteins expressed by vaccinia virus recombinants.

Genes encoding Brucella abortus Cu/Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54. Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the beta-gal reporter gene but no B. abortus DNA were also developed. Recombinants were analyzed in Western blotting using a polyclonal B. abortus immune serum. vUBSOD expressed a protein of apparent molecular weight of 28 kDa, composed of the 20 kDa B. abortus Cu/Zn-SOD and a protein approximately 8 kDa encoded by a portion of the vaccinia virus TK gene. vSB54 expressed a 54 kDa protein corresponding to the 54 kDa HtrA homologue. Recombinants vUSV-1 and vSC11 did not express B. abortus proteins. Groups of mice were inoculated intraperitoneally with 10(7) TCID50 of 1 of the 4 different recombinant vaccinia viruses and 5 weeks later their sera were analyzed for antibodies against vaccinia virus and B. abortus proteins. Each group of mice responded with antibodies to vaccinia virus. Sera of vSB54-inoculated mice recognized the 54 kDa HtrA homologue. vUBSOD did not induce a humoral immune response. These results represent the first report on the expression of B. abortus proteins by vaccinia virus recombinants and the first demonstrated immune response against a B. abortus protein expressed by such a recombinant.

Animals↗
PubMed1994

Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant.

In order to evaluate the biological function of the Brucella abortus high-temperature-requirement A (HtrA) stress response protein homolog, the majority of the htrA gene was deleted from the chromosome of B. abortus 2308 via gene replacement. In contrast to the parental strain, the resulting htrA deletion mutant, designated PHE1, failed to grow on solid medium at 40 degrees C and demonstrated increased sensitivity to killing by H2O2 and O2- in disk sensitivity assays. BALB/c mice were infected with strains 2308 and PHE1 to assess the effect of the htrA mutation on virulence, and significantly fewer brucellae were recovered from the spleens of mice infected with PHE1 than from those of mice infected with 2308 at 1 week postinfection. Genetic complementation studies were performed to confirm the relationship between the htrA mutation and the phenotype observed for PHE1. Plasmid pRIE1 was constructed by inserting a 1.9-kb EcoRI fragment encoding the B. abortus htrA gene into the broad-host-range plasmid pBBR1MCS. Introduction of pRIE1 into PHE1 relieved the temperature- and H2O2-sensitive phenotypes of this mutant in vitro, and PHE1(pRIE1) colonized the spleens of BALB/c mice at levels equivalent to those of the parental 2308 strain at 1 week postinfection. These results support our previous proposal that the B. abortus htrA gene product functions as a stress response protein and further suggest that this protein contributes to virulence. These studies also demonstrate the utility of the broad-host-range plasmid pBBR1MCS for genetic complementation studies in Brucella spp., establishing a key reagent for more detailed genetic analysis of this important zoonotic pathogen.

Animals↗
PubMed1994

Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog.

An 11-kb fragment of Brucella abortus genomic DNA cloned into the BamHI site of pUC9 expressed a 60-kDa protein in Escherichia coli DH5-alpha. Antibodies reactive with this 60-kDa protein were detected by Western blot (immunoblot) analysis in sera from mice, cattle, and goats experimentally infected with B. abortus, in sera from mice experimentally infected with Brucella melitensis, and in serum from a dog experimentally infected with Brucella canis. Similar results were seen with sera obtained from cattle and dogs with naturally acquired brucellosis. The gene encoding the 60-kDa Brucella protein was localized to a 2-kb EcoRI fragment which was also reactive in Southern blots with genomic DNA from other strains of B. abortus as well as with genomic DNA from B. melitensis and B. canis. Nucleotide sequence analysis of the cloned EcoRI fragment revealed an open reading frame encoding a protein with a predicted molecular mass of 51,847 Da and an isoelectric point of 5.15. Comparison of the deduced amino acid sequence of the immunoreactive Brucella protein with the SWISS-PROT protein sequence data base revealed that it shares > 40% amino acid sequence identity with the E. coli and Salmonella typhimurium HtrA stress response proteins. Computer-assisted analysis of this amino acid sequence also predicted that the putative Brucella HtrA homolog contains an export signal sequence and a serine protease active site, two structural features characteristic of previously described HtrA proteins. A potential sigma E type heat shock promoter sequence was detected upstream of the cloned Brucella htrA gene, and Northern (RNA) blot analysis demonstrated that exposure of B. abortus 2308 to heat shock conditions resulted in a transient elevation of htrA transcription. These results strongly suggest that the immunoreactive 60-kDa Brucella protein is a member of the HtrA class of stress response proteins.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp60 protein, BA60K.

A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B. abortus and a dog experimentally infected with B. canis. Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K. The gene encoding BA60K was localized within an 18 kb B. abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments. The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57515). Solid phase micro-sequencing of BA60K eluted from two-dimensional polyacrylamide gels confirmed the predicted amino acid sequence. Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67.9% identity with the GroEL protein of E. coli, a member of the Hsp60 family of chaperonins. The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein. The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections. It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis.

Amino Acid Sequence↗
PubMed1992

Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.

The gene encoding the Cu/Zn superoxide dismutase (SOD) of Brucella abortus strain 2308 was identified in a Brucella genomic library utilizing a combination of Western blotting and native gel electrophoresis. The Cu/Zn SOD gene was inactivated in vitro by ligation of a kanamycin resistance gene into the open reading frame encoding SOD. The plasmid born construct was introduced back into B. abortus by electroporation. Replacement of the wild-type Cu/Zn SOD by recombination was demonstrated by showing that both the KnR gene and the Cu/Zn SOD gene hybridized to the same band in a Southern analysis of genomic DNA. In addition, KnR strains were deficient in Cu/Zn SOD activity as assessed by lack of Cu/Zn SOD activity on a native gel and by lack of reactivity with specific serum in a Western analysis. Either strain 2308 or the Cu/Zn SOD deficient mutant injected intraperitoneally into BALB/c mice, exhibited no differences in their ability to colonize the spleen at 7 and 28 days post-inoculation. Thus, the inability to produce Cu/Zn SOD by B. abortus does not significantly impair its virulence in mice.

Animals↗
PubMed1992

Identification and purification of a cpn60 heat shock protein homolog from Helicobacter pylori.

Helicobacter pylori is associated with gastritis and peptic ulcer disease in humans. We have identified a homolog of the chaperonin cpn60 family of heat shock proteins in H. pylori, referred to as Hp54K. Hp54K, purified from water-extractable H. pylori proteins, migrated as a single band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its native molecular mass was 740 kDa; thus, Hp54K apparently comprises a 14-mer. The N-terminal 33 residues of Hp54K exhibited 60.6, 57.6, 54.5, 54.5, 51.5, and 51.5% identity with corresponding sequences in the following cpn60 homologs: HtpB (Legionella pneumophila), P1 (human mitochondria), GroEL (Escherichia coli), BA60K (Brucella abortus), HypB (Chlamydia trachomatis), and the 65-kDa immunodominant protein of Mycobacterium bovis BCG, respectively. Hp54K was the only protein recognized in whole-cell preparations of H. pylori by immunoblotting using monospecific antisera against cpn60 homologs from L. pneumophila, E. coli, C. trachomatis, and M. bovis BCG. Antiserum against Hp54K recognized proteins with molecular masses of 50 to 60 kDa in a large number of gram-negative bacteria, consistent with the known highly conserved nature of cpn60 proteins. Hp54K is a major protein and is immunogenic in humans infected with H. pylori. Thus, Hp54K shares many similarities with known cpn60 homologs. On the basis of the proposed role of other cpn60 proteins in induction of chronic inflammation, immune cross-reactivity between Hp54K and gastric tissue may provide an important link between H. pylori infection and gastritis.

Amino Acid Sequence↗
PubMed1991

Experimental infection of goat fetuses in utero with a stable, rough mutant of Brucella abortus.

Fetuses of goats in their last trimester of pregnancy were experimentally infected with Brucella abortus strain RB51, a stable rough mutant deficient in the perosamine O-chain content of its lipopolysaccharide. RB51 maintained its rough phenotype in vivo and did not induce abortion. Infection with RB51 resulted in the production of significant levels of IgG type antibodies specific for B abortus cellular antigens distinct from the perosamine O-chain. These findings suggest that strain RB51 will be useful in the pregnant goat for studying the role of brucella antigens other than the lipopolysaccharide O-chain in the immune response to brucellosis.

Animals↗
PubMed1991

Biological properties of RB51; a stable rough strain of Brucella abortus.

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.

Animals↗
PubMed1991

Serum sensitivity and lipopolysaccharide characteristics in Bordetella bronchiseptica, B. pertussis and B. parapertussis.

The viability of four strains of Bordetella bronchiseptica, two strains of B. pertussis and one strain of B. parapertussis exposed to hyperimmune and pre-colostrum porcine serum was examined. Viable cell numbers (cfu/ml) of the B. pertussis strains and a rough strain of B. bronchiseptica (CSU-P-1) decreased by 99% and 99.99%, respectively, after exposure for 1 h to porcine hyperimmune serum. In contrast, smooth B. bronchiseptica strains and the B. parapertussis strain showed no significant decrease in viable cell numbers after the same treatment. B. bronchiseptica strain CSU-P-1 also showed a 99% decrease in viable cell numbers after exposure to pre-colostrum porcine serum for 1 h whereas the other strains tested showed no decrease in viable numbers under the same conditions. Heating the hyperimmune and pre-colostrum serum at 56 degrees C for 30 min resulted in the loss of bactericidal activity suggesting the involvement of complement in both systems. Analysis of silver-stained SDS-PAGE profiles of lipopolysaccharide (LPS) extracted from the bacterial cells indicated that the smooth strains of B. bronchiseptica and the B. parapertussis strain possessed high mol. wt O-side chain-like material, whereas the B. pertussis strains and B. bronchiseptica strain CSU-P-1 did not. Gel filtration of acid-hydrolysed LPS samples indicated two distinct carbohydrate peaks for the strains with high mol. wt O-side chain-like material, whereas the other strains each yielded one distinct peak. Western-blot analysis indicated a positive reaction for anti-B. bronchiseptica antibodies to the high mol. wt O-side chain-like material of all serum-resistant strains used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗