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

Roop, R. Martin; Price, Michelle L.; Dunn, Bruce E.; Boyle, Stephen M.; Sriranganathan, Nammalwar; Schurig, Gerhardt G.

doi:10.1016/0882-4010(92)90065-v

Cited by 33 publications

(29 citation statements)

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“…5). This could be due to serological cross-reactivity between HSP65 and Brucella GroEL, since there is ϳ60% amino acid sequence homology between these two proteins (29). Similar cross-reactivity has been reported in the literature for other bacterial GroEL proteins and HSP65 (19,32).…”

Section: Discussionsupporting

confidence: 52%

Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

Vemulapalli

Boyle

et al. 2000

Infect Immun

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Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, ␤-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for ␤-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of ␤-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of ␤-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the ␤-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with ␤-galactosidase induced the secretion of gamma interferon (IFN-␥), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-␥, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of ␤-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy against B. abortus 2308 challenge.Brucella abortus is a faculatatively intracellular, gram-negative bacterial pathogen that can cause abortion in pregnant cattle and undulant fever in humans (1). In the infected host, B. abortus multiplies within the endosomes of phagocytic cells by inhibiting the phago-lysosome fusion (13). Rough mutants which do not contain the O antigen (O polysaccharide chain of the smooth lipopolysaccharide) are attenuated in their virulence compared to their smooth virulent parent B. abortus strains (3, 24, 30, 39). Similar to most of the intracellular bacterial infections, cell-mediated immunity (CMI) appears to play a major role in acquired resistance to brucellosis, although antibodies to surface antigens, especially to the O antigen, can confer a certain level of protection against a challenge infection in some host species, such as mice (4, 5, 13). Attenuated, live B. abortus vaccines have been highly successful in protecting against bovine brucellosis. Recent studies demonstrated that B. abortus induces a Th1 type of immune responses...

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Section: Discussionsupporting

confidence: 52%

Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

Vemulapalli

Boyle

et al. 2000

Infect Immun

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“…Our results indicated that the DnaK protein may be recognized by sera from sheep either infected by Brucella species or vaccinated with an attenuated living Brucella strain. Recently, it was reported that another member of the hsp family, the B. abortus hsp60, homologous to E. coli GroEL, was a major antigen during naturally acquired and experimentally induced brucellosis (32). These results may indicate that Brucella cells react by a stress response during their development in the host.…”

Section: Discussionmentioning

confidence: 55%

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli

et al. 1992

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“…Cloning, subcloning and the isolation of plasmid DNA were performed using standard procedures (Sambrook et al ., 1989). Genomic DNA was isolated from B. abortus cells, washed and suspended in phosphate buffered saline (PBS), using a modification of a previously described procedure (Roop et al ., 1992). Cells were extracted with chloroform and then treated with 18 mg ml −1 lysozyme for 20 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The Brucella abortus host factor I (HF‐I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice

Robertson

Roop

1999

Molecular Microbiology

174

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SummaryBrucella abortus is a facultative intracellular pathogen that causes abortion and infertility in domestic animals and a severe debilitating febrile illness in humans. The mechanisms that this highly successful intracellular pathogen uses to adapt to, and survive within, the harsh intracellular environment of the host macrophage are presently unknown. Maintenance of the stationary phase growth state has been proposed to be critical for the virulence of several mammalian pathogens, but analysis of this relationship for the brucellae has not been undertaken. In order to evaluate this relationship, we examined the in vitro and in vivo characteristics of an isogenic hfq mutant constructed from virulent Brucella abortus 2308. In Escherichia coli, the hfq gene product is an RNA-binding protein that participates in the regulation of stationary phase stress resistance, at least partly by enhancing translation of the stationary phase-speci®c sigma factor RpoS. As expected, the Brucella abortus hfq mutant, designated Hfq3, showed increased sensitivity to H 2 O 2, and decreased survival under acidic conditions (pH 4.0), during stationary phase growth compared with 2308. Hfq3 was also less able to withstand prolonged starvation than 2308. The Brucella abortus hfq mutant, unlike its parental strain 2308, fails to replicate in cultured murine macrophages, and is rapidly cleared from the spleens and livers of experimentally infected BALB/c mice. These ®ndings suggest that the Brucella abortus hfq gene product makes an essential contribution to pathogenesis in mice, probably by allowing the brucellae to adapt appropriately to the harsh environmental conditions encountered within the host macrophage.

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

Cited by 33 publications

References 30 publications

Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli

The Brucella abortus host factor I (HF‐I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice

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