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

ABSTRACT: 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.

SUBMITTER: Roop RM

PROVIDER: S-EPMC186216 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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

Roop R M RM Fletcher T W TW Sriranganathan N M NM Boyle S M SM Schurig G G GG

Infection and immunity 19940301 3

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 ...[more]

PMID: 8112833

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Project description:The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (?wrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ?wrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family.Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

Project description:Virulence of pathogenic bacteria is often determined by their ability to adapt to stress.The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis.The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection.This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the ?-proteobacterial general stress response (GSR) system, PhyR and ?(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ?phyR and ?rpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ?phyR and ?rpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Dataset Information

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

Publications

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

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