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The Brucella abortus - The Journal of Biological .The B. abortus general stress response 3 Oxidative

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The B. abortus general stress response


The Brucella abortus general stress response system regulates chronic mammalian infection, and is controlled by phosphorylation and proteolysis

Hye-Sook Kim1,2 , Clayton C. Caswell3, Robert Foreman2,4, R. Martin Roop II3,

and Sean Crosson1,2,4

1 Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

2 Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Argonne, IL, 60439, USA.

3 Department of Microbiology and Immunology, East Carolina University School of Medicine,

Greenville, NC 27834, USA.

4 The Committee on Microbiology, The University of Chicago, Chicago, IL 60637, USA.

Running title: The B. abortus general stress response

To whom correspondence should be addressed: Sean Crosson, Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E. 57th St. GCIS W138, Chicago, IL 60637, USA.

Tel. (773) 834-1926; Fax (773) 702-0439. E-mail: scrosson@uchicago.edu. Keywords: Brucella, general stress response, PhyR, signaling, proteolysis, sigma Background: Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. Result: The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection, and is regulated by phosphorylation and proteolysis. Conclusion: The B. abortus GSR signaling pathway has multiple layers of post-translational control, and is a determinant of chronic infection. Significance: This study provides new, molecular-level insight into chronic Brucella infection. SUMMARY

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 B. 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 one month post-infection. 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. INTRODUCTION

Bacteria of the genus Brucella are the causative agents of brucellosis, which is among the most common zoonotic infections globally (1). Within the environment of a mammalian host,

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.459305The latest version is at JBC Papers in Press. Published on April 1, 2013 as Manuscript M113.459305

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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The B. abortus general stress response


brucelleae largely inhabit professional and non-professional phagocytic cells (2), where they occupy a Brucella-containing vacuole (3-5). Brucelleae replicate within this vacuole and, if left untreated, eventually spread and cause a chronic infection of multiple tissues. Even after antibiotic treatment the rate of relapse is high (6,7), and in a minority of patients chronic brucellosis persists for years despite multiple courses of treatment (8).

The capacity of Brucella spp. to survive the harsh environmental stressors encountered en route to, and within, their replicative niche, including reactive oxygen species, acidic pH, and nutrient deprivation is a key virulence determinant (reviewed in refs. (2,9)). In this study, we present a genetic and molecular analysis of the Brucella abortus general stress response (GSR) system, which integrates features of two-component signal transduction (TCS) and extracytoplasmic function (ECF) sigma () transcriptional regulation (10,11). The central regulator in this pathway is the anti-anti- factor PhyR, which is composed of an ECF -like (SL) domain positioned amino-terminally to a two-component receiver domain (Figure 1A). Studies in related -proteobacteria have shown that stress-induced phosphorylation of the PhyR receiver domain promotes binding of its SL domain to the anti- factor, NepR (10,12-15). PhyR binding titrates the NepR anti- factor away from a bona fide ECF factor (16), thereby initiating transcription of genes that mediate bacterial cell survival during stress (Figure 1A).

The core genetic components of this regulatory system including phyR, nepR, and the ECF sigma factor rpoE1 are conserved within the Brucellaceae (11,17). It has been previously reported that a B. melitensis 16M rpoE1 mutant is attenuated in BALB/c mice at 4 weeks post infection (17) and exhibits derepressed flagellar biogenesis (17,18). However, this rpoE1 mutant did not have a survival defect in a hydrogen peroxide disk diffusion assay (17). Herein, we report that the B. abortus 2308 phyR-nepR-rpoE1 system is required for survival under acute oxidative and acid stress in vitro and for maintenance of B. abortus within murine spleens during the chronic phase of infection (i.e. > 1 month post-infection). Additionally, we present a detailed molecular analysis of purified B. abortus GSR proteins that defines unusual features of

PhyR-NepR-E1 protein-protein interactions, including high-affinity binding between unphosphorylated PhyR and NepR and a PhyR~P/NepR complex with an extremely slow dissociation half-time. The cellular concentration of PhyR is regulated post-translationally in B. abortus: PhyR is degraded in the absence of stress, likely by the essential (19) ClpXP protease, and is stabilized when the cell is exposed to either oxidative or acid stress. A B. abortus mutant strain encoding a stable version of PhyR (PhyRVAA/ADD) exhibits derepressed transcription of GSR target genes. Thus, PhyR proteolysis helps to ensure proper control of GSR-dependent transcription in B. abortus. In contrast, we did not observe PhyR protein turnover in the related -proteobacterium, Caulobacter crescentus; this provides evidence for diverse modes of GSR regulation among -proteobacteria. Diversity in GSR regulation likely reflects the broad range of ecological niches inhabited by -proteobacterial species, from dilute freshwater, to plant surfaces, to mammalian phagocytic cells. EXPERIMENTAL PROCEDURES Bacterial strains and culture conditions. All studies in live B. abortus were performed at Biosafety Level 3 (BSL3) as per CDC select agent regulations at the University of Chicago H.T Ricketts Laboratory or East Carolina University School of Medicine. A detailed description of bacterial strains and culture conditions is provided in Supplemental Materials and Methods. Construction of B. abortus mutants and genetic complementation. All primers used in this study are listed in Table S4. A double recombination strategy described previously (20,21) was used to create unmarked B. abortus 2308 mutant strains in which phyR, rpoE1, and nepR-rpoE1 were deleted, or in which the wild-type phyR allele was replaced with phyRVAA/ADD, phyR(D191A), and phyR(D191E) mutant alleles. The phyR and nepR-rpoE1 deletion mutations were complemented by plasmid integration of a copy of phyR or nepR-rpoE1 plus native promoter back into B. abortus chromosome 1. A detailed description of strain and plasmid constructions is provided in Supplemental Materials and Methods.

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The B. abortus general stress response


Oxidative and acid stress assays. Brucella cells grown on Schaedler blood agar (SBA) for 48 hrs were inoculated at either 103 or 106 CFU/ml into Brucella broth and grown at 37C for 24 hr. Final cell density was adjusted to 108 CFU/ml before oxidative and acid stress assays. For PhyR stability experiments, B. abortus strains grown on SBA supplemented with appropriate antibiotics for 48 h were harvested into Gerhardt's minimal medium (GMM) (22) and then adjusted to a cell density of 108 CFU/ml. Cells were subjected to oxidative and acid stresses by the addition of 5 mM H2O2 (final concentration) or addition of acid to adjust the medium to pH 3.9, respectively. Cell survival was measured at 1 or 2 hrs after the H2O2 or low pH exposures in a shaking incubator at 37 C by enumerating CFU/ml. For analysis of protein turnover under stress conditions, 500 g ml-1 of chloramphenicol was added to inhibit protein synthesis 10 min after oxidative or acid stress induction; protein samples were subsequently harvested at 0, 30, 60, and 120 min for immunoblot analysis. Microarray analysis. After 10 min exposure to 5 mM H2O2, B. abortus wild type and rpoE1 cells were harvested, RNA was isolated and hybridized to URMC_Dunman_Brucella_abortus_

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