Characterization of Brucella suis clpB and clpAB Mutants and Participation of the Genes in Stress Responses

Abstract

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42°C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.Brucella suis is a gram-negative, facultative intracellular bacterium and one of the causative agents of brucellosis in humans and animals (2). Oral infection, which is considered a natural route of infection by brucellae, and localization of the bacteria inside the host cell phagosome most likely expose the pathogen to various stresses. In response to environmental stress conditions such as elevated temperatures, variation in pH, and starvation, a certain number of proteins are induced or repressed in Brucella spp., among which are the GroE and DnaK heat shock proteins (12,16,24). In intracellular brucellae, which encounter stressful conditions such as acidic pH (23) and possibly oxidative stress due to the presence of oxygen radicals, the known stress proteins GroEL and DnaK are induced during infection, and DnaK is essential for multiplication of B. suis in macrophage-like cells (12,17). The HtrA (high-temperature requirement) stress protein is involved in high-level spleen colonization with Brucella abortus in mice (5). Several other stress response proteins including ClpA, ClpB, ClpC, and ClpX, are members of a family of proteins called the Clp ATPases (HSP100 proteins), represented in prokaryotes and eukaryotes with a high degree of conservation (28). Clp stress proteins can be induced by high temperature, oxidative stress, high salt or ethanol concentration, and iron limitation (14,26,28). In addition to being sensitive to various stresses, Listeria monocytogenes clpC mutants are attenuated for virulence in mice (26).Our article describes the isolation and characterization of a B. suis gene encoding a homolog of the ClpB stress response proteins. An isogenic clpB mutant and the complemented strain were constructed and compared with the wild type and a clpA mutant (4) for survival at high temperature, at different ethanol concentrations, at acid pH, and in the presence of hydrogen peroxide. In addition, the effect of hydrogen peroxide on a clpAB double mutant was studied, and the possible participation of ClpA and ClpB in protein degradation was analyzed....

clpB, a Novel Member of theListeria monocytogenesCtsR Regulon, Is Involved in Virulence but Not in General Stress Tolerance

“…In agreement with this activity, ClpB has been shown to have three closely related functions in several gram-negative bacteria and in eukaryotes: (i) resistance to high-temperature stress in H. pylori, B. suis, and E. coli (1,12,29,62); (ii) cold acclimatization in Synechococcus sp. ; and (iii) induced thermotolerance to lethal stress in the cyanobacterium Synechococcus sp.…”

Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress

“…Despite the fact that these molecules are required for resistance to high-temperature stress, cold acclimation, and induced thermotolerance to lethal stress (8,9,18,24), these in vitro studies were performed mostly under nonstress conditions. Moreover, unlike gramnegative bacteria and eukaryotes, little is known about the structure and function of gram-positive bacterial ClpBs; this is despite the fact that, with the exception of Bacillus subtilis, these molecules are carried on the genomes of all gram-positive bacteria (21).…”

“…In gram-negative bacteria and eukaryotes, the disaggregating activities of ClpB and HSP104 have been reported to be important for resistance to high-temperature stress, cold acclimation, and induced thermotolerance to lethal stress (8,9,18,24). In gram-positive bacteria, a few reports have described the in vivo functions of ClpB: (i) the mutation of clpB did not affect the resistance of Lactococcus lactis MG1363 to high-temperature, salt, and puromycin stress (13), and (ii) ClpB was required for the induced thermotolerance and virulence of Listeria monocytogenes (4).…”

Induction ofdnaKthrough Its Native Heat Shock Promoter Is Necessary for Intramacrophagic Replication ofBrucella suis

“…Additional phenotypic characteristics that might be linked to a modification of dnaK expression, such as sensitivity to acid pH and to oxidative stress, were investigated according to previously published protocols (8,12). Briefly, for pH sensitivity experiments B. suis 1330 and the dnaK mut strain were grown to stationary phase (OD 600 ϭ 1.5) in TS broth, and the number of CFU per milliliter was determined prior to dilution 1:50 in TS broth adjusted to pH 4.5.…”

“…Each strain (75 l) was plated on TS agar, and sterile paper disks saturated with 10 l of H 2 O 2 at concentrations of 1.8, 2.9, and 4.4 M were layered on top prior to incubation at 30°C for 2 days and measurement of inhibition zone diameters (8). Interestingly, both the dnaK null mutant and the dnaK mut strain were significantly more sensitive to the three concentrations of H 2 O 2 than the parental strain ( Fig.…”