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

Abstract

The heat shock protein DnaK is essential for intramacrophagic replication of Brucella suis. The replacement of the stress-inducible, native dnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a dnaK null mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutive dnaK mutant, which showed high sensitivity to H 2 O 2 -mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of dnaK is a crucial event in the intracellular replication of B. suis.We have described the important role of the heat shock protein and molecular chaperone DnaK in intramacrophagic growth of Brucella suis and its acid-induced expression (12). Brucella spp., facultative intracellular, gram-negative bacteria which are the causative agents of brucellosis in humans and animals (6), resist intracellular killing and replicate within the phagosome of macrophages (3,11,16). It has been observed recently that this phagosome is acidic (pH 4.0 to 4.5), suggesting a stressful environment, and that early acidification is essential for intracellular multiplication of B. suis (19). Work on the identification of proteins induced under stress conditions in Brucella abortus and Brucella melitensis confirmed our results (17,20,23). From previous data (12), we concluded that DnaK from B. suis may play an essential role as part of protein repair systems in protecting the bacteria from the environment encountered in the phagosome. Another hypothesis is that DnaK may be directly involved in folding and proper localization of virulence factors, as intracellular multiplication is abolished in the null mutant.The earlier work was done with a dnaK null mutant, allowing us to conclude only that the chaperone participated in intracellular multiplication of B. suis (12). On the other hand, dnaJ, which is located downstream of dnaK and forms an operon with the latter, is not involved in resistance to acid stress and intracellular multiplication of the pathogen (12). As a dnaJ knockout mutant of B. suis behaves like the wild-type strain with respect to these properties, we concluded that dnaJ was of no relevance in the context of the work presented here. Western blot analysis showed induction of dnaK during heat and acid shock and under intramacrophagic growth conditions (12). We therefore hypothesized that low-level, constitutive expression of dnaK was not sufficient for B. suis to resist macrophage attack. To verify this hypothesis, we replaced the original dnaK promoter on the B. suis chromosome by the constitutive promoter of the ␤-lactamase gene bla TEM (P bla ) from pUC18, and we studied the phenotype of the mutant obtained. P bla was chosen, as we and others (13,14) have observed that bla is expressed in brucellae, conferring ampicillin resistance to strains bearin...

Modulation of Adherence, Invasion, and Tumor Necrosis Factor Alpha Secretion during the Early Stages of Infection by Streptococcus pneumoniae ClpL

“…HSP60 (GroEL) and HSP70 (DnaK) have been shown to be required for virulence modulation (6,42), adherence (12,14,37), intracellular replication (25), invasion (44), and expression of TNF-␣ and adhesion molecules (15,30). Modulation of virulence by HSP100 family members, such as ClpB (8), ClpC (20,33,40), ClpE (34), ClpX (13), and ClpP (28,39), has also been documented.…”

The mglA gene and its flanking regions in Brucella: The role of mglA in tolerance to hostile environments, Fe-metabolism and in vivo persistence

“…The increased tolerance of the deletion mutant in a galactose-containing hyperosmotic medium ( Fig. 5) as well as to oxidative stress (H 2 O 2 ) ( Table 2) may be taken as a hint that the region is involved in tolerance to adverse environmental conditions ( Köhler et al, 2002 andKöhler et al, 2003).…”

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus

“…These observations are consistent with findings in Bac. subtilis, E. coli, Brucella suis, Listeria monocytogenes and other bacterial species, where inactivation of dnaK has been shown to result in poor growth at higher temperatures (Bukau & Walker, 1990;Hanawa et al, 1999;Kohler et al, 2002;Schulz et al, 1995). In addition to heat, the DnaK system has also been implicated to have a role in other stress conditions, such as ethanol, low pH, salt and oxidative stress (Mogk et al, 1998;Echave et al, 2002;Periago et al, 2002).…”

“…Similar results have been reported in Bru. suis, where DnaK has been shown to be important in the resistance of the organism to oxidative stress, as well as to acidic pH (Kohler et al, 2002). In L. monocytogenes however, a dnaK mutant has been shown to be more susceptible to acidic conditions than the wild-type strain, but not more susceptible to oxidative stress (Hanawa et al, 1999).…”