Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937‐derived phagocytes

Abstract

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37 degrees C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30 degrees C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37 degrees C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.

The analysis of the intramacrophagic virulome ofBrucella suisdeciphers the environment encountered by the pathogen inside the macrophage host cell

“…To this end, a previously described strong promoter isolated from B. suis (8) was cloned upstream of the gfp-cat reporter tandem into pBBR1-GFP (12), reisolated in fusion with gfp-cat, and inserted into the unique EcoRV site of bcsp31 cloned in pUC18. The final construct, which is a suicide vector in brucellae, was electroporated into B. suis 1330 (13), and clones that were fluorescent were further verified, confirming the insertion of gfp into chromosomal bcsp31. A spontaneous Nal R mutant of this strain was used as B. suis acceptor strain for transposon mutagenesis performed as described (14).…”

“…Previous work has demonstrated that an acid phagosomal pH of 3.5-4 is required for early steps of infection within the cell, and neutralization of the phagosome after 6 h of infection has no effect on intracellular growth of B. suis (49). Synthesis of stress proteins such as DnaK, Lon, or Host Factor I are probably required at this stage (13,25,27).…”

Genomic analysis of the original Elberg Brucella melitensis Rev.1 vaccine strain reveals insights into virulence attenuation

“…During this process, molecular chaperones, peroxiredoxins, and siderophores were shown to play an important role in Brucella survival [33–35]. Our SNP analysis revealed mutations in the ORFs BMEI2002 (c.25A> C; a missense mutation), BMEII1048 (c.850T> C; a missense mutation), and BMEI1619 (c.738T> A; a missense mutation), which encode the molecular chaperones DnaK, GroEL, and Hsp33, respectively.…”

“…Our SNP analysis revealed mutations in the ORFs BMEI2002 (c.25A> C; a missense mutation), BMEII1048 (c.850T> C; a missense mutation), and BMEI1619 (c.738T> A; a missense mutation), which encode the molecular chaperones DnaK, GroEL, and Hsp33, respectively. The induction of DnaK has been reported in B. suis [36], and the induced DnaK enabled Brucella to adapt to the hostile environment of the macrophage and was essential for the intracellular multiplication of B. suis [33,36]. We also found missense mutations in the ORFs BMEI1049 (c.167C> A) and BMEII0078 (c.485A> C), which encode a peroxiredoxin and the 2,3-dihydroxybenzoate-AMP ligase EntE, respectively.…”

Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

“…The DnaK/DnaJ chaperone machinery of Salmonella enterica has been shown to be involved in bacterial invasion of epithelial cells, and the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages (11). In the case of the pathogen Brucella suis it was shown that DnaK but not DnaJ is required for intracellular multiplication (12). DnaK of Listeria monocytogenes is essentially required for survival under high temperatures and acidic conditions, and it is essential for the efficient phagocytosis of the facultative intracellular pathogen with macrophages (13).…”