Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice The GenBank accession number for the sequence reported in this paper is AJ224881.

Abstract

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70 % identity to ClpA of Rhodobacter blasticus, was identified and sequenced. Following heterologous expression in Escherichia coli strains SG1126 (∆clpA) and SG1127 (∆lon ∆clpA), b-ClpA replaced the function of E. coli ClpA, participating in the degradation of abnormal proteins. A b-clpA null mutant of B. suis was constructed, and growth experiments at 37 and 42 SC showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 SC. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B. suis overexpressing b-clpA behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of B. suis. These results showed that in B. suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.

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

“…Statistical analysis showed that, based on the assumption that B. suis 1330 possesses Ϸ3,200 genes, the maximal probability to pick up a gene at least once was 96.5%. The resulting mutants were screened for their participation in virulence by infection of the macrophage cell line THP-1, as described (15,18), and by individual analysis of intracellular multiplication by using fluorescence microscopy, as described in Materials and Methods. The disrupted genes were identified by sequencing the chromosomal DNA flanking Tn5 and homology search by using BLAST. One hundred thirty-one mutants unable to multiply in THP-1 cells were selected.…”

“…Wells containing attenuated mutants showed no intracellular fluorescence, allowing their rapid identification. Attenuation of the selected mutants was validated in THP-1 infection experiments performed in duplicates in 24-well plates where the number of intracellular bacteria was determined at 90 min and 7, 24, and 48 h postinfection (15).…”

“…Macrophage-like THP-1 cells were infected by individual B. suis mutants and the acceptor strain as a control at a multiplicity of infection of 50 in 96-well plates (15). At 24 and 48 h postinfection, the plates were examined by using fluorescence microscopy.…”

Brucellosis Vaccines: An Overview

“…But for the absence of an O-PS linked to the remaining LPS molecule, it can be predicted that not all these mutants are equivalent and they can be hypothetically grouped as follows: (i), R mutants have a complete lipid A-core plus a cytoplasmic O-PS, the incorporation of which to the LPS is blocked (at least the wzm/wzt and possibly the WaaL mutants); (ii), R mutants have a complete lipid A-core but no O-PS (mutants in wb** glycosyltransferases, in wecA, and in genes coding for enzymes necessary to synthesise some precursors, such as manBOAg, gmd and per) and (iii), R mutants have progressive deficiencies in the core and that may or may not accumulate cytoplasmic O-PS (mutants in some wa** genes and in some precursor genes such as manBcore). Mutants of each of these three groups have in fact been described, and the question then arises as to what extent they are equivalent in attenuation and immunizing abilities (27,28,29,30). Numerous outer and inner membranes, cytoplasmic, and periplasmic antigenic proteins have also been characterized.…”

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

“…In the construct containing the 3Ј half of clpB, a 400-bp EcoRV-Bsu15I fragment located within the open reading frame encoding ClpB was replaced by the kanamycin resistance gene from pUC4K. The whole insert was blunted and recloned into the Brucella suicide vector pCVD442 (3) prior to electroporation of B. suis (4). clpB inactivation by double recombination was confirmed by Southern blot analysis (not shown) and by Western blot using polyclonal anti-E. coli ClpB cross-reacting with the homologous protein of B. suis (Fig.…”