Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice.
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 (DeltaclpA) and SG1127 (Deltalon DeltaclpA), 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 degrees C 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 degrees C. 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.
Ekaza
Euloge
E
Institut National de la Santé et de la Recherche Médicale U-431, Université Montpellier II, CC 100, Pl. E. Bataillon, 34095 Montpellier, France1.
Guilloteau
Laurence
L
Laboratoire de Pathologie Infectieuse et d'Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France2.
Teyssier
Jacques
J
Institut National de la Santé et de la Recherche Médicale U-431, Université Montpellier II, CC 100, Pl. E. Bataillon, 34095 Montpellier, France1.
Liautard
Jean-Pierre
JP
Institut National de la Santé et de la Recherche Médicale U-431, Université Montpellier II, CC 100, Pl. E. Bataillon, 34095 Montpellier, France1.
Köhler
Stephan
S
Institut National de la Santé et de la Recherche Médicale U-431, Université Montpellier II, CC 100, Pl. E. Bataillon, 34095 Montpellier, France1.
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002002:Brucella / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D002006:Brucellosis / Q000382:microbiology; D002460:Cell Line; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D013154:Spleen / Q000382:microbiology; D013696:Temperature
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases