Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis

doi:10.1128/IAI.68.3.1297-1303.2000

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Infection and Immunity, March 2000, p. 1297-1303, Vol. 68, No. 3
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis

Vincent Foulongne, Giséle Bourg, Chantal Cazevieille, Sylvie Michaux-Charachon, and David O'Callaghan^*

INSERM U431, Faculté de Médecine, 30900 Nîmes, France

Received 7 October 1999/Returned for modification 22 November 1999/Accepted 6 December 1999

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiplyin professional and nonprofessional phagocytes. The genetic basisof this aspect of Brucella virulence is still poorly understood.To identify new virulence factors, we have adapted signature-taggedtransposon mutagenesis, which has been used essentially in animalmodels, to an in vitro human macrophage infection model. A libraryof 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12pools, was screened for intracellular survival and multiplicationin vitamin D₃-differentiated THP1 cells. Eighteen mutants wereidentified, and their attenuation was confirmed in THP1 macrophagesand HeLa cells. For each avirulent mutant, a genomic fragmentcontaining the transposon was cloned. The genomic DNA sequenceflanking the transposon allowed us to assign functions to allof the inactivated genes. Transposon integration had occurredin 14 different genes, some of which were known virulence genesinvolved in intracellular survival or biosynthesis of smooth lipopolysaccharide(the virB operon and manB), thus validating the model. Other genesidentified encoded factors involved in the regulation of geneexpression and enzymes involved in biosynthetic or metabolic pathways.Possible roles in the virulence of Brucella for the differentfactors identified arediscussed.

^* Corresponding author. Mailing address: INSERM U431, Faculté de Médecine, Avenue Kennedy, 30900 Nîmes, France. Phone: (33)4 66 23 48 99. Fax: (33) 4 66 23 49 28. E-mail: docallaghan{at}zeus.sc.univ-montpl.fr.

Infection and Immunity, March 2000, p. 1297-1303, Vol. 68, No. 3
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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