THE Cgt PROTEIN OF Brucella abortus REQUIRES AN INTACT MONONUCLEOTIDE BINDING DOMAIN TO FUNCTION IN TRANSPORT OF CYCLIC b-1,2-GLUCAN VIRULENCE FACTOR

ROSET MS, CIOCCHINI AE, UGALDE RA, IÑON DE IANNINO N.

Pinamar, Buenos Aires, Argentina

Congreso; XLI Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB 2005); 2005

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB)

Brucella abortus Cgt protein responsible for the transport of cyclic b-1,2-glucan to the periplasm is required for intracellular replication and full expression of virulence (Roset et al., 2004, Infect. Immun. 72: 2263-2271). The predicted membrane protein of 66 KDa, Cgt, has in the C-terminal domain all the conserved features of a typical ABC transporter such as the Walker site A (GxxGxGKS/T), the Walker site B (hhhhD), and the ABC signature, suggesting that this protein couple energy by NTP hidrolysis to transport the cyclic b-1,2-glucan. The Lys-374, found within the conserved Walker site A nucleotide binding motif of Cgt was changed to Ala by site-directed mutagenesis. The cgt gene altered in NTP-binding was unable to complement the cgt null mutant for either cyclic b-1,2-glucan transport or intracellular replication and virulence. These results demonstrated that an intact NTP-binding domain is critical for Cgt function in cyclic glucan transport and confirm that the cyclic b-1,2-glucan is necessary in the periplasm and/or extracellular to exert is action as virulence factor. On the other hand, the transport of the cyclic b-1,2-glucan to the periplasm was inhibited in wild type B. abortus after introducing a plasmid expressing a mutant cgt altered in the NTP-binding region. The dominant negative phenotype suggests that Cgt function as a multimer.