Interaction network among Brucella abortus membrane proteins involved in the synthesis, transport and succinylation of cyclic â-1,2-glucans

GUIDOLIN L. S.; CIOCCHINI A. E.; IñON DE IANNINO N.; COMERCI D. J

Buenos Aires

Conferencia; 2011 Brucellosis International Research Conference; 2011

Asociación Argentina de Microbiología

Cyclic b-1,2-glucans (CbG) are periplasmic homopolysaccharides that play an important role in several symbiotic and pathogenic relationships. The CbG of Brucella abortus are involved in controlling host cell functions to prevent fusion of the Brucella-containing vacuole with lysosomes, probably by interfering directly with lipid raft microdomains of the phagosome. B. abortus CbG synthase (Cgs) is the integral inner membrane (IIM) protein which catalyzes the four different activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CbG. Once synthesized in the cytoplasm, CbG are transported to the periplasm by the CâG transporter (Cgt) and succinylated by the CâG modifier enzyme (Cgm). Cgt and Cgm, as well as Cgs, are IIM proteins. In this work, we used a bacterial two-hybrid system (BACTH) to study the interaction network between these three IIM proteins. Our results indicate that Cgs interacts with Cgt and Cgm, and that Cgt interacts with Cgm. We have also observed that each one of these proteins form homotypic complexes. We propose that Cgs, Cgt and Cgm form an inner membrane protein complex where the interactions between these proteins may provide a mechanism to coordinate the synthesis, transport to periplasm and succinylation of CbG. Furthermore, interaction analyses carried out with Cgs C-terminally truncated and in-frame pentapeptide insertion mutants allowed us to identify a novel functional domain of the enzyme that is required for Cgs-dimerization as well as Cgs interactions with Cgt and Cgmb-1,2-glucans (CbG) are periplasmic homopolysaccharides that play an important role in several symbiotic and pathogenic relationships. The CbG of Brucella abortus are involved in controlling host cell functions to prevent fusion of the Brucella-containing vacuole with lysosomes, probably by interfering directly with lipid raft microdomains of the phagosome. B. abortus CbG synthase (Cgs) is the integral inner membrane (IIM) protein which catalyzes the four different activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CbG. Once synthesized in the cytoplasm, CbG are transported to the periplasm by the CâG transporter (Cgt) and succinylated by the CâG modifier enzyme (Cgm). Cgt and Cgm, as well as Cgs, are IIM proteins. In this work, we used a bacterial two-hybrid system (BACTH) to study the interaction network between these three IIM proteins. Our results indicate that Cgs interacts with Cgt and Cgm, and that Cgt interacts with Cgm. We have also observed that each one of these proteins form homotypic complexes. We propose that Cgs, Cgt and Cgm form an inner membrane protein complex where the interactions between these proteins may provide a mechanism to coordinate the synthesis, transport to periplasm and succinylation of CbG. Furthermore, interaction analyses carried out with Cgs C-terminally truncated and in-frame pentapeptide insertion mutants allowed us to identify a novel functional domain of the enzyme that is required for Cgs-dimerization as well as Cgs interactions with Cgt and Cgm