¬Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Biosynthesis, Transport and Succinylation of Cyclic beta-1,2-Glucans

L. SOLEDAD GUIDOLIN; SUSANA MORRONE; FRANCISCO F. GUAIMAS; DIEGO J. COMERCI; ANDRÉS E. CIOCCHINI

Berlin

Congreso; Brucellosis 2014. International Research Conference; 2014

Cyclic beta-1,2-glucans (CBG) are periplasmic homopolysaccharides that play an important role in several symbiotic and pathogenic relationships. Brucella abortus CBG synthase (Cgs) is a polytopic integral inner membrane (IIM) protein of 320-kDa (2,867-amino acid residues) with six transmembrane-spanning segments. Cgs itself acts as a protein intermediate and catalyzes the four reactions (initiation, elongation, phosphorolysis and cyclization) required for the synthesis and control of the degree of polymerization of CBG. Once synthesized in the cytoplasm, CBG are transported to the periplasm by the CBG transporter (Cgt) and succinylated by the CBG modifier enzyme (Cgm). Cgt and Cgm as well as Cgs are polytopic IIM proteins. In this work, we used a bacterial two-hybrid system (BACTH) and co-immunoprecipitation techniques 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 also observed that each one of these proteins form homotypic complexes. Analyses carried out with Cgs in-frame pentapeptide insertion mutants and Cgs deletion mutants revealed that a coiled-coil motif located in the N-terminal domain of the protein (Cgs-region 1 to 418) is required to sustain the interactions of Cgs with Cgt and Cgm as well as with itself, although other regions may also be implicated. Finally, to analyze the subcellular localization of the protein complexes we performed fluorescence confocal microscopy studies fusing Cgs, Cgt and Cgm to mCherry, eYFP or eCFP and found that these proteins are localized at the cell poles in B. abortus. Taken together, these results demonstrate that Cgs, Cgt and Cgm interacts with itself (homotypic interactions) and with each other (heterotypic interactions) forming a membrane-associated biosynthetic complex located at the poles of the bacterial cell. Biosynthesis of CBG is predicted to require strict coordination of the initiation, elongation, phosphorolysis and cyclization reactions as well of its export to the periplasm and succinylation. We propose that the formation of the Cgs-Cgt-Cgm membrane complex at cell poles could serve as a mechanism to maintain the fidelity of cyclic B-1,2-glucans biosynthesis and coordinate the synthesis with its transport and modification.