The Brucella abortus High Affinity Choline Transporter is Involved in Virulence

BUKATA, L.; HERRMANN, C. K.; COMERCI D. J

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular SAIB; 2010

Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular

Phosphatidylcholine (PC) is one of the major Brucella cell envelope lipids, which is involved in virulence. In a previous work, we demonstrated that in Brucella, PC synthesis occurs exclusively through the pcs pathway, implying that exogenous choline uptake is a prerequisite for its synthesis. However, a choline transporter has not been described for this pathogen to date. Bioinformatic and biochemical approaches were carried out in order to characterize choline transporter candidates. Three putative choline ABC transporters were detected by in silico screening of the B. abortus genome. Single and double deletion mutants were generated in the periplasmic binding protein of each system. By using synthesis of PC as a read-through of choline uptake, we detected that a mutant in BAB1_1593 was unable to form PC when cultured in cholinesupplemented media. Radiolabeled choline uptake confirmed that BAB1_1593 is the only high affinity periplasmic choline binding protein encoded by B. abortus. Mice and cell culture infection assays showed that the absence of choline transport impairs virulence of Brucella. This phenotype paralleled the defect of a mutant unable to form PC, which confirmed the relevance of PC for the intracellular stage and, hence, for virulence of this pathogen. These results open up the possibility to evaluate choline transport as a new therapeutic target for brucellosis.Brucella cell envelope lipids, which is involved in virulence. In a previous work, we demonstrated that in Brucella, PC synthesis occurs exclusively through the pcs pathway, implying that exogenous choline uptake is a prerequisite for its synthesis. However, a choline transporter has not been described for this pathogen to date. Bioinformatic and biochemical approaches were carried out in order to characterize choline transporter candidates. Three putative choline ABC transporters were detected by in silico screening of the B. abortus genome. Single and double deletion mutants were generated in the periplasmic binding protein of each system. By using synthesis of PC as a read-through of choline uptake, we detected that a mutant in BAB1_1593 was unable to form PC when cultured in cholinesupplemented media. Radiolabeled choline uptake confirmed that BAB1_1593 is the only high affinity periplasmic choline binding protein encoded by B. abortus. Mice and cell culture infection assays showed that the absence of choline transport impairs virulence of Brucella. This phenotype paralleled the defect of a mutant unable to form PC, which confirmed the relevance of PC for the intracellular stage and, hence, for virulence of this pathogen. These results open up the possibility to evaluate choline transport as a new therapeutic target for brucellosis.