Characterization of the Brucella abortus high affinity choline transporter

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

Buenos Aires

Conferencia; 2011 Brucellosis International Research Conference; 2011

Asociación Argentina de Microbiología

In recent years there have been an increasing number of reports recognizing the importance of membrane lipids for the complex symbiotic or pathogenic interaction of intracellular microorganism with the host cell. In Brucella spp., phosphatidylcholine (PC) is one of the most abundant cell envelope lipids, which is necessary for sustaining a chronic infection process in mice. PC synthesis occurs exclusively through the PCS pathway (since the endogenous methylation pathway for PC formation is not functional), suggesting that Brucella intracellular survival depends on choline synthesis by the host cell and its subsequent uptake by a bacterial transporter. However, a specific transporter for choline has not yet been described for this pathogen. Bioinformatic and biochemical approaches were carried out in order to identify choline transporter candidates. Three putative choline ABC transporters were detected by an in silico screening of the B. abortus 2308 genome. Each ABC transporter consists of three proteins: an inner-membrane protein, an ATPase and a periplasmic-binding protein (BP) which gives the system the substrate specificity. For this reason, single and double deletion mutants were generated in the periplasmic-BP of each putative transporter. By using synthesis of PC as a read-through of choline uptake, we found that a mutant in BAB1_1593 gene was unable to form PC when cultured in cholinesupplemented media. Further experiments using radiolabeled choline were carried out to evaluate the kinetics of choline incorporation. In this regard, we demonstrated that BAB1_1593 is the only high affinity periplasmic choline binding protein encoded by B. abortus. To determine the role of the transporter in virulence, a deletion mutant was evaluated for its ability to survive and replicate inside macrophages. We observed that the mutant strain exhibited a conditional replication defect at 24hs pi depending on the concentration of choline in the culture medium previous to cell infection. These results are consistent with the idea that BAB1_1593 is required for the intracellular uptake under low availability of choline and suggests the presence of another yet uncharacterized low affinity transport system which is induced under higher concentration of the vitamin. Further experiments are being carried out for thorough characterization of this high affinity choline transporter in terms of biochemical activity and its significance for the intracellular stages of Brucella.Brucella spp., phosphatidylcholine (PC) is one of the most abundant cell envelope lipids, which is necessary for sustaining a chronic infection process in mice. PC synthesis occurs exclusively through the PCS pathway (since the endogenous methylation pathway for PC formation is not functional), suggesting that Brucella intracellular survival depends on choline synthesis by the host cell and its subsequent uptake by a bacterial transporter. However, a specific transporter for choline has not yet been described for this pathogen. Bioinformatic and biochemical approaches were carried out in order to identify choline transporter candidates. Three putative choline ABC transporters were detected by an in silico screening of the B. abortus 2308 genome. Each ABC transporter consists of three proteins: an inner-membrane protein, an ATPase and a periplasmic-binding protein (BP) which gives the system the substrate specificity. For this reason, single and double deletion mutants were generated in the periplasmic-BP of each putative transporter. By using synthesis of PC as a read-through of choline uptake, we found that a mutant in BAB1_1593 gene was unable to form PC when cultured in cholinesupplemented media. Further experiments using radiolabeled choline were carried out to evaluate the kinetics of choline incorporation. In this regard, we demonstrated that BAB1_1593 is the only high affinity periplasmic choline binding protein encoded by B. abortus. To determine the role of the transporter in virulence, a deletion mutant was evaluated for its ability to survive and replicate inside macrophages. We observed that the mutant strain exhibited a conditional replication defect at 24hs pi depending on the concentration of choline in the culture medium previous to cell infection. These results are consistent with the idea that BAB1_1593 is required for the intracellular uptake under low availability of choline and suggests the presence of another yet uncharacterized low affinity transport system which is induced under higher concentration of the vitamin. Further experiments are being carried out for thorough characterization of this high affinity choline transporter in terms of biochemical activity and its significance for the intracellular stages of Brucella.