Dihydrolipoamide dehydrogenase: A regulator of ABC transport in Streptococcus pneumoniae
Tyx, Robert Edward
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Streptococcus pneumoniae (pneumococcus) is a leading cause of morbidity and mortality in both children and elderly worldwide. Insufficiencies of the current vaccine and increasing prevalence of drug resistant strains warrant more investigation into the mechanisms of virulence of this important human pathogen. We have identified a membrane-bound enzyme, dihydrolipoamide dehydrogenase (DLDH) in pneumococci, that when mutated, renders the bacteria unable to survive in an animal host. In the pneumococcus, DLDH does not function in its usual role as an electron carrier in 2-oxo-acid dehydrogenase enzyme complexes; instead it is necessary for both utilization and uptake of raffinose, galactose, and other substrates. The objective of this project is to understand how DLDH activates ATP binding cassette (ABC) transport, using the raffinose transporter in S. pneumoniae as a model system. Immunoprecipitations showed that DLDH binds to the raffinose ABC transporter ATP-binding protein, RafK, in vitro and in vivo. We are working to determine the mechanism of DLDH regulation in order to characterize the functional relevance in vivo. We are also looking at other ABC transporters in the pneumococcus and E. coli that feature either a consensus lipoyl domain, where the DLDH substrate lipoamide group is normally attached, or a conserved lipoyl domain protein fold. These sites potentially function as specific binding sites for DLDH. Our hypothesis is that DLDH regulates ABC transport in S. pneumoniae both on a transcriptional level and through an inducer exclusion mechanism involving direct binding to the transporter protein. The specific aims of this project are to 1) investigate how DLDH regulates transport of raffinose in S. pneumoniae , 2) identify the functional role of the regulated transporter proteins in the virulence of pneumococcus. ABC transporters are spread throughout the bacterial and eukaryotic kingdoms, but regulation through a C-terminal lipoyl domain seems to be solely a prokaryotic phenomenon and is therefore an attractive target for drugs that could interrupt this interaction. Through this dissertation, we will characterize an important regulatory mechanism for ABC transport systems and bring to light this novel potential drug target.