B-262. Dihydrolipoamide Dehydrogenase Regulates ABC-Transport in Streptococcus pneumoniae
Streptococcus pneumoniae (pneumococcus) is a leading cause of morbidity and mortality in both children and elderly worldwide. The insufficiencies of the current vaccines and the increasing prevalence of drug resistant strains warrant more investigation into the mechanisms of virulence of this important human pathogen. We have identified an enzyme, dihydrolipoamide dehydrogenase (DLDH) in pneumococci, that when mutated, renders the bacteria unable to colonize or infect mice. In S. pneumoniae, 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 galactose and possibly other carbohydrates, through ATP-binding cassette (ABC) transporters. Sequence analysis of the galactose ABC transport proteins MglA and MglB, revealed a lipoyl-binding motif, the substrate for DLDH, in the C-terminus of MglA. This suggested that DLDH could potentially regulate galactose transport through a direct interaction with MglA. DLDH and MglA were expressed recombinantly, with different tags and binding was assessed in a modified ELISA assay, using antibody to the prey to detect binding. This method showed strong binding between the proteins, irrespective of which protein was used as bait or prey. To determine the role of the C-terminal lipoyl-binding motif in MglA, MglA protein lacking the last 53 amino acids (MglAΔlip) was expressed in E. coli and tested for binding to DLDH. This domain was not necessary for MglA’s ATPase activity as MglA and MglAΔlip showed similar ATP degradation activity. However, MglAΔlip showed a 80% reduction in binding to DLDH, suggesting that this domain is responsible for the major interaction with DLDH. Our results suggest that DLDH may stimulate galactose transport in pneumococci through a direct binding to MglA. Analysis of the pneumococcal genome as well as genomes of other organisms has identified lipoyl-binding motifs in several other ABC transport proteins. This indicates that DLDH may exert a broad regulation of ABC transport events in pneumococci and potentially in other species, including humans.