Characterization of the KpsMT transporter complex in Escherichia coli K1

The K1 capsule is an important virulence factor of pathogenic Escherichia coli. It is composed of an α(2,8)-linked polymer of sialic acid (polySia) and has been shown to be the most prevalent capsular antigen isolated from E. coli cases of neonatal meningitis. The 17-kb kps cluster of E. coli encodes the information necessary for capsule expression at the bacterial surface. Region 3 of the cluster consists of two genes, kpsM and kpsT. kpsM encodes a 29.8 kDa hydrophobic protein, and kpsT encodes a 25 kDa hydrophilic protein containing a consensus ATP-binding domain. Both proteins belong to the ATP-binding cassette (ABC) transporter superfamily. Proposed models postulate that KpsM and KpsT play a role in the transport of capsular polysaccharide across the cytoplasmic membrane, utilizing the energy from ATP hydrolysis. To characterize the KpsMT transporter complex, KpsT was purified in its native form and tested for functionality. It was determined that purified KpsT binds ATP resulting in a conformational change in the protein. Finally, it was shown that KpsT possesses intrinsic ATPase activity. The interaction between KpsM and KpsT was also examined. A mutation (ΔV 100) in the first cytoplasmic loop of chromosomally encoded KpsM caused a complete loss of polymer transport. This defect was suppressed by introducing kpsT, in trans, on a multicopy plasmid. To further analyze the interaction between KpsM and KpsT, a ΔkpsMT double deletion strain, and a plasmid encoding both proteins in tandem were constructed. KpsM and KpsT were stably encoded by the ensuing plasmid as they restored capsule expression to ΔkpsMT. Using ΔkpsMT, the protease accessibility of KpsM in the presence versus absence of KpsT was assayed in spheroplasts. KpsM was degraded faster in the presence of KpsT suggesting an interaction between the proteins. This interaction was also studied in a non-K1 background. Both proteins were expressed in a K12 strain and protease accessibility studies conducted. The proteins did not appear to interact as KpsM was not degraded more rapidly in the presence of KpsT. This implies that capsule synthesis and/or the presence of other kps proteins may be required for a KpsM-KpsT interaction.