Biophysical Studies of the Lpt Pathway
George, Alexander Hall
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AbstractThe outer membrane (OM) of Gram-negative bacteria is impermeable to many antibiotics because its outer leaflet is composed entirely of lipopolysaccharide (LPS), a large glycolipid with an extracellular saccharide that is between a dozen and hundreds of sugars long. LPS forms tight associations, preventing entry of many small molecules. LPS is synthesized at the inner membrane (IM), and is transported to the OM by the seven-protein Lipopolysaccharide transport (Lpt) pathway. While the Lpt proteins of the IM are readily identified as an ABC transporter, and the ATP dependence of the early steps of LPS transport are well characterized, the mechanism of the OM components of the Lpt pathway remains unclear.
This thesis describes efforts towards improving our model of the mechanism of LptD/E, the translocon in the OM responsible for flipping and inserting LPS across and into the OM. LptE is a lipoprotein that sits within the membrane-integral β-barrel of LptD. LptE had previously been shown to function as a chaperone for LptD folding, but had no known function in LPS transport. We used surface plasmon resonance (SPR) to show that LptE not only binds LPS, but disrupts and solubilizes LPS aggregates, and that mutants of LptE that lead to a compromised OM are also deficient in their ability to disaggregate LPS. We also used SPR to measure dissociation constants between LptE and six different soluble fragments of LPS, showing that LptE binds LPS by the di-glucosamine-di-phosphate, with neither the core oligosaccharide nor the acyl chains making notable contributions to binding. This, combined with the structural work of others, suggests a model for LptE function in which, by binding the charged headgroup, it disrupts LPS-LPS interactions and guides the sugars of LPS through the hydrophilic lumen of LptD while the lipids transfer directly from a periplasmic aggregate to the interior of the OM. A desire to understand the energy requirements of this transfer led us to build on a reconstitution of LPS transport between liposomes. We used a combination of fluorescent flow cytometry and confocal microscopy to show that two populations of liposomes, each containing either the IM or OM complex, associate only in the presence of LptA, indicating that LPS transport occurs via a bridge of Lpt proteins that could transduce energy from the IM ATPase LptB to the OM. This reconstitution will allow for future studies of how different Lpt pathway mutants and differently modified forms of LPS alter LPS transport efficiency.
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