Investigating the LPS Translocation Role of LptE in Escherichia coli

Abstract

The resilience of Gram-negative bacteria against toxins and antibiotics is in large part due to their unique cell envelope composition. This essential barrier is composed of an inner membrane, an aqueous periplasmic space and an outer membrane. The outer membrane is an asymmetric bilayer with a surface-exposed leaflet consisting of a molecule called lipopolysaccharide (LPS) that is responsible for the membrane’s fundamental permeability barrier function. LPS is transported from the inner membrane to the outer membrane by the essential Lpt pathway. In the final step of this pathway, the LptDE complex, formed by the integral β-barrel protein LptD and the lipoprotein LptE, guides LPS into the outer leaflet of the outer membrane. Studies suggest that LptE plays a direct role in LPS binding and translocation. However, how LptE performs this function remains unknown. Moreover, LptE has a second function in assisting the folding of its partner LptD that complicates the study of LptE-LPS interaction. Structure-guided site- directed mutagenesis and antibiotic sensitivity assays were conducted to isolate mutations in LptE that disrupt LptE-LPS interactions without affecting LptDE complex formation and integrity. Mutations at three sites, A87, S88 and E97 were identified that disrupted the outer membrane barrier function. At these sites, the introduction of a positively charged residue (A87R, S88R and E97R) caused increased sensitivity to antibiotics, while other substitutions were well tolerated. The positive charge mutations did not impair the ability of LptE to assist in LptD folding, suggesting that these mutations likely disrupt outer membrane integrity by affecting the LPS translocation function of LptE. This study identifies a critical region of LptE that is involved in LPS interaction and provides insight for further biochemical characterization of LPS translocation.