Molecular basis for bile sensitivity of Escherichia coli with defective LPS O-antigen assembly

ABSTRACT The periplasmic space between the inner (IM) and outer membranes (OM) of diderm gram-negative bacteria is densely filled with proteins and peptidoglycan cell wall. Lipopolysaccharide (LPS) is transported through this crowded compartment and then installed at the cell surface. While laboratory-adapted Escherichia coli K-12 strains produce a truncated “rough” LPS, clinical E. coli strains decorate rough LPS with an O-antigen (O-Ag) polysaccharide. At the periplasmic leaflet of the IM, WaaL ligates O-Ag chains onto LPS once it has transited the IM, yielding long “smooth” LPS molecules that are subsequently transported to the OM. Recent reports claim that periplasmic O-Ag chains in Δ waaL cells disrupt cell envelope integrity to cause bile sensitivity. The presence of long periplasmic O-Ag chains is suggested to stress envelope architecture. Notably, this bile sensitivity is suppressed by disrupting OM–peptidoglycan interactions. Here, we show that even O-Ag repeat units trapped in the cytosol cause bile sensitivity that can be similarly suppressed by disruption of OM–peptidoglycan interactions, despite O-Ag never entering the periplasm. We show that stalled O-Ag chains cause bile sensitivity because they sequester a lipid carrier, limiting its availability for other biosynthetic processes, especially peptidoglycan cell wall synthesis. Indeed, providing more lipid carrier effectively suppressed severe bile sensitivity in Δ waaL even while increasing the accumulation of long O-Ag chains in the periplasm. Our study clearly shows that periplasmic O-Ag chains do not inherently damage the cell envelope. Rather, stalled O-Ag biogenesis causes a detrimental lipid carrier sequestration that presents as bile sensitivity. IMPORTANCE Lipopolysaccharide O-antigen (O-Ag) covers the cell surface of wild-type Escherichia coli strains. These long molecules are made inside the crowded periplasmic space and must be accommodated in this compartment. Recent studies reported that long O-Ag chains are toxic molecules in the periplasm, stressing the cell envelope architecture to cause bile sensitivity. We demonstrate that long O-Ag chains in the periplasm are not inherently toxic and that short cytosolic O-Ag repeat units similarly cause bile sensitivity. The accumulation of these O-Ag repeat units or chains acts to sequester an essential lipid carrier, which causes bile sensitivity by impinging on cell wall synthesis. Our study shows that enteric bacteria are vulnerable when lipid carrier availability is reduced, making them more susceptible to the antibacterial effects of bile.