Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Abstract

The gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. In Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella, Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPG contributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, in Escherichia coli K-12, the reaction synthesizing ECAPG from ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPG and provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor. IMPORTANCEThe outer membrane of gram-negative bacteria is a permeability barrier that prevents the entry of many antibiotics into the cell. However, the pathways responsible for outer membrane biogenesis are potential targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane of Enterobacterales such as Escherichia, Salmonella, Klebsiella, and Yersinia. ECAPG consists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate--ECA is the headgroup of a phospholipid. The details of the reaction forming this molecule from ECA linked to an isoprenoid carrier are unknown. We determined that the lipid donor that provides the phospholipid moiety to ECAPG is phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPG provides the opportunity for the development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat gram-negative infections.