LPS O-antigen polysaccharide length impacts outer membrane permeability of enteric gram-negative bacteria

The Gram-negative outer membrane (OM) forms the bacterial cell surface and acts as a barrier against antibiotic influx. In enteric species, the OM is covered by lipopolysaccharides (LPS) decorated with varying lengths of O-antigen (O-Ag) polysaccharide that protect bacteria against mammalian host defenses. Studies of lab-adapted Escherichia coli K-12 strains have proven instrumental in unravelling the essential processes of LPS synthesis, transport, and assembly into the OM. However, O-Ag synthesis was inactivated in K-12 strains during their lab adaption, and these cells produce a non-native, truncated LPS form. Surprisingly, we found that re-activating O-Ag synthesis in K-12 permeabilizes the OM to diverse antibiotics, causing susceptibility. The O-Ag that modifies LPS is directly responsible for the compromised OM barrier. Lengthening the O-Ag polysaccharide worsens antibiotic sensitivity; while shortening it, or removing it entirely, improves antibiotic resistance in both E. coli and the human pathogen, Shigella flexneri. Our data show that OM antibiotic barrier integrity is maintaining by a balanced production of long and short LPS forms, and that this balance is dysfunctional in model E. coli K-12 strains. Our findings reveal that long O-Ag polysaccharides are a double-edged sword: while well-recognized as critical for protection against external host assaults, their transport and assembly onto the surface comes at the inherent price of compromising the OM barrier. Hence, LPS production balances between competing needs in host defense and OM integrity. Moreover, we identify an inherent advantage for species that produce O-Ag-lacking lipooligosaccharide (LOS), rather than LPS.