Sequestration of dead-end undecaprenyl phosphate-linked oligosaccharide intermediate

Abstract

Most Gram-negative bacteria synthesize a plethora of cell surface polysaccharides that play key roles in immune evasion, cell envelope structural integrity and host-pathogen interactions. In the predominant polysaccharide Wzx/Wzy-dependent pathway, synthesis is divided between the cytoplasmic and periplasmic faces of the membrane. Initially, an oligosaccharide composed of 3-8 sugars is synthesized on a membrane-embedded lipid carrier, undecaprenyl pyrophosphate, within the cytoplasmic face of the membrane. This lipid-linked oligosaccharide is then translocated to the periplasmic face by the Wzx flippase, where it is polymerized into a repeat-unit polysaccharide. Structural alterations to the O-antigen repeating oligosaccharide significantly reduce polysaccharide yield and lead to cell death or morphological abnormalities. These effects are attributed to the substrate recognition function of the Wzx flippase, which we postulated to act as a gatekeeper to ensure that only complete substrates are translocated to the periplasmic face. Here, we labelled Salmonella enterica serovar Typhimurium group B1 with [¹⁴C] d-galactose. Our results showed that strains unable to synthesize the full O-antigen repeat unit accumulate significantly higher levels of Und-P-linked material (~10-fold). Importantly, this sequestration is alleviated by membrane disruption which opens the lipid-linked oligosaccharide at the cytosolic face to periplasmic ligation to support accumulation occurs at the cytosolic face of the membrane.

Keywords: O-antigen; Wzx flippase; polysaccharide; substrate fidelity; substrate preference; undecaprenyl phosphate.

Fig. 1.. The structure of S. enterica group B1 LPS and controlled d-galactose metabolism. (a) Group B1 O:4 LPS structure. (b) d-Gal metabolism pathway. (c) Deactivation of galE to control d-Gal metabolism. d-Gal residue was circled in red to indicate biosynthetic steps blocked by the inaccessibility to the sugar precursor (pH indicator neutral red detects mixed acid fermentation and agar near bacterial growth would turn red if d-Gal is utilized). Abe, abequose; Man, mannose; Rha, rhamnose; Gal, galactose; GlcN, glucosamine.

Fig. 2.. [¹⁴C] d-Gal uptake in S. enterica group B1 WT and ∆abe. (a) % [¹⁴C] d-Gal counts of the whole cell; (b) % [¹⁴C] d-Gal counts in purified LPS; (c) % [¹⁴C] d-Gal counts in n-butanol extracts. Data from independent repeats are shown, and the error bar uses sem.

Fig. 3.. Reduction of [¹⁴C] d-Gal counts by membrane rearrangements in the n-butanol extractable pool from S. enterica group B1 ∆abe but not that from the WT pool. a Light microscopy image of WT and ∆abe samples after sonication. (b) Viability reduction by sonication. Both sonicated and untreated samples were adjusted to OD₆₀₀ 1.0 and diluted in a tenfold series with fresh growth medium. Five microlitres from 10⁰ to 10⁻⁶ dilutions were spotted onto M9-glycerol medium supplemented with casamino acids, tryptophan and thiamine [14]. (c) Sonication does not affect the n-butanol extractable pool in the WT. (d) Sonication significantly reduced the n-butanol extractable pool in the ∆abe mutant. Note that the unsonicated n-butanol extractable pool counts in (c, d) were the same datasets as Fig. 2c. Growth and sonication treatment of cells in (a, b) were executed as described for the [¹⁴C] work except only cold d-Gal was used. Data from three independent repeats are shown and the error bar uses sem.