Sequestration of dead-end undecaprenyl phosphate-linked oligosaccharide intermediate

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Gram-negative bacteria synthesize cell surface polysaccharides crucial for immune evasion and structural integrity.
• The Wzx/Wzy-dependent pathway involves cytoplasmic and periplasmic stages of polysaccharide synthesis.
• The Wzx flippase is hypothesized to regulate substrate translocation to ensure complete O-antigen synthesis.

Purpose of the Study:

• To investigate the role of the Wzx flippase in O-antigen polysaccharide synthesis.
• To determine the consequences of incomplete O-antigen synthesis on lipid-linked oligosaccharide accumulation.
• To elucidate the gatekeeper function of Wzx in substrate translocation.

Main Methods:

• Labeling *Salmonella enterica* serovar Typhimurium with [¹⁴C] d-galactose.
• Analyzing the accumulation of undecaprenyl pyrophosphate (Und-P)-linked oligosaccharides in bacterial strains with altered O-antigen synthesis.
• Investigating the effect of membrane disruption on lipid-linked oligosaccharide sequestration.

Main Results:

• Strains with defects in full O-antigen repeat unit synthesis showed a ~10-fold increase in Und-P-linked material.
• This accumulation of Und-P-linked oligosaccharides occurred at the cytosolic face of the membrane.
• Membrane disruption alleviated the sequestration, allowing periplasmic ligation.

Conclusions:

• The Wzx flippase acts as a gatekeeper, preventing the translocation of incomplete O-antigen precursors.
• Defective O-antigen synthesis leads to significant accumulation of lipid-linked oligosaccharides.
• Membrane integrity is critical for the proper functioning of the Wzx/Wzy pathway and polysaccharide export.