Hong Yaoqin, Qin Jilong, Doyle Matthew Thomas, Reeves Peter Richard
Biomedical Sciences and Molecular Biology, College of Medicine and Dentistry, James Cook University, Douglas, Queensland, Australia.
School of Life and Environmental Sciences, The University of Sydney, Camperdown, New South Wales, Australia.
Microbiology (Reading). 2025 Jan;171(1). doi: 10.1099/mic.0.001530.
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 serovar Typhimurium group B1 with [C] 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.
大多数革兰氏阴性菌会合成大量细胞表面多糖，这些多糖在免疫逃逸、细胞包膜结构完整性以及宿主-病原体相互作用中发挥关键作用。在主要的多糖Wzx/Wzy依赖性合成途径中，合成过程在细胞膜的细胞质面和周质面之间进行。最初，由3至8个糖组成的寡糖在细胞膜细胞质面的一种膜嵌入脂质载体——十一异戊烯焦磷酸上合成。然后，这种脂质连接的寡糖通过Wzx翻转酶转运至周质面，在那里它会聚合成重复单元多糖。O抗原重复寡糖的结构改变会显著降低多糖产量，并导致细胞死亡或形态异常。这些影响归因于Wzx翻转酶的底物识别功能，我们推测它充当把关者，以确保只有完整的底物才会转运至周质面。在此，我们用[C] d-半乳糖标记了鼠伤寒血清型B1。我们的结果表明，无法合成完整O抗原重复单元的菌株积累的十一异戊烯磷酸连接物质水平显著更高（约10倍）。重要的是，这种隔离现象会因膜破坏而得到缓解，膜破坏会使脂质连接的寡糖在胞质面打开，以便与周质连接，从而支持在细胞膜胞质面发生积累。
