Subcellular localization of the enterobacterial common antigen GT-E-like glycosyltransferase, WecG.

Enterobacterales have developed a specialized outer membrane polysaccharide (enterobacterial common antigen [ECA]). ECA biosynthesis begins on the cytoplasmic side of the inner membrane (IM) where glycosyltransferases sequentially add sugar moieties to form a complete repeat unit which is then translocated across the IM by WzxE before being polymerized into short linear chains by WzyE/WzzE. Research into WecG, the enzyme responsible for generating ECA lipid-II, has not progressed beyond Barr et al. (1988) who described WecG as a membrane protein. Here we revise our understanding of WecG and re-characterize it as a peripherally associated membrane protein. Through the use of Western immunoblotting we show that WecG in Shigella flexneri is maintained to the IM via its three C-terminal helices and further identify key residues in helix II which are critical for this interaction which has allowed us to identify WecG as a GT-E glycosyltransferase. We investigate the possibility of protein complexes and ultimately show that ECA lipid-I maintains WecG to the membrane which is crucial for its function. This research is the first since Barr et al. (1988) to investigate the biochemistry of WecG and reveals possible novel drug targets to inhibit WecG and thus ECA function and cell viability.

Interdependence of Shigella flexneri O Antigen and Enterobacterial Common Antigen Biosynthetic Pathways.

Outer membrane (OM) polysaccharides allow bacteria to resist harsh environmental conditions and antimicrobial agents, traffic to and persist in pathogenic niches, and evade immune responses. Shigella flexneri has two OM polysaccharide populations, being enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) O antigen (Oag); both are polymerized into chains by separate homologs of the Wzy-dependent pathway. The two polysaccharide pathways, along with peptidoglycan (PG) biosynthesis, compete for the universal biosynthetic membrane anchor, undecaprenyl phosphate (Und-P), as the finite pool of available Und-P is critical in all three cell wall biosynthetic pathways. Interactions between the two OM polysaccharide pathways have been proposed in the past where, through the use of mutants in both pathways, various perturbations have been observed. Here, we show for the first time that mutations in one of the two OM polysaccharide pathways can affect each other, dependent on where the mutation lies along the pathway, while the second pathway remains genetically intact. We then expand on this and show that the mutations also affect PG biosynthesis pathways and provide data which supports that the classical mutant phenotypes of cell wall mutants are due to a lack of available Und-P. Our work here provides another layer in understanding the complex intricacies of the cell wall biosynthetic pathways and demonstrates their interdependence on Und-P, the universal biosynthetic membrane anchor. IMPORTANCE Bacterial outer membrane polysaccharides play key roles in a range of bacterial activities from homeostasis to virulence. Two such OM polysaccharide populations are ECA and LPS Oag, which are synthesized by separate homologs of the Wzy-dependent pathway. Both ECA and LPS Oag biosynthesis join with PG biosynthesis to form the cell wall biosynthetic pathways, which all are interdependent on the availability of Und-P for proper function. Our data show the direct effects of cell wall pathway mutations affecting all related pathways when they themselves remain genetically unchanged. This work furthers our understanding of the complexities and interdependence of the three cell wall pathways.

Topology of the Shigella flexneri Enterobacterial Common Antigen polymerase WzyE.

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.