Wzy-dependent pathway

Figure 2 Pathways for O antigen assembly, (a) Wzy-dependent pathway. After the initiation reaction, glycosyltransferases extend the O antigen until the completion of the repeating unit, which is translocated across the membrane by Wzz. In the periplasmic side of the membrane nascent units are polymerized by Wzy and the control of the chain length distribution is carried out by Wzz. (b) ABC transporter-dependent pathway. In this pathway the polymer is formed intracellular and terminated by the addition of a termination signal (black square), which also couples the polymer to the Wzt protein. ATP hydrolysis is required for export across the membrane, (c) Synthase-dependent pathway. After initiation, one more adaptor sugar is added (black circle) and the WbbF bifunctional enzyme that is also responsible for its vectorial translocation across the inner membrane extends the rest of the polymer.

Hi) Are the components of the Wzy-dependent pathway in a membrane complex Several authors have suggested that the proteins of the Wzy-dependent pathway function as multiprotein com-plexes. " Also, it is possible that protein components for the assembly of the EGA, which is similar to the Wzy-dependent pathway, exist together in the plasma membrane as a complex. Direct evidence exists for oligomerization in vivo of at least one of these proteins, Wzz, in S. flexneri, E. coli... 

Wzy-dependent pathway is most widespread in O polysaccharide biosynthesis. Following the assembly of O repeating units, the individual und-PP-linked O units are exported to the site of polymerization at the periplasmic face of the plasma membrane (Figure 2). This process requires a Wzx protein, the O unit transporter (flippase), which is highly hydrophobic with 12 potential transmembrane domains. Although the Wzx proteins share little primary sequence similarity, they do share structural features with bacterial permeases... 

Mechanisms of secretion in Gram negative bacteria have been elucidated (with either a Wzx-Wzy-dependent pathway or an ABC transporter-dependent pathway [58]) but very little is known in fungi. Even in the rather well-studied A. pullulans, mechanisms of pullulan secretion remain unclear and no secretory enzyme has been identified [59,60]. Yoneda et al. [49] demonstrated only recently that the cryptococcal capsule was synthesized intracellularly and secreted via exocytosis. It is clear from these few examples that the wide diversity of the molecular structures formd in EPS implies a wide variety of synthetic routes, difficult to identify. 

In contrast to the Wzy/Wzx-independent O antigens (see Section 6.10.2.2.2), no obvious ABC transporters have been identified in wzy-dependent systems. At least three proteins (Wzx, Wzy, and Wzz) are involved in this export pathway but currently, there is no information concerning the manner in which these proteins interact with one another to facilitate the formation of predicted functional complexes. Once the individual Und-PP-linked O subunits are formed, they must be exported to the site of polymerization at the periplasmic face of the plasma membrane (Figure 2(a)). 

The third component in the Wzy/Wzx-dependent pathway is the Wzz protein. Wzz generates the strain-specific chain length distribution of 0 polysaccharide chain lengths as reflected in characteristic clusters of bands following gel electrophoresis of LPS samples.The function of the Wzz protein is not required for bacterial growth in the laboratory setting, as polymerization can proceed in the absence of this protein. However, the distribution of the 0 polysaccharide chain length is critical for virulence. 

Figure 13 Combination of the LPS pathway and a protein glycosylation system in the engineered Escherichia coli. Left panel shows a polymerase(Wzy)-dependent LPS pathway and the right panel is an engineered coli expressing exogenous polysaccharides, a protein acceptor (e.g., pilin or AcrA), and an OTase. Inset in the right panel shows Western-blot analysis of unglycosylated protein acceptor (lane 1) and glycosylated protein with an O antigen polysaccharide (lane 2).

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