Detection of Wzy/Wzz interaction in Shigella flexneri

Abstract

The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by the Wzy-dependent mechanism. S. flexneri Wzy has 12 transmembrane segments and two large periplasmic loops. The modal chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-GFP-His 8 and detected the presence of a dimeric form. In vivo cross-linking was then performed in an S. flexneri wzy mutant strain carrying plasmids encoding Wzy-GFP-His 8 and untagged Wzz. Following solubilization with n-dodecyl-b-D-maltopyranoside (DDM) and affinity purification of Wzy-GFP-His 8 , Western immunoblotting with Wzz antibody detected copurification of Wzz; this was supported by MS analysis. To the best of our knowledge, this is the first reported isolation of a complex between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K) whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational alterations did not affect the interaction of Wzy with Wzz. Thus, the interaction may involve many regions of Wzy.

Full-length, Oligomeric Structure of Wzz Determined by Cryoelectron Microscopy Reveals Insights into Membrane-Bound States

“…Recently, Nath and Morona (2015) reported the formation of a stable complex between Wzz/Wzy in S. flexneri using in vivo crosslinking studies. There is no structural information about Wzy proteins, but topological predictions revealed that most homologs consist of 10-12 TMHs and only one large periplasmic domain, predicted by several prediction tools (TOPCONS and TMHMM) (Tsirigos et al, 2015; Krogh et al, 2001).…”

“…In the absence of Wzz activity, only short Oag chains are produced suggesting a loss of processivity in the Wzy polymerase (Islam and Lam, 2014;Kalynych et al, 2014;Raetz and Whitfield, 2002). Wzz and Wzy interactions have been detected after crosslinking but the nature and functional consequences of this are unresolved (Nath and Morona, 2015).…”

“…In the absence of Wzz, the glycan is still polymerized but there is no defined range and the profile favors shorter molecules. Wzz and Wzy proteins appear to interact in Shigella flexneri (Nath and Morona, 2015). However, while Wzz is clearly implicated as a critical factor in chain-length determination, the mechanism by which it modulates the polymerase remains unknown.…”

A novel role of VisP and the Wzz system during O-antigen assembly inSalmonellaTyphimurium pathogenesis

“…Typhimurium, although the specific stimuli were not identified (31). The length of longer OAg forms is controlled by the PCP proteins Wzz ST and Wzz fepE in a possible interaction with Wzy polymerase, as previously described in Shigella flexneri (106). However, the underlying mechanism remains unclear.…”

Unique Regions of the Polysaccharide Copolymerase Wzz 2 from Pseudomonas aeruginosa Are Essential for O-Specific Antigen Chain Length Control

“…This results in the TMDs resembling an open claw in the oligomer (25). The authors proposed that Wzy might interact with Wzz between these TMDs, which is supported by biochemical and genetic evidence of Wzy-Wzz interaction (26)(27)(28)(29).…”