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Type: Journal article
Title: Detection of Wzy/Wzz interaction in Shigella flexneri
Author: Nath, P.
Morona, R.
Citation: Microbiology, 2015; 161(9):1797-1805
Publisher: Microbiology Society
Issue Date: 2015
ISSN: 1350-0872
Statement of
Pratiti Nath, Renato Morona
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-His8 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-His8 and untagged Wzz. Following solubilization with n-dodecyl-β-D-maltopyranoside (DDM) and affinity purification of Wzy-GFP-His8, Western immunoblotting with Wzz antibody detected co-purification 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.
Keywords: Shigella flexneri; Peptides; Bacterial Proteins; Recombinant Fusion Proteins; Protein Binding; Mutation; Mass Spectrometry
Rights: © 2015 The Authors | Published by the Microbiology Society
RMID: 0030036021
DOI: 10.1099/mic.0.000132
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Appears in Collections:Molecular and Biomedical Science publications

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