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Type: Journal article
Title: LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles
Author: Tran, N.
Doyle, M.
Morona, R.
Citation: PLoS One, 2013; 8(7):1-16
Publisher: Public Library of Science
Issue Date: 2013
ISSN: 1932-6203
Statement of
Elizabeth Ngoc Hoa Tran, Matthew Thomas Doyle, Renato Morona
Abstract: The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.
Keywords: Cell Membrane; Shigella flexneri; O Antigens; Bacterial Proteins; DNA-Binding Proteins; Transcription Factors; Tunicamycin; Hemagglutinins; Fluorescent Antibody Technique; Staining and Labeling; Genetic Complementation Test; Protein Engineering; Mutagenesis, Insertional; Sequence Alignment; Quantum Dots; Gene Expression Regulation, Bacterial; Amino Acid Sequence; Sequence Homology, Amino Acid; Models, Molecular; Molecular Sequence Data
Rights: © 2013 Tran et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RMID: 0020130937
DOI: 10.1371/journal.pone.0070508
Appears in Collections:Molecular and Biomedical Science publications

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