LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles

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dc.contributor.authorTran, N.
dc.contributor.authorDoyle, M.
dc.contributor.authorMorona, R.
dc.contributor.editorHozbor, D.F.
dc.date.issued2013
dc.description.abstractThe 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.
dc.description.statementofresponsibilityElizabeth Ngoc Hoa Tran, Matthew Thomas Doyle, Renato Morona
dc.identifier.citationPLoS One, 2013; 8(7):1-16
dc.identifier.doi10.1371/journal.pone.0070508
dc.identifier.issn1932-6203
dc.identifier.issn1932-6203
dc.identifier.orcidTran, N. [0000-0003-1644-2287]
dc.identifier.orcidMorona, R. [0000-0001-7009-7440]
dc.identifier.urihttp://hdl.handle.net/2440/80039
dc.language.isoen
dc.publisherPublic Library of Science
dc.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.
dc.source.urihttps://doi.org/10.1371/journal.pone.0070508
dc.subjectCell Membrane
dc.subjectShigella flexneri
dc.subjectO Antigens
dc.subjectBacterial Proteins
dc.subjectDNA-Binding Proteins
dc.subjectTranscription Factors
dc.subjectTunicamycin
dc.subjectHemagglutinins
dc.subjectFluorescent Antibody Technique
dc.subjectStaining and Labeling
dc.subjectGenetic Complementation Test
dc.subjectProtein Engineering
dc.subjectMutagenesis, Insertional
dc.subjectSequence Alignment
dc.subjectQuantum Dots
dc.subjectGene Expression Regulation, Bacterial
dc.subjectAmino Acid Sequence
dc.subjectSequence Homology, Amino Acid
dc.subjectModels, Molecular
dc.subjectMolecular Sequence Data
dc.titleLPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles
dc.typeJournal article
pubs.publication-statusPublished

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