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

Elizabeth Ngoc Hoa Tran,Matthew Thomas Doyle,Renato Morona

doi:10.1371/journal.pone.0070508

Elizabeth Ngoc Hoa Tran, Matthew Thomas Doyle + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0070508

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Journal: PloS one	Publication Date: Jul 25, 2013
Citations: 16	License type: CC BY 4.0

Affiliation: University of Adelaide

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 IcsPHA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsPHA 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 IcsPHA 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 IcsPHA and IcsA showed that IcsPHA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsPHA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsPHA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsPHA 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.

Highlights

Shigella flexneri is an intracellular pathogen which causes bacillary dysentery, a disease characterised by the presence of severe mucoid bloody diarrhoea and by invasion of the gut epithelium [1,2]
We investigated the distribution of IcsP by cell surface quantum dot (QD) IF labelling of functional, HA-tagged IcsP (IcsPHA) in S. flexneri 2457T and establish that LPS O antigen (Oag) masks detection of IcsPHA on the cell surface by using tunicamycin to inhibit Oag synthesis
When IcsPHA was expressed at native IcsP equivalent levels (Fig. 1A & C), it was undetectable in the outer membrane (OM) via Quantum Dot (QD) IF microscopy in smooth LPS S. flexneri (Fig. S1) but detectable on both LPS Oagdepleted and rough LPS Shigella bacteria (Fig. 4, 5 & 6)

Summary

Introduction

Shigella flexneri is an intracellular pathogen which causes bacillary dysentery, a disease characterised by the presence of severe mucoid bloody diarrhoea and by invasion of the gut epithelium [1,2]. IcsA (VirG) is a 120 kDa outer membrane (OM) protein localised at the cell pole [3]. It mediates intracellular cytoplasmic movement of S. flexneri in epithelial cells, and cell-to-cell spread, by the assembly of an F-actin comet-tail at one pole of the bacterium [4,5,6]. This type of movement is described as actin-based motility (ABM). Over-expression of IcsP results in the complete removal of IcsA from the cell surface [13]

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