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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.

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles