Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles (#314)

Elizabeth N. H. Tran 1 , Matthew Doyle 1 , Renato Morona 1
  1. University of Adelaide, Adelaide, SA, Australia
The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases, and cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP in the OM, a haemagglutinin (HA) epitope was inserted into the non-essential OM loop 5 of IcsP. Quantum Dot (QD) immunofluorescence (IF) surface labelling to detect IcsPHA was then undertaken. Quantitative IF microscopic 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 surfaces of both 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 an S. flexneri 2457T strain lacking Oag was also investigated and an equivalent 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. The asymmetrical distribution of IcsP suggests that the efficiency of IcsA cleavage is also asymmetrical which supports the role of IcsP in establishing and maintaining IcsA polarity.