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.