Molecular characterisation of Shigella flexneri outer membrane protease IcsP.
Date
2008
Authors
Tran, Elizabeth Ngoc Hoa.
Editors
Advisors
Morona, Renato
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Thesis
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Abstract
Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans.
Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing
countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane
(OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella
virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA,
however the significance of IcsP in Shigella virulence is incompletely understood.
In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP
mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it
was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell
spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey
kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the
mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella
intercellular spread.
The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a
haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell
surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide
(LPS), the distribution of IcsPHA was found to be punctate across the cell surface.
Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS
backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also
constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail
spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During
these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a
helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate,
banded distribution across the cell surface.
The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK,
rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no
change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to
wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by
Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK
was not essential for Shigella intercellular spread (contradicting the published data on this
gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA
into culture supernatants.
Finally alternative substrates for the protease activity of IcsP were investigated against known
Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins).
However, IcsP appeared to have no effect on these substrates as determined by proteolytic
cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri,
and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed.
School/Discipline
School of Molecular and Biomedical Science
Dissertation Note
Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008
Provenance
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.