Study of expression and function of SepL, a regulator of type 3 secretion in enterohaemorrhagic escherichia coli O157
Abstract
Enterohaemorrhagic Escherichia coli (EHEC) are a recently emerged group of
pathogens that can cause fatal infections in the young and elderly. EHEC
utilize a virulence factor delivery organelle called a ’Type 3 secretion system’
that results in the formation of characteristic ‘pedestal structures’ on epithelial
cells allowing colonization in the human or ruminant gastrointestinal tract. To
achieve this, effector proteins have to be injected into host cells. The SepL-SepD
complex has been shown to be key for controlling T3-related protein secretion in
EHEC. Lack of either protein results in effector hypersecretion and strongly
impaired secretion of EspADB translocon proteins. Therefore, the expression
and function of SepL was the focus of my PhD research. The expression of SepL
was shown to be heterogeneous and co-expressed with EspA filaments in EHEC
O157 strains. My work revealed two transcriptional regulators (Ler and SepD)
and two putative posttranscriptional regulators (Hfq and CsrA) of SepL
expression. Further experiments mapped a key mRNA region required for
heterogeneous expression of SepL. This sequence forms a predicted hairpin
structure around the Shine-Dalgarno (SD) site of sepL. A model has been
formed based on my data in which Hfq and CsrABCD bind to the mRNA
potentially competing to control translation. Functionally, the C-terminus of
SepL was found to be expendable for 1) SepD binding; 2) SepL membrane
localization and 3) translocon export, however it was required for 1) limiting
effector secretion via (2) a Tir interaction which might be disassociated by (3) an
EscD interaction once host cell signals are sensed. Previously, the concept of
two different types of T3 secretion signal were demonstrated in Yersinia spp, I
tested this hypothesis in EHEC using both wild type and SepL/SepD deficient
EHEC strains. SepL/SepD is required for the N-terminal signal pathway but not
a chaperone binding domain signal pathway. A 12aa NleA which only contained
an N-terminal signal was shown to bind to SepD and so did the multi-functional
T3 chaperone ― CesT. Finally, Far-Western assays demonstrated that SepL
only interacted with Tir while SepD could bind other effector proteins indicating
that SepL/SepD may act as a targeting hub for effector protein secretion.