2 research outputs found
N‑Terminus of IpaB Provides a Potential Anchor to the <i>Shigella</i> Type III Secretion System Tip Complex Protein IpaD
The type III secretion system (T3SS)
is an essential virulence
factor for Shigella flexneri, providing
a conduit through which host-altering effectors are injected directly
into a host cell to promote uptake. The type III secretion apparatus
(T3SA) is composed of a basal body, external needle, and regulatory
tip complex. The nascent needle is a polymer of MxiH capped by a pentamer
of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g.,
deoxycholate) causes a conformational change in IpaD and promotes
recruitment of IpaB to the needle tip. It has been proposed that IpaB
senses contact with host cell membranes, recruiting IpaC and inducing
full secretion of T3SS effectors. Although the steps of T3SA maturation
and their external triggers have been identified, details of specific
protein interactions and mechanisms have remained difficult to study
because of the hydrophobic nature of the IpaB and IpaC translocator
proteins. Here, we explored the ability for a series of soluble N-terminal
IpaB peptides to interact with IpaD. We found that DOC is required
for the interaction and that a region of IpaB between residues 11–27
is required for maximum binding, which was confirmed in vivo. Furthermore,
intramolecular FRET measurements indicated that movement of the IpaD
distal domain away from the protein core accompanied the binding of
IpaB<sup>11–226</sup>. Together, these new findings provide
important new insight into the interactions and potential mechanisms
that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies
probing T3SS activation
Binding Affects the Tertiary and Quaternary Structures of the <i>Shigella</i> Translocator Protein IpaB and Its Chaperone IpgC
<i>Shigella flexneri</i> uses its type III
secretion system (T3SS) to promote invasion of human intestinal epithelial
cells as the first step in causing shigellosis, a life-threatening
form of dysentery. The <i>Shigella</i> type III secretion
apparatus (T3SA) consists of a basal body that spans the bacterial
envelope and an exposed needle that injects effector proteins into
target cells. The nascent <i>Shigella</i> T3SA needle is
topped with a pentamer of the needle tip protein invasion plasmid
antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic
translocator protein, IpaB, to the tip complex where it senses contact
with a host membrane. In the bacterial cytoplasm, IpaB exists in a
complex with its chaperone IpgC. Several structures of IpgC have been
determined, and we recently reported the 2.1 Ã… crystal structure
of the N-terminal domain (IpaB<sup>74.224</sup>) of IpaB. Like IpgC,
the IpaB N-terminal domain exists as a homodimer in solution. We now
report that when the two are mixed, these homodimers dissociate and
form heterodimers having a nanomolar dissociation constant. This is
consistent with the equivalent complexes copurified after they had
been co-expressed in <i>Escherichia coli</i>. Fluorescence
data presented here also indicate that the N-terminal domain of IpaB
possesses two regions that appear to contribute additively to chaperone
binding. It is also likely that the N-terminus of IpaB adopts an alternative
conformation as a result of chaperone binding. The importance of these
findings within the functional context of these proteins is discussed