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basal body and export apparatus. This complex then secretes the parts necessary
for completion of the injectisome (so-called early/mid substrates) including the
inner-rod, needle, tip, and translocon.
Recent studies in Salmonella typhimurium ( Wagner et al., 2010 ) and
Yersinia ( Diepold et al., 2011 ) have suggested a broadly conserved sequence
of assembly in these two different systems. Based on observations of the Sal-
monella SPI-1 T3SS injectisome, assembly was proposed to proceed in an
'inside-out' manner, starting with the formation of the inner-membrane export
apparatus with SpaP (EscR), SpaQ (EscS), and SpaR (EscT) forming a stable
complex even in the absence of the needle complex ( Figure 14.3 A). This core
complex acts as a nucleation point for the assembly of the remaining export
apparatus proteins SpaS (EscU) and InvA (EscV) (the export apparatus pro-
teins are presumed to be exported by the general secretory (sec) pathway) fol-
lowed by the formation of the inner-membrane rings PrgK (EscJ) and PrgH
(EscD), and outer-membrane secretin InvG (EscC) (the inner- and outer-rings
are assembled via sec-mediated secretion). Independent studies into the assem-
bly of the Yersinia injectisome revealed a similar pathway. Here, assembly is
initiated by the oligomerization of the largest export apparatus protein YscV
(EscV). The exact nature of this oligomer is unclear but, like in Salmonella
typhimurium , several of the smaller export apparatus proteins are required for
its assembly, namely YscRST (EscRST). This export apparatus complex is then
capable of recruiting YscJ (EscJ), the smaller of the inner-membrane rings,
which assembles into an oligomeric ring around YscV (EscV). In a separate
process, the outer-membrane secretin YscC (EscC) assembles into a ring. These
inner- and outer-membrane complexes are then connected via the larger of the
inner-membrane rings, YscD (EscD), which envelops YscJ (EscJ) and interacts
with the secretin ( Figure 14.3 B).
This nascent injectisome then recruits the ATPase and its associated proteins
( Figure 14.3 C). At this stage, it becomes secretion-competent, and the compo-
nents required to complete assembly are secreted in a T3SS ATPase-dependent
manner. Initially, early substrates including the inner-rod, which traverses the
basal body, and the extracellular needle are secreted ( Figure 14.3 D). After ter-
mination of the needle, the 'mid' substrates are secreted; initially the tip protein
is translocated and forms an oligomeric cap at the distal end of the needle, or
in the case of EPEC EspA, a filamentous extension of the needle (see above).
The two translocon proteins are then secreted ( Figure 14.3 E) and upon contact
with a target cell, form a pore inside its membrane, attached to the needle tip,
completing the assembly of the injectisome and forming a continuous channel
from the bacterial cytoplasm to the host cell cytoplasm ( Figure 14.3 F) for the
'late' substrates, or effectors, to pass.
This hierarchical assembly requires a high degree of spatial and temporal
regulation to ensure the correct formation of the injectisome substructures and
order of component secretion. The first point of regulation is the substrate speci-
ficity switch between the secretion of the early and mid substrates, or between
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