Type 3 secretion systems - Escherichia coli: Pathotypes and Principles of Pathogenesis - page 413

In addition to these structural components, analysis in the Salmonella SPI-1
system suggests the conserved but less well-characterized proteins PrgJ (EscI
in EPEC) and InvJ (EscP in EPEC) are also essential components of the gen-
eralized T3SS basal body, with both proteins co-purifying with isolated injec-
tisomes in vitro ( Marlovits et al., 2004, 2006 ). These proteins are localized to
the periplasmic region of the basal body, and are proposed to interact with the
secretin and needle T3SS components, as well as with components of the inner
membrane export apparatus ( Kimbrough and Miller, 2000 ; Sukhan et al., 2003 ;
Sal-Man et al., 2012 ). EscI (PrgJ in the SPI-I T3SS) is hypothesized (although
not yet verified) to be a polymerized hollow rod that spans the T3SS basal body
to form a continuous path with the downstream needle component ( Marlovits
et al., 2006 ; Sal-Man et al., 2012 ). This so-called 'inner-rod' has also been
implicated in the transition from secretion of the needle protein to tip and trans-
locon proteins (EspABD in EPEC), a process known as substrate switching
(discussed below) ( Marlovits et al., 2004 ; Sal-Man et al., 2012 ). NMR studies
of a refolded, recombinant form of the EscI ortholog PrgJ shows that this protein
largely lacks a defined structure ( Zhong et al., 2012 ), although the relevance of
this observation within the assembled injectisome remains to be determined.
The biochemical role of InvJ/EscP is better characterized: its deletion creates
T3SS variants with abnormal needle length ( Tamano et al., 2002 ; Journet et al.,
2003 ; Mota et al., 2005 ; Marlovits et al., 2006 ; Monjaras Feria et al., 2012 ),
leading to the hypothesis that this protein acts as a 'molecular ruler' to regulate
T3SS needle formation, although the mechanism by which such measurement
is achieved remains unexplained.
The extracellular components: the needle, filament,
and translocon
The T3SS needle is the portion of the apparatus extending away from the extra-
cellular surface of the basal body; it is a ∼70 Å wide superhelical structure,
with a ∼30 Å hollow channel in the middle ( Figure 14.2 ), and varies in length
depending on the system (∼23 nm for the EPEC T3SS ( Monjaras Feria et al.,
2012 ), ∼45 nm for the Shigella T3SS ( Tamano et al., 2002 )). The needle is
composed of multiple copies of a single protein (EscF in EPEC), which is well
conserved between species. EM helical reconstructions of the needle from the
Shigella (MxiH) ( Cordes et al., 2003 ; Fujii et al., 2012 ) and Salmonella SPI-1
(PrgI) T3SSs ( Galkin et al., 2010 ) suggested that the needle helical arrangement
possesses a pitch of 25 Å, and contains approximately 5.5 subunits per turn,
which is similar to the flagellum filament.
Purified needle proteins spontaneously form extended needle-like oligo-
meric assemblies of several µm in length ( Poyraz et al., 2010 ), which has
impaired high-resolution X-ray crystallographic characterization in the context
of the assembled needle; nonetheless, several structures of point and truncation
mutants that inhibit oligomerization, or of needle monomers complexed with