Type 3 secretion effectors - Escherichia coli: Pathotypes and Principles of Pathogenesis - page 450

intimate attachment of the bacteria to the host membrane and actin polymerization
underneath the bacterial attachment site. In cultured cells the actin accumulation
is visualized as raised pedestal-like structures underneath adherent bacteria. EPEC
and EHEC have derived an efficient way to ensure this important step in coloni-
zation occurs by expressing an outer-membrane adhesin, intimin, as well as the
translocated intimin receptor (Tir), which the bacteria deliver into the host cell via
the T3SS. Tir inserts into the host cell membrane in a hairpin loop topology with the
extracellular loop interacting with intimin; the bacteria therefore provide both the
adhesin and the 'host cell' receptor required for attachment ( Figure 15.1 A).
Intimin is encoded by the eae gene on the LEE PI and is secreted by the
general secretory pathway to be inserted into the bacterial outer membrane
( McDaniel et al., 1995 ; Touze et al., 2004 ). In addition to binding the T3SS
effector Tir, intimin can also interact with endogenous host cell proteins, includ-
ing β1-chain integrins ( Isberg and Leong, 1990 ; Frankel et al., 1996 ) and nucle-
olin, which is up-regulated by Shiga toxin production ( Robinson et al., 2006 ).
The T3SS effector Tir not only acts as the host cell receptor for intimin but,
upon binding, intimin initiates Tir clustering which mediates protein signaling
within epithelial cells. Diverse EPEC and EHEC strains have evolved different
mechanisms of Tir signaling, all of which result in actin accumulation under-
neath the bacterial attachment site ( Figure 15.1 A). In some EPEC strains, Tir is
phosphorylated at tyrosine 474 (Y474p) by host tyrosine kinases, (the specific
kinases involved remain unclear) ( Phillips et al., 2004 ; Swimm et al., 2004 ).
Tir phosphorylation promotes its interaction with the SH2 domain of the adap-
tor protein Nck leading to the recruitment of neural Wiskott-Aldrich syndrome
protein (N-WASP) via the Nck SH3 domain ( Gruenheid et al., 2001 ; Phillips
et al., 2004 ). Activation of N-WASP occurs by relieving its autoinhibition fold,
allowing interaction with the actin-related protein 2/3 (Arp2/3) complex and
initiation of actin polymerization ( Gruenheid et al., 2001 ; Campellone et al.,
2002 ). Proteins normally involved in endocytosis, clathrin ( Veiga et al., 2007 ),
CD-2-associated protein (CD2AP) ( Guttman et al., 2010 ), and dynamin-2
( Unsworth et al., 2007 ), are also involved in pedestal formation via the Nck-
dependent pathway, the significance of which requires further investigation.
EHEC Tir lacks a tyrosine 474 equivalent and the process of actin polymeriza-
tion is mediated via the non-LEE encoded T3SS translocated effector protein,
TccP (Tir-cytoskeleton coupling protein) ( Garmendia et al., 2004 ) also known
as EspF U ( E. coli secreted protein F in prophage U) ( Campellone et al., 2004 ).
TccP/EspF U interacts with the IRSp53/MIM proteins, IRTKS and IRSp53, which
also bind Tir at an Asn-Pro-Tyr (NPY458) tripeptide in the Tir C-terminal
domain thereby linking TccP/EspF U indirectly to Tir ( Campellone and Leong,
2005 ; Brady et al., 2007 ; Vingadassalom et al., 2009 ). TccP/EspF U also inter-
acts with and activates N-WASP to initiate Arp2/3 recruitment and actin
polymerization ( Cheng et al., 2008 ). In mammalian cells TccP/EspF U was
also shown to induce Arp2/3 complex-dependent actin polymerization in the
absence of N-WASP suggesting that alternative proteins can act in this pathway