Type 1 and 5 secretion systems and associated toxins - Escherichia coli: Pathotypes and Principles of Pathogenesis - page 489

Secretion of HlyA via the TolC channel
TolC (55 kDa) is a multifunctional OMP in E. coli that can serve as an outer
membrane component for several processes, including T1SS and drug export
via RND (resistance, nodulation, division) systems ( Wandersman and Delepe-
laire, 1990; Zgurskaya and Nikaido, 2000 ). Crystal structures of TolC reveal
that it is a homotrimer in the outer membrane of E. coli where it forms a trans-
periplasmic channel-tunnel. The trimer is about 140 Å in length, comprising a
40 Å β-barrel (channel domain) and a 100 Å long α-helical barrel that projects
across the periplasmic space (tunnel domain) ( Koronakis et al., 2000 ). The mol-
ecule forms a tapering tube that is almost closed at the periplasm end and wide
open at the outer-membrane surface. The internal diameter of the channel is
around 35 Å and consists of a large water-filled cavity open to the extracellular
medium ( Koronakis et al., 2000 ).
It is proposed that TolC opens at the proximal end by an iris-like mecha-
nism, by unwinding the coiled-coiled helices so as to move the inner ring to the
exterior. This would open the periplasmic end of the channel going from 5 Å in
diameter to a maximal opening of around 20 Å ( Figure 16.2 ) ( Koronakis et al.,
2000; Bavro et al., 2008 ). Crystal structures of TolC mutants either stuck in
closed or semi-open states support this hypothesis ( Bavro et al., 2008 ). Opening
of TolC is triggered by members of the MFP class (HlyD in the case of hemo-
lysin) ( Janganan et al., 2011 ). Thus, HlyA binds HlyB, triggering a change in
conformation in the HlyB/HlyD complex. HlyD then binds to TolC, triggering
the opening of the periplasmic end of the TolC channel and making a complete
pore from cytoplasm to extracellular medium. The HlyA protein is then secreted
through the pore to the extracellular medium.
Mechanisms of action and role in virulence
Once secreted, E. coli HlyA acts as a pore-forming RTX cytotoxin, with a
broad range of cytocidal activity on a wide spectrum of cells from a variety of
species, including erythrocytes, leukocytes, granulocytes, monocytes, endothe-
lial cells, and renal epithelial cells ( Gadeberg and Orskov, 1984; Keane et al.,
1987; Bhakdi et al., 1989, 1990; Mobley et al., 1990; Suttorp et al., 1990 ).
HlyA alteration of membrane permeability causes lysis and death, which may
provide iron and prevent phagocytosis. Once inserted, HlyA behaves as an
integral membrane protein and causes target cell lysis by forming transmem-
brane pores that are cation-selective, pH-dependent, and apparently asym-
metric ( Bhakdi et al., 1986; Menestrina et al., 1995 ). Membrane insertion
of HlyA is thought to be through a monomolecular mechanism. It has been
estimated that only 1-3 HlyA molecules form the pore, with oligimerization
occurring by subsequent addition of monomers within the membrane ( Bhakdi
et al., 1986; Benz et al., 1989 ). A conserved region toward the N-terminus
of HlyA is essential for lysis and is predicted to be involved in pore forma-
tion, as it spans the only pronounced hydrophobic sequences in the otherwise