Type 1 and 5 secretion systems and associated toxins - Escherichia coli: Pathotypes and Principles of Pathogenesis - page 488

et al., 1996; Leeds and Welch, 1997 ). RfaH is a bacterial elongation factor
that increases expression of distal genes in several long, horizontally acquired
operons, including those encoding lipopolysaccharide (LPS) and F (fertility)
pilus biogenesis ( Bailey et al., 1997 ). Loss of RfaH or the ops element shows
a distinctive effect on hlyCABD transcription, modestly decreasing transcrip-
tion of the first two genes, hlyC and A, but virtually abolishing transcription
of hlyB and D ( Bailey et al., 1992, 1996; Nieto et al., 1996 ). The ops element
is required for RfaH recruitment to RNA polymerase (RNAP) ( Bailey et al.,
1996; Artsimovitch and Landick, 2002; Belogurov et al., 2009 ); it induces isom-
erization of the transcription elongation complex (TEC) into a distinct state
necessary for the sequence-specific recruitment of RfaH to the non-template
DNA strand. Following recruitment, RfaH remains bound to RNAP and acts as
an antiterminator by reducing RNAP pausing and termination at some factor-
independent and Rho-dependent signals ( Bailey et al., 1996 ).
After translation of HlyA, the protein is inactive until matured intracellu-
larly by the cotranslated HlyC, a fatty acid acyltransferase ( Issartel et al., 1991 ).
HlyC forms a homodimer that uses acyl-acyl carrier protein (ACP) as the fatty
acid donor to acylate two internal lysine residues, K564 (K1) and K690 (KII)
of HlyA. Although both lysines are acylated independently, both are required
for in vivo toxin activity. However, acylation is only required for the hemo-
lytic activity; the secretion of HlyA is independent of HlyC ( Koronakis and
Hughes, 1996 ).
After acylation HlyA is secreted via a T1SS formed by the proteins HlyB,
HlyD, and TolC. HlyB, the ABC component of secretion, couples ATP hydro-
lysis to the secretion of HlyA ( Koronakis et al., 1995 ). The topology of HlyB
(707 aa) has been determined, suggesting that the protein is inserted in the inner
membrane by eight hydrophobic, α-helical transmembrane domains (TMDs)
extending from the amino acid positions 38-432 ( Wang et al., 1991 ). The cyto-
plasmic loops of HlyB are large with many positively charged amino acids,
whereas in contrast the periplasmic loops are quite small. HlyD is a prototypical
and well-characterized member of the MFP family ( Dinh et al., 1994 ). HlyD
is anchored in the cytoplasmic membrane by a single TMD and possesses a
large 100 aa C-terminal periplasmic domain ( Schulein et al., 1992 ). This peri-
plasmic domain is highly conserved not only within the MFP family but also
within the superfamily of periplasmic efflux proteins (PEP) ( Schulein et al.,
1992 ). Contact between the HlyB:HlyD inner membrane complex and the final
component, TolC in the outer membrane, is primarily mediated by HlyD in
response to engagement by the HlyA substrate ( Thanabalu et al., 1998 ). The
fully active complex contains the substrate and all three export proteins, all of
which undergo conformational change. The complex is transient however; once
the substrate leaves the cell the inner-membrane and outer-membrane compo-
nents disengage ( Thanabalu et al., 1998 ). As TolC is involved with at least four
different export systems, the mechanism behind forming the physical bridge
across the periplasm has been well studied.