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HlyD-dependent secretion of toxins by Gran-negative bacteria
FEMS MicrobiologyImmunology105 (1992)45-54 © 1992 Federation of European MicrobiologicalSocieties 0920-8534/92/$05.00 Published by Elsevier
45
FEMSIM 00230
The HlyB/HlyD-dependent secretion of toxins by Gram-negative bacteria * Vassilis K o r o n a k i s , P e t e r Stanley, E v a K o r o n a k i s a n d Colin H u g h e s
Department of Pathology, CambridgeUniversity, Cambridge, UK
Key words: Protein export; Escherichia coli hemolysin; R T X toxin; Membrane translocation
1. S U M M A R Y Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer m e m b r a n e s of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate. It is directed by an uncleaved C-terminal targetting signal and the HIyD and HIyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the H l y B / H l y D - d e p e n d e n t translocation allow a discussion of the hemolysin export process in the wider context of protein translocation.
Correspondence to: V. Koronakis, Cambridge University Department of Pathology,CB2 1QP Cambridge, UK. * Figures in this article were reprinted or modified, by permission, from our publications in Mol. Microbiol. 5, 23912403, Blackwell Scientific Publications and EMBO J. 10, 3263-3272, Oxford UniversityPress.
2. S E C R E T I O N O F H E M O L Y S I N ACROSS B A C T E R I A L CYTOPLASMIC AND O U T E R MEMBRANES Export of proteins across the bacterial cytoplasmic membrane is characteristically directed by an N-terminal leader signal sequence and is dependent upon several cellular sec genes [1], Further movement of proteins across the outer membrane of Gram-negative bacteria is rare and is generally achieved by periplasmic intermediates which are exported by distinct outer membrane-translocation mechanisms e.g. IgA protease, aerolysin or cholera toxin (Fig. 1) [2-4]. Secretion of the l l 0 - k D a hemolysin protein (HlyA) by E. coli is exceptional in that it occurs without an N-terminal signal, and proceeds across both membranes into the surrounding medium without employing a periplasmic intermediate [57]. The HlyA secretion process is a model for the closely related export of toxins and proteases by other Gram-negative bacterial pathogens of man, animals and plants~ including Proteus, Actinobacillus, Pasteurella, Erwinia, Serratia, Pseudomonas, and Bordetella pertussis, and probably the NodO plant nodulation protein by Rhizobium [8-14].
46
contiguous and co-expressed ~vith the hlyC and hlyA genes responsible for synthesis of cytolyti-
IgA protease JII choleratoxin Or01"-::.
conventional
I~ c
HEMOLYSIN
~..'.;~
Fig. 1. Protein secretion across the Gram-negative bacterial outer membrane (OM). Hemolysin does not employ a two-step process based on initial use of the conventional translocation across the cytoplasmic membrane (CM), i.e. involving secA and an N-terminal signal.
3. HEMOLYSIN SECRETION IS DIRECTED BY THE HIyB AND HIyD MEMBRANE PROTEINS The hemolysin export process is directed by two specific membrane-located secretion proteins HIyB and HlyD, which are encoded by genes
terminator
HIyT
0"~.~.~ hlyR
I 973 I
cally active toxin (Fig. 2) [15,16]. Hemolysin secretion is also influenced by a further 'unlinked' locus, tolC, which encodes an outer membrane protein that has substantial pleiotropic effects on the bacterial envelope [17]. HIyB and HIyD are predicted to be 80 and 54 kDa respectively, and are present at very low levels in the cell membrane fraction, primarily due to down regulation of secretion gene expression. This is achieved by uncoupling operon transcription of the synthesis and secretion genes via transcript termination at a rho-independent terminator within the operon [16,18], an effect probably accentuated by differential stability of the truncated and elongated transcripts. Expression of the specific hemolysin export function is mediated by transcript antitermination at the hlyA-hlyB intergenic terminator which allows elongation of mRNA into hlyB and hlyD. This is dependent on structural elements, including the AT-rich hlyT locus, which are positioned upstream of the hly operon promoter sequences and which are thought to act as binding sites for regulatory proteins involved in both h/y
=11" "
tolC
secretion
synthesis
II ~ ' ~ , = ~ % . ~ ' ~ ! i l s C ~ . f , ~
IT~F1
hlyC
hlyA .~. . . ~ IS ...hlyB~
Re~ion 1
secretion si~lnal
- - - -- "--II1||1|111~
hlyD
Re(lion 2
~
.--I
990 I
10110
1 02. 3
LN P LIN EISKIISAAGSF[DVKE ER~AASLLQLSG NASDFSYGRNISITLTTSAI e charged cluster
unchargedregion
hydroxylated
cluster
potential a - helix potential amphipathJc helices Fig. 2. The h/y operon encoding synthesis and secretion of hemolysin. The gene hlyA encodes the inactive prohemolysin which is activated to cytolytic HlyA by HlyC. HIyA is secreted by a process dependent upon HIyB, HIyD and also TolC. Two h/y transcripts are synthesized, truncated (Tt) and elongated (Te), as a result primarily of transcription termination at the hlyA-hlyB intergenic space and antitermination determined by the interaction of trans-acting protein(s), e.g. HIyT and structural elements e.g. hlyR, upstream of the hly promoter. Secretion is directed by the C-terminal secretion signal of the 1024 amino acid HlyA, which is proposed to have a tripartite structure.
47 transcription activation and antitermination. [1820]; unpublished results). The HlyB secretion protein is thought to comprise a number of cytoplasmic membrane-spanning regions and an extensive C-terminal domain located in the cytoplasm [21-23]. The cytoplasmic domain in particular shares close homology with a wide range of proteins (Table 1) which are involved or implicated in the transport of various relatively small molecules such as sugars, antitumour drugs, ions, peptides and pheromones across membranes of bacteria, yeast, parasites and mammalian cells, but HlyB shares even more extensive homology with mammalian members such as the Mdr proteins. Discovery of the close homology with the known secretion protein HIyB prompted models for the action of Mdr [24] and other proteins as membrane pumps, and the genetically-derided HlyB-dependent transport may prove valuable in explaining how diverse substrates are recognised by these translocators, possibly involving a HlyA-type intermediary and successive lipid and translocator interactions. The common protein domains are thought, and in some cases have been shown, to bind NTPs and it is felt that a capacity to hydrolyse ATP or use it for phosphorylation is central to the ability of these translocation proteins to act as membrane pumps. Indeed they have been grouped as mem-
bers of the ATP-binding cassette (ABC) superfamily [23]. ATP is required for in vitro bacterial uptake of amino acids and sugars by members of this family and ATP hydrolysis occurs during this import [25]. The belief that HlyB-directed ATP hydrolysis or phosphorylation is necessary for secretion of the large hemolysin protein has not yet been supported by any direct evidence, although amino acid substitutions in the ATP-binding region of HIyB do debilitate secretion [26].
4. TARGETTING TO THE CYTOPLASMIC MEMBRANE BY THE UNCLEAVED HIyA C-TERMINAL SIGNAL The 1024-residue HIyA protein has an unprocessed secretion signal within the final 48 amino acids of its C-terminus (Fig. 2) [6,27,28]. HlyA C-terminal peptides of ca. 200 amino acids are recognised and secreted by HIyB/HIyD and neither these nor full HIyA derivatives carrying intact or defective secretion signals appear in the periplasm (Fig. 3) [6,7]. This supports the possibility that HlyA is exported directly into the medium via a HlyB/HIyD membrane contact site and without a periplasmic intermediate, and thus suggests possible analogies with the import of protein to the mitochondrial matrix [29]. Mutational
Table 1 HIyBand related proteins of prokaryoticand eukaryoticcells HIyBrelative HIyB,LktB,CyaB PrtD, NodI ColV HisP Pst Mall(
OppD ChvA,NdvA STE.6 P-glyeoprotein(MDR) PFMDR CFrR WB loci p69 HAM, mtp
Cell Bacteria Bacterm Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Yeast Human tumour Plasmodium
Human epithelial Drosophila Myceplasma HumanER
'Function/phenotype' Protein toxinexport Protein protease export Protein export?,noduleformation Bacteriocinexport Aminoacid import Phosphate import Sugar import Oligopeptide import Polysaccharideexport Pheromoneexport Drug export, resistance Drug export,resistance Ion pump?,cysticfibrosis Pteridine transport?,pigmentation Receptor? Peptide transport, MHC linked
48 (i) e x t r a c e l l u l a r
pmc
pmc
pmc
Fig. 3. (i) HlyB/HlyD-secretion into the extracellularmedium of the 1024 residue HlyA protein (wt) and C-terminal truncated derivativeslacking the number of residues indicated. (ii) Cellular location of the wt and truncated secretion-defective HlyA proteins identified by immunoblots; p, periplasm; m, membrane; and c, cytoplasm[6].
analyses suggest that the secretion signal has no primary sequence requirement and supports a tripartite structure (Fig. 2) in which the major feature is a flexible, amphipathic helix approximately 22 residues in length, fused to an uncharged sequence and a tail of primarily small and hydroxylated residues [6,28]. All the toxins and proteases secreted to the cell-free medium by this pathway carry C-terminal signals and these presumably share higher order structural similarities since many have been shown to be recognised and secreted by the E. coli HIyB and HlyD proteins [8,9,11]. Indeed, all have a potential amphipathic sequence of between 10 and 14 amino
(a)
(b)
IM N
HlyA
acids analogous to that of E. coli HlyA, with a central section of hydrophobic and small, hydroxylated residues. A model for the function of the HIyA C-terminal secretion signal (Fig. 4) suggests that the region I helix fuses with the inner membrane by inserting as a loop, presenting its hydrophobic face to the hydrophobic core of the bilayer, with the two ends of the helix kept at the membrane surface by the interaction of the charged residues with membrane phospholipids. Region I would unloop and span the inner membrane with the central region maintaining a 310 helix in order to cross the non-polar part of the bilayer [28]. Hess et al. [30] have suggested that region I could act as a 'spacer' sequence separating the bulk of the transported protein from the actual targetting signal, and the observation that the length of region I is critical would be compatible with it acting primarly as a transmembrane section. Once the region I helix unloops across the membrane, the uncharged region distal to region I forms a second transmembrane domain (Fig. 4b). There would be a loss in free energy associated with moving the charged groups in the Region I helix across the non-polar bilayer, but this may in part be compensated by a local disruption of the phospholipid bilayer caused by the initial binding of the looped helix [31], and the net negative charge at the bend between the two transmembrane sections may aid insertion by interacting with the membrane potential or proton gradient across the membrane. In support of this, the removal of all acidic residues from the HlyA charge cluster
~ IM e
N
(~_. ~
HlyA
~ B.
IM HlyA
e
Fig. 4. Possible steps in the interactions of the C-terminal signal with HIyBand the inner and outer membranes during the export of HlyA (see text for reference to steps (a), (b) and (c)). The N-terminal and C-terminal ends of HlyA are labelled N and C,
respectively. Positions of positive (+) and negative ( - ) charge are indicated but do not signifyquantity. Residues are numbered from the C-terminus. (IM, inner membrane; OM, outer membrane; P, periplasm).
49
drastically reduces secretion, and (see later), the early stage of HlyB/HlyD-dependent secretion is dependent on electrochemical potential [7]. The HIyA structure exposed to the periplasm in stage (II) of this model might act as a trigger for the fusion of the outer and inner membranes. Once fusion occurs, a second unlooping of the HlyA C-terminal sequence spans both membranes and exposes HIyA to the medium (Fig. 4c) and the second unlooping could bring either the region I or the region II transmembrane section into the outer membrane depending on whether the C-terminus remains on the cytoplasmic side or not. As presented in Fig. 4c, the HlyA Cterminus itself would provide a bridge across both membranes, but such a surface may be provided by the HlyB/HIyD complex. Either way, no periplasmic intermediate is formed and the model would accommodate data which show that in the absence of HIyB, HIyA becomes associated with the inner membrane, whereas in the presence of HIyB but in the absence of HIyD, HIyA is associated with both membranes with part of it exposed on the cell surface [32]. The hlyB and hlyD secretion genes of this alternative secretion system can be deleted without causing pleiotropic effects on the cell, in
contrast to genes which are central to conventional inner membrane secretion by bacteria and mitochondrial import [1,33]. It therefore allows experimental differentiation in vivo between binding of translocated proteins to the lipid bilayer and to the specific secretion machinery (possibly a membrane contact site) formed by HIyB and HIyD. Our proposal that the HIyA signal is involved in two stages of membrane recognition, with first the lipid bilayer and then the secretion protein(s), is supported by initial investigations with fusions of the HIyA C-terminal signal to non-translocatable globular structure proteins, which appear to be targetted to the membrane in the absence of HIyB and HIyD (unpublished results) and thus could represent intermediates at the initial stage of membrane targetting.
5. E N E R G E T I C A L L Y - D I S T I N C T E A R L Y AND LATE STAGES OF HIyB/D-DEPENDENT SECRETION We have differentiated two stages in the HlyB/D-dependent translocation process [7]: (i) an early stage which requires AP; and (ii) a late KCI (mM) 0 2 10 50 120
valinomycin-K+
co-T
v
Val-K +
post-T
c~,,.~ ~ AP ' ~ ~ A
pH
Nigcricin KCI(mM) valinomycin-K++ CCCP
~
0
2 10 50 120
post-T
Fig. 5. Dissipation of the total proton motive force (electrochemical potential, AP) and its components, the proton gradient (pH gradient, ApH) and the membrane potential (Aqt), by the potassium ionophore valinomycin, the proton ionophore carbonylcyanide m-chlorophenylhydrazone ( ~ ) and the K + / H + exchanger nigericin. The upper inset shows inhibition of HlyB/HlyD-dependent secretion of a HIyA C-terminal peptide Actp by valinomycin in what is termed the co-translational assay [7] reflecting the entire secretion process, but virtually no inhibition of secretion in the post-translational assay, which reflects only the late stage(s) of secretion. The lower inset shows no inhibition of the late stage in HlyB/HlyD-dependent secretion in the combined presence of valinomycin and CCCP, indicating that there is at this stage no requirement for AP, and possibly also no need for ATP hydrolysis.
50
stage of secretion, which is strongly dependent upon pH and temperature but which does not require AP (Fig. 5). The early AP-requirement can be met by either of the Ap components, the membrane potential (A~) and the pH gradient A pH, and thus equates to the requirement (which is distinct from the need for ATP in the translocation of subsequent intermediates [35,36]) of the conventional processing and transfer of /3-1actamase across the E. coli cytoplasmic membrane [34]. An electrochemical gradient is also required for an early stage of protein import to the mitochondrial inner membrane or matrix but the need here is specifically for the A~ component [37]. The AP requirement of the early stage of HlyB/D-directed secretion is suggested to reflect the needs of binding or some other interaction at the cytoplasmic membrane. It may be that the AP requirement in the early stage of HlyB/D-dependent secretion applies when the HIyA protein enters a close association with the HIyB/HIyD secretion machinery after release from the initial inner membrane complex, in a way similar to that outlined for protein passage into the periplasm during conventional SecA-dependent bacterial secretion [35]. Another possibly, less tenable, alternative to the prospect of HIyA entering a preformed membrane 'pore' (with or without preliminary association with the lipid bilayer) is to postulate an earlier cytosolic association between HIyA and one or both HlyB/D proteins which then insert into the inner membrane. In this case the AP-dependence could reflect the energy requirement for HlyB insertion into the inner membrane (the specificity of HlyD to the bacterial outer membrane systems suggests HIyB as t h e common 'recognition protein'). In vitro and in vivo experiments [35,36,38] have indicated that protein translocation across the cytoplasmic membrane comprises the membrane association of the pre-protein needing only the energy of the 'translocase'-bound SecA ATPase, while AP is a requirement for a subsequent step once the translocation intermediate has been released from SecA. Secretion of M13 procoat, which does not utilize the SecA/Y translocase, requires Ap for insertion into the membrane [39]. It seems likely that one or more of these requirements for Ap is
shared by the early stage of hemolysin export but release of protein into the periplasm is replaced in this case by entry into some form of energetically-primed membrane fusion channel or complex, independent of a periplasmic intermediate. In this way the outer membrane barrier is not confronted directly, in contrast to the movement Ao
+ / - CCCP
treatment
{~,
"
J J
t rlrYaPtSinn t
• trypsin
inhibitor
• secretion assay
secretion assay +
trypsin control (O.Ot mg/ml)
SPHEROPLASTS
B. IPt
spheropl, + B/D 2 3 4 5 6
whole ceils -I- BID 1 2 3 4 5 6
spheropl. - - BID 1 2 3 4
ccc trypsin treatmel Irypsin conlo
Fig. 6. Trypsin-accessibility of the AP-independent Actp transiocation intermediate in the late stage of HlyB/HlyD-dependent secretion. The lower half of the figure shows the presence or absence of radiolabelled Actp secreted from whole cells and spheroplasts following treatment as indicated in the cage and the diagram. 1P is an immunoprecipitated control. The data show that in both whole cells and spheroplasts the inherently trypsin-sensitive translocating Actp is inaccessible (in the diagram, possible protected sites are indicated by filled circles, accessible sites by open circles), indicating that the intermediate is located in a translocation complex at the inner membrane (IM) or a membrane contact site (MC)
[7].
51
Oh
HlyC-acyl i N (i) release ~ maintainaneo of unfolded state ~ ' ~
'
(ii) bindingto lipid bilayer
N/
(iii) bindingto HlyB/D translocatorat IM
~ ~N
(iv) looping, translocationthroughOM
I "C
I J
~
_/1
/1~! ~
~ ( ~
/
C~+ 0
(v) bind 0=2 +, targetting,to rnammalianrnembranesf / Fig. 7. Summary model of the process directing secretion of intraceliular HIyA (IN) out into the extracellular medium (OUT), where it is targetted to mammalian cells. FA, fatty acid; IM, inner membrane; OM, outer membrane; P, periplasm, MM, mamrnalian membrane.
of periplasmic aerolysin across this membrane, which is suggested to need Ap [4]. A translocation intermediate identified in the AP-independent late stage of HlyB/HlyD-dependent secretion was not accessible to trypsin in either whole cells or spheroplasts (Fig. 6), indicating that it is located at the inside of the cytoplasmic membrane in an early translocation complex, which could be a HlyB/D-induced double membrane contact site. Late translocation to the outer membrane and beyond could be driven directly by HIyB function, e.g. by ATP binding and possibly hydrolysis or phosphorylation, but it is also possible that this transfer is energetically favourable, requiring no additional energy (in the early stage of mitochondrial protein import for example, once the targetting sequence has traversed the inner membrane the bulk of the protein is translocated independently of A ~ [29]). The persistence of Actp secretion in the presence of high levels of AP-uncouplers would appear to support the second possibility as intracellular ATP would
be depleted under these circumstances. That HIyB is not energetically involved in the late stage of protein secretion is also suggested by the function of HIyB analogues in translocation across a single eukaryotic membrane. This is also supported by analogy with conventional export from the bacterial cytosol which requires ATP hydrolysis by the SecA ATPase, apparently for chaperone function and early translocation [36,40], and also the import of pre-pro-alpha factor and A D P / A T P carrier into microsomes and mitochondria, respectively [41,42] which are suggested to need ATP hydrolysis for maintaining translocation competence. The data allow one to suggest a summary of the secretion process as presented in Fig. 7.
ACKNOWLEDGEMENTS We thank the MRC, Wellcome Trust and AFRC for financial support.
52
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C-terminal secretion signal of haemolysin. EMBO J. 6, 2835-2841. Stanley, P., Koronakis, V. and Hughes, C. (1991) Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli hemolysin. Mol. Microbiol. 5, 2391-2403. Schleyer, M. and Neupert, W. (1985) Transport of proteins into mitochondria: translocation intermediates spanning contact sites between outer and inner membranes. Cell 43, 339-350. Hess, J., Gentshav, I., Goebel, W. and Jarchau, T. (1990) Analysis of the hemolysin secretion by phoA-HlyA fusion proteins. Mol. Gen. Genet. 224, 201-208. De Vrije, G.J., Batenburg, A.M., Killian, J.A. and De Kruijff, B. (1990) Lipid involvement in protein translocation in Escherichia coli. Mol. Microbiol. 4, 143-150. Oropeza-Wekerle R.L., Speth, W., Imhof, B., Gentshev, I. and Goebel, W. (1990) Translocation and compartmentalization of Escherichia coli hemolysin (HIyA). J. Bacteriol. 172, 3711-3717. Baker, K.P., Schaniel, A., Vestweber, D. and Schatz, G. (1990) A yeast mitochondrial outer membrane protein essential for protein import and soil viability. Nature 348, 605-609. Bakker, E.P. and Randall, L.L. (1984) The requirement for energy during export of /3-1actamase in Escherichia coli is fulfilled by the total proton motive force. EMBO J. 3, 895-900.
[35] Geller, B. (1990) Electrochemical potential releases a membrane-bound secretion intermediate of maltosebinding protein in Escherichia coli. J. Bacteriol. 172, 4870-4876. [36] Schiebel, E., Driessen, A.J., Hartl, F-U. and Wickner, W. (1991) A/~H + and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell 64, 927-939. [37] Planner, N., and Neupert, W. (1985) Transport of protein into mitocbondria: a potassium diffusion potential is able to drive the import of A D P / A T P carrier. EMBO J. 4, 2819-2825. [38] Thorn, J.R. and Randall, L.L. (1988) Role of the leader peptide of maltose-binding protein in two steps of the export process. J. Bacteriol. 170, 5654-5661. [39] Date, T., Goodman, J.M. and Wickner, W. (1980) Procoat, the precursor of M13 coat protein, requires an electrochemical potential for membrane insertion. Proc. Natl. Acad. Sci. USA 77, 4669-4673. [40] Geller, B. and Green, H.M. (1989) Translocation of ProOmpA across inner membrane vesicles of E. coli occurs in two consecutive energetically-distinct steps., J. Biol. Chem. 264, 16465-16469. [41] Chirico, W.J., Waters, M.G. and Blobel, G. (1988) 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 332, 805-810. [42] Planner, N., Tropschug, M. and Neupert, W. (1987) Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors. Cell 49, 815-823.
45
FEMSIM 00230
The HlyB/HlyD-dependent secretion of toxins by Gram-negative bacteria * Vassilis K o r o n a k i s , P e t e r Stanley, E v a K o r o n a k i s a n d Colin H u g h e s
Department of Pathology, CambridgeUniversity, Cambridge, UK
Key words: Protein export; Escherichia coli hemolysin; R T X toxin; Membrane translocation
1. S U M M A R Y Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer m e m b r a n e s of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate. It is directed by an uncleaved C-terminal targetting signal and the HIyD and HIyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the H l y B / H l y D - d e p e n d e n t translocation allow a discussion of the hemolysin export process in the wider context of protein translocation.
Correspondence to: V. Koronakis, Cambridge University Department of Pathology,CB2 1QP Cambridge, UK. * Figures in this article were reprinted or modified, by permission, from our publications in Mol. Microbiol. 5, 23912403, Blackwell Scientific Publications and EMBO J. 10, 3263-3272, Oxford UniversityPress.
2. S E C R E T I O N O F H E M O L Y S I N ACROSS B A C T E R I A L CYTOPLASMIC AND O U T E R MEMBRANES Export of proteins across the bacterial cytoplasmic membrane is characteristically directed by an N-terminal leader signal sequence and is dependent upon several cellular sec genes [1], Further movement of proteins across the outer membrane of Gram-negative bacteria is rare and is generally achieved by periplasmic intermediates which are exported by distinct outer membrane-translocation mechanisms e.g. IgA protease, aerolysin or cholera toxin (Fig. 1) [2-4]. Secretion of the l l 0 - k D a hemolysin protein (HlyA) by E. coli is exceptional in that it occurs without an N-terminal signal, and proceeds across both membranes into the surrounding medium without employing a periplasmic intermediate [57]. The HlyA secretion process is a model for the closely related export of toxins and proteases by other Gram-negative bacterial pathogens of man, animals and plants~ including Proteus, Actinobacillus, Pasteurella, Erwinia, Serratia, Pseudomonas, and Bordetella pertussis, and probably the NodO plant nodulation protein by Rhizobium [8-14].
46
contiguous and co-expressed ~vith the hlyC and hlyA genes responsible for synthesis of cytolyti-
IgA protease JII choleratoxin Or01"-::.
conventional
I~ c
HEMOLYSIN
~..'.;~
Fig. 1. Protein secretion across the Gram-negative bacterial outer membrane (OM). Hemolysin does not employ a two-step process based on initial use of the conventional translocation across the cytoplasmic membrane (CM), i.e. involving secA and an N-terminal signal.
3. HEMOLYSIN SECRETION IS DIRECTED BY THE HIyB AND HIyD MEMBRANE PROTEINS The hemolysin export process is directed by two specific membrane-located secretion proteins HIyB and HlyD, which are encoded by genes
terminator
HIyT
0"~.~.~ hlyR
I 973 I
cally active toxin (Fig. 2) [15,16]. Hemolysin secretion is also influenced by a further 'unlinked' locus, tolC, which encodes an outer membrane protein that has substantial pleiotropic effects on the bacterial envelope [17]. HIyB and HIyD are predicted to be 80 and 54 kDa respectively, and are present at very low levels in the cell membrane fraction, primarily due to down regulation of secretion gene expression. This is achieved by uncoupling operon transcription of the synthesis and secretion genes via transcript termination at a rho-independent terminator within the operon [16,18], an effect probably accentuated by differential stability of the truncated and elongated transcripts. Expression of the specific hemolysin export function is mediated by transcript antitermination at the hlyA-hlyB intergenic terminator which allows elongation of mRNA into hlyB and hlyD. This is dependent on structural elements, including the AT-rich hlyT locus, which are positioned upstream of the hly operon promoter sequences and which are thought to act as binding sites for regulatory proteins involved in both h/y
=11" "
tolC
secretion
synthesis
II ~ ' ~ , = ~ % . ~ ' ~ ! i l s C ~ . f , ~
IT~F1
hlyC
hlyA .~. . . ~ IS ...hlyB~
Re~ion 1
secretion si~lnal
- - - -- "--II1||1|111~
hlyD
Re(lion 2
~
.--I
990 I
10110
1 02. 3
LN P LIN EISKIISAAGSF[DVKE ER~AASLLQLSG NASDFSYGRNISITLTTSAI e charged cluster
unchargedregion
hydroxylated
cluster
potential a - helix potential amphipathJc helices Fig. 2. The h/y operon encoding synthesis and secretion of hemolysin. The gene hlyA encodes the inactive prohemolysin which is activated to cytolytic HlyA by HlyC. HIyA is secreted by a process dependent upon HIyB, HIyD and also TolC. Two h/y transcripts are synthesized, truncated (Tt) and elongated (Te), as a result primarily of transcription termination at the hlyA-hlyB intergenic space and antitermination determined by the interaction of trans-acting protein(s), e.g. HIyT and structural elements e.g. hlyR, upstream of the hly promoter. Secretion is directed by the C-terminal secretion signal of the 1024 amino acid HlyA, which is proposed to have a tripartite structure.
47 transcription activation and antitermination. [1820]; unpublished results). The HlyB secretion protein is thought to comprise a number of cytoplasmic membrane-spanning regions and an extensive C-terminal domain located in the cytoplasm [21-23]. The cytoplasmic domain in particular shares close homology with a wide range of proteins (Table 1) which are involved or implicated in the transport of various relatively small molecules such as sugars, antitumour drugs, ions, peptides and pheromones across membranes of bacteria, yeast, parasites and mammalian cells, but HlyB shares even more extensive homology with mammalian members such as the Mdr proteins. Discovery of the close homology with the known secretion protein HIyB prompted models for the action of Mdr [24] and other proteins as membrane pumps, and the genetically-derided HlyB-dependent transport may prove valuable in explaining how diverse substrates are recognised by these translocators, possibly involving a HlyA-type intermediary and successive lipid and translocator interactions. The common protein domains are thought, and in some cases have been shown, to bind NTPs and it is felt that a capacity to hydrolyse ATP or use it for phosphorylation is central to the ability of these translocation proteins to act as membrane pumps. Indeed they have been grouped as mem-
bers of the ATP-binding cassette (ABC) superfamily [23]. ATP is required for in vitro bacterial uptake of amino acids and sugars by members of this family and ATP hydrolysis occurs during this import [25]. The belief that HlyB-directed ATP hydrolysis or phosphorylation is necessary for secretion of the large hemolysin protein has not yet been supported by any direct evidence, although amino acid substitutions in the ATP-binding region of HIyB do debilitate secretion [26].
4. TARGETTING TO THE CYTOPLASMIC MEMBRANE BY THE UNCLEAVED HIyA C-TERMINAL SIGNAL The 1024-residue HIyA protein has an unprocessed secretion signal within the final 48 amino acids of its C-terminus (Fig. 2) [6,27,28]. HlyA C-terminal peptides of ca. 200 amino acids are recognised and secreted by HIyB/HIyD and neither these nor full HIyA derivatives carrying intact or defective secretion signals appear in the periplasm (Fig. 3) [6,7]. This supports the possibility that HlyA is exported directly into the medium via a HlyB/HIyD membrane contact site and without a periplasmic intermediate, and thus suggests possible analogies with the import of protein to the mitochondrial matrix [29]. Mutational
Table 1 HIyBand related proteins of prokaryoticand eukaryoticcells HIyBrelative HIyB,LktB,CyaB PrtD, NodI ColV HisP Pst Mall(
OppD ChvA,NdvA STE.6 P-glyeoprotein(MDR) PFMDR CFrR WB loci p69 HAM, mtp
Cell Bacteria Bacterm Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Yeast Human tumour Plasmodium
Human epithelial Drosophila Myceplasma HumanER
'Function/phenotype' Protein toxinexport Protein protease export Protein export?,noduleformation Bacteriocinexport Aminoacid import Phosphate import Sugar import Oligopeptide import Polysaccharideexport Pheromoneexport Drug export, resistance Drug export,resistance Ion pump?,cysticfibrosis Pteridine transport?,pigmentation Receptor? Peptide transport, MHC linked
48 (i) e x t r a c e l l u l a r
pmc
pmc
pmc
Fig. 3. (i) HlyB/HlyD-secretion into the extracellularmedium of the 1024 residue HlyA protein (wt) and C-terminal truncated derivativeslacking the number of residues indicated. (ii) Cellular location of the wt and truncated secretion-defective HlyA proteins identified by immunoblots; p, periplasm; m, membrane; and c, cytoplasm[6].
analyses suggest that the secretion signal has no primary sequence requirement and supports a tripartite structure (Fig. 2) in which the major feature is a flexible, amphipathic helix approximately 22 residues in length, fused to an uncharged sequence and a tail of primarily small and hydroxylated residues [6,28]. All the toxins and proteases secreted to the cell-free medium by this pathway carry C-terminal signals and these presumably share higher order structural similarities since many have been shown to be recognised and secreted by the E. coli HIyB and HlyD proteins [8,9,11]. Indeed, all have a potential amphipathic sequence of between 10 and 14 amino
(a)
(b)
IM N
HlyA
acids analogous to that of E. coli HlyA, with a central section of hydrophobic and small, hydroxylated residues. A model for the function of the HIyA C-terminal secretion signal (Fig. 4) suggests that the region I helix fuses with the inner membrane by inserting as a loop, presenting its hydrophobic face to the hydrophobic core of the bilayer, with the two ends of the helix kept at the membrane surface by the interaction of the charged residues with membrane phospholipids. Region I would unloop and span the inner membrane with the central region maintaining a 310 helix in order to cross the non-polar part of the bilayer [28]. Hess et al. [30] have suggested that region I could act as a 'spacer' sequence separating the bulk of the transported protein from the actual targetting signal, and the observation that the length of region I is critical would be compatible with it acting primarly as a transmembrane section. Once the region I helix unloops across the membrane, the uncharged region distal to region I forms a second transmembrane domain (Fig. 4b). There would be a loss in free energy associated with moving the charged groups in the Region I helix across the non-polar bilayer, but this may in part be compensated by a local disruption of the phospholipid bilayer caused by the initial binding of the looped helix [31], and the net negative charge at the bend between the two transmembrane sections may aid insertion by interacting with the membrane potential or proton gradient across the membrane. In support of this, the removal of all acidic residues from the HlyA charge cluster
~ IM e
N
(~_. ~
HlyA
~ B.
IM HlyA
e
Fig. 4. Possible steps in the interactions of the C-terminal signal with HIyBand the inner and outer membranes during the export of HlyA (see text for reference to steps (a), (b) and (c)). The N-terminal and C-terminal ends of HlyA are labelled N and C,
respectively. Positions of positive (+) and negative ( - ) charge are indicated but do not signifyquantity. Residues are numbered from the C-terminus. (IM, inner membrane; OM, outer membrane; P, periplasm).
49
drastically reduces secretion, and (see later), the early stage of HlyB/HlyD-dependent secretion is dependent on electrochemical potential [7]. The HIyA structure exposed to the periplasm in stage (II) of this model might act as a trigger for the fusion of the outer and inner membranes. Once fusion occurs, a second unlooping of the HlyA C-terminal sequence spans both membranes and exposes HIyA to the medium (Fig. 4c) and the second unlooping could bring either the region I or the region II transmembrane section into the outer membrane depending on whether the C-terminus remains on the cytoplasmic side or not. As presented in Fig. 4c, the HlyA Cterminus itself would provide a bridge across both membranes, but such a surface may be provided by the HlyB/HIyD complex. Either way, no periplasmic intermediate is formed and the model would accommodate data which show that in the absence of HIyB, HIyA becomes associated with the inner membrane, whereas in the presence of HIyB but in the absence of HIyD, HIyA is associated with both membranes with part of it exposed on the cell surface [32]. The hlyB and hlyD secretion genes of this alternative secretion system can be deleted without causing pleiotropic effects on the cell, in
contrast to genes which are central to conventional inner membrane secretion by bacteria and mitochondrial import [1,33]. It therefore allows experimental differentiation in vivo between binding of translocated proteins to the lipid bilayer and to the specific secretion machinery (possibly a membrane contact site) formed by HIyB and HIyD. Our proposal that the HIyA signal is involved in two stages of membrane recognition, with first the lipid bilayer and then the secretion protein(s), is supported by initial investigations with fusions of the HIyA C-terminal signal to non-translocatable globular structure proteins, which appear to be targetted to the membrane in the absence of HIyB and HIyD (unpublished results) and thus could represent intermediates at the initial stage of membrane targetting.
5. E N E R G E T I C A L L Y - D I S T I N C T E A R L Y AND LATE STAGES OF HIyB/D-DEPENDENT SECRETION We have differentiated two stages in the HlyB/D-dependent translocation process [7]: (i) an early stage which requires AP; and (ii) a late KCI (mM) 0 2 10 50 120
valinomycin-K+
co-T
v
Val-K +
post-T
c~,,.~ ~ AP ' ~ ~ A
pH
Nigcricin KCI(mM) valinomycin-K++ CCCP
~
0
2 10 50 120
post-T
Fig. 5. Dissipation of the total proton motive force (electrochemical potential, AP) and its components, the proton gradient (pH gradient, ApH) and the membrane potential (Aqt), by the potassium ionophore valinomycin, the proton ionophore carbonylcyanide m-chlorophenylhydrazone ( ~ ) and the K + / H + exchanger nigericin. The upper inset shows inhibition of HlyB/HlyD-dependent secretion of a HIyA C-terminal peptide Actp by valinomycin in what is termed the co-translational assay [7] reflecting the entire secretion process, but virtually no inhibition of secretion in the post-translational assay, which reflects only the late stage(s) of secretion. The lower inset shows no inhibition of the late stage in HlyB/HlyD-dependent secretion in the combined presence of valinomycin and CCCP, indicating that there is at this stage no requirement for AP, and possibly also no need for ATP hydrolysis.
50
stage of secretion, which is strongly dependent upon pH and temperature but which does not require AP (Fig. 5). The early AP-requirement can be met by either of the Ap components, the membrane potential (A~) and the pH gradient A pH, and thus equates to the requirement (which is distinct from the need for ATP in the translocation of subsequent intermediates [35,36]) of the conventional processing and transfer of /3-1actamase across the E. coli cytoplasmic membrane [34]. An electrochemical gradient is also required for an early stage of protein import to the mitochondrial inner membrane or matrix but the need here is specifically for the A~ component [37]. The AP requirement of the early stage of HlyB/D-directed secretion is suggested to reflect the needs of binding or some other interaction at the cytoplasmic membrane. It may be that the AP requirement in the early stage of HlyB/D-dependent secretion applies when the HIyA protein enters a close association with the HIyB/HIyD secretion machinery after release from the initial inner membrane complex, in a way similar to that outlined for protein passage into the periplasm during conventional SecA-dependent bacterial secretion [35]. Another possibly, less tenable, alternative to the prospect of HIyA entering a preformed membrane 'pore' (with or without preliminary association with the lipid bilayer) is to postulate an earlier cytosolic association between HIyA and one or both HlyB/D proteins which then insert into the inner membrane. In this case the AP-dependence could reflect the energy requirement for HlyB insertion into the inner membrane (the specificity of HlyD to the bacterial outer membrane systems suggests HIyB as t h e common 'recognition protein'). In vitro and in vivo experiments [35,36,38] have indicated that protein translocation across the cytoplasmic membrane comprises the membrane association of the pre-protein needing only the energy of the 'translocase'-bound SecA ATPase, while AP is a requirement for a subsequent step once the translocation intermediate has been released from SecA. Secretion of M13 procoat, which does not utilize the SecA/Y translocase, requires Ap for insertion into the membrane [39]. It seems likely that one or more of these requirements for Ap is
shared by the early stage of hemolysin export but release of protein into the periplasm is replaced in this case by entry into some form of energetically-primed membrane fusion channel or complex, independent of a periplasmic intermediate. In this way the outer membrane barrier is not confronted directly, in contrast to the movement Ao
+ / - CCCP
treatment
{~,
"
J J
t rlrYaPtSinn t
• trypsin
inhibitor
• secretion assay
secretion assay +
trypsin control (O.Ot mg/ml)
SPHEROPLASTS
B. IPt
spheropl, + B/D 2 3 4 5 6
whole ceils -I- BID 1 2 3 4 5 6
spheropl. - - BID 1 2 3 4
ccc trypsin treatmel Irypsin conlo
Fig. 6. Trypsin-accessibility of the AP-independent Actp transiocation intermediate in the late stage of HlyB/HlyD-dependent secretion. The lower half of the figure shows the presence or absence of radiolabelled Actp secreted from whole cells and spheroplasts following treatment as indicated in the cage and the diagram. 1P is an immunoprecipitated control. The data show that in both whole cells and spheroplasts the inherently trypsin-sensitive translocating Actp is inaccessible (in the diagram, possible protected sites are indicated by filled circles, accessible sites by open circles), indicating that the intermediate is located in a translocation complex at the inner membrane (IM) or a membrane contact site (MC)
[7].
51
Oh
HlyC-acyl i N (i) release ~ maintainaneo of unfolded state ~ ' ~
'
(ii) bindingto lipid bilayer
N/
(iii) bindingto HlyB/D translocatorat IM
~ ~N
(iv) looping, translocationthroughOM
I "C
I J
~
_/1
/1~! ~
~ ( ~
/
C~+ 0
(v) bind 0=2 +, targetting,to rnammalianrnembranesf / Fig. 7. Summary model of the process directing secretion of intraceliular HIyA (IN) out into the extracellular medium (OUT), where it is targetted to mammalian cells. FA, fatty acid; IM, inner membrane; OM, outer membrane; P, periplasm, MM, mamrnalian membrane.
of periplasmic aerolysin across this membrane, which is suggested to need Ap [4]. A translocation intermediate identified in the AP-independent late stage of HlyB/HlyD-dependent secretion was not accessible to trypsin in either whole cells or spheroplasts (Fig. 6), indicating that it is located at the inside of the cytoplasmic membrane in an early translocation complex, which could be a HlyB/D-induced double membrane contact site. Late translocation to the outer membrane and beyond could be driven directly by HIyB function, e.g. by ATP binding and possibly hydrolysis or phosphorylation, but it is also possible that this transfer is energetically favourable, requiring no additional energy (in the early stage of mitochondrial protein import for example, once the targetting sequence has traversed the inner membrane the bulk of the protein is translocated independently of A ~ [29]). The persistence of Actp secretion in the presence of high levels of AP-uncouplers would appear to support the second possibility as intracellular ATP would
be depleted under these circumstances. That HIyB is not energetically involved in the late stage of protein secretion is also suggested by the function of HIyB analogues in translocation across a single eukaryotic membrane. This is also supported by analogy with conventional export from the bacterial cytosol which requires ATP hydrolysis by the SecA ATPase, apparently for chaperone function and early translocation [36,40], and also the import of pre-pro-alpha factor and A D P / A T P carrier into microsomes and mitochondria, respectively [41,42] which are suggested to need ATP hydrolysis for maintaining translocation competence. The data allow one to suggest a summary of the secretion process as presented in Fig. 7.
ACKNOWLEDGEMENTS We thank the MRC, Wellcome Trust and AFRC for financial support.
52
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