Bacterial secretins form constitutively open pores akin to general

36 Pages·2013·0.74 MB·English

JB Accepts, published online ahead of print on 18 October 2013
J. Bacteriol. doi:10.1128/JB.00750-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
Bacterial secretins form constitutively open pores akin to general
(cid:883)
porins
(cid:884)
Elena Disconzi1,2,3,4, Ingrid Guilvout3,4, Mohamed Chami5, Muriel Masi1,2, Gerard H. M.
(cid:885) Huysmans3,4, Anthony P. Pugsley3,4 and Nicolas Bayan1,2#
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1 Université de Paris-Sud, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, e
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Orsay, France m

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2 Centre National de la Recherche Scientifique UMR 8619, Orsay, France. tp
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3 Institut Pasteur, Molecular Genetics Unit, rue du Dr. Roux Paris 15, France. a
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4 Centre National de Recherche Scientifique ERA3625, rue du Dr. Roux Paris 15, France. r
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5 C-CINA Center For Imaging and NanoAnalytics, Biozentrum, University of Basel, CH n
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(cid:883)(cid:883) 4058 Basel, Switzerland c
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(cid:883)(cid:885) 5
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#To whom correspondence should be addressed: Nicolas Bayan, IBBMC, Bât 430, Université
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(cid:883)(cid:886) de Paris-Sud, 91405 Orsay cedex. Tel: [phone], Fax: [phone], e-mail: 8

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(cid:883)(cid:887) [email protected] y
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Running title: Secretin pore size
(cid:883)(cid:890)
Keywords: liposomes, pore forming protein, reconstitution, leakage assay, cell-free protein
(cid:883)(cid:891) synthesis
(cid:884)(cid:882)
(cid:883)
(cid:884)(cid:883)
Abstract
(cid:884)(cid:884)
Proteins called secretins form large multimeric complexes that are essential for
(cid:884)(cid:885) macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence D
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(cid:884)(cid:886) suggests that the channels formed by some secretin complexes are not tightly closed, but their n
lo
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(cid:884)(cid:887) permeability properties have not been well characterized. Here, we used cell-free synthesis d
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(cid:884)(cid:888) coupled with spontaneous insertion into liposomes to investigate the permeability of the
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(cid:884)(cid:889) secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the

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(cid:884)(cid:890) efflux of small fluorescent molecules with a permeation cut-off similar to that of general tp
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(cid:884)(cid:891) porins. Other secretins were also found to form similar pores. To define the polypeptide jb
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a
s
(cid:885)(cid:882) region involved in determining the pore size, we analyzed a collection of PulD variants and m
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(cid:885)(cid:883) studied the roles of Gates 1 and 2, previously reported to affect the pore size of filamentous rg
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(cid:885)(cid:884) phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo n

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(cid:885)(cid:885) assay showed that substitution of the conserved proline residue at position 443 in PulD by e
c
e
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(cid:885)(cid:886) leucine increased the apparent size of the pore. The in vitro approach described here could be
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(cid:885)(cid:887) used to study the pore properties of membrane proteins whose production in vivo is toxic.
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(cid:885)(cid:888) 2
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(cid:885)(cid:889) Abbreviations b
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(cid:885)(cid:890) u
OM: outer membrane; T2SS: type II secretion system; T3SS: type III secretion system; T4P: e
s
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(cid:885)(cid:891) type 4 pilus assembly; PulDfl: full-length PulD; PulD28-42/259-660: truncated PulD consisting of
(cid:886)(cid:882) residues 28-42/259-660.
(cid:886)(cid:883)
(cid:886)(cid:884)
(cid:886)(cid:885)
(cid:884)
(cid:886)(cid:886)
(cid:886)(cid:887)
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Introduction
(cid:886)(cid:888)
Multi-domain proteins called secretins form large outer membrane (OM) complexes that act
(cid:886)(cid:889) as portals for protein (e.g., PulD, OutD, XcpQ) and filamentous bacteriophage (e.g., pIV)
(cid:886)(cid:890) secretion, for DNA uptake and type IV pilus assembly (T4P; e.g., PilQ) and for needle
(cid:886)(cid:891) assembly and protein secretion in type III secretion systems (T3SS) in Gram-negative bacteria D
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(cid:887)(cid:882) (1). Cryo-electron microscopy of the archetypical type II secretion system (T2SS) secretin n
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(cid:887)(cid:883) PulD from Klebsiella oxytoca revealed how twelve protomers arrange in a barrel-like d
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(cid:887)(cid:884) complex of dodecameric symmetry with an open, outwards-facing ring connected to a second
f
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(cid:887)(cid:885) ring creating a vestibule deep into the periplasm (2). A plug closes off the barrel near its m

h
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(cid:887)(cid:886) center. Other secretins have a similar architecture (3-5), but the structure of the membrane- tp
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(cid:887)(cid:887) embedded part of the complex, including the plug, has not been reported at atomic resolution. jb
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s
(cid:887)(cid:888) m
Other approaches have begun to reveal molecular details of secretin architecture. All secretins .
o
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(cid:887)(cid:889) share a common domain organization comprising a well-conserved C domain, which includes /

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(cid:887)(cid:890) the afore-mentioned gated membrane channel, and a less well conserved N domain that
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(cid:887)(cid:891) consists of up to four globular domains named N0 to N3, all of which are present in the ce
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(cid:888)(cid:882) prototype secretin PulD (Fig. 1A). The N domain is located in the periplasm and interacts b
e
r

(cid:888)(cid:883) with inner membrane components of the secretion machinery (6-9). The atomic resolution 2
5
,

2
(cid:888)(cid:884) structures of part of the N domains of several secretins have been solved by X-ray 0
1
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(cid:888)(cid:885) crystallography (T3SS: EscC (10) and T2SS: GspD (11) and XcpQ (12)) and NMR
b
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(cid:888)(cid:886) spectroscopy (T4P: EscC (13)). Some secretins, including PulD, possess a C-terminal g
u
e
s
(cid:888)(cid:887) extension (S domain; Fig. 1A) that interacts with a dedicated chaperone (PulS in the case of t
(cid:888)(cid:888) PulD) that protects it from degradation and promotes correct localization (14, 15). In PulD,
(cid:888)(cid:889) the C and S domains and the last of three periplasmic repeat domains (N ) (Fig. 1A) are
3
(cid:888)(cid:890) sufficient for targeting to- and insertion as a multimer into the OM (16).
(cid:888)(cid:891)
(cid:886)
The plug in the ≥6 nm-wide secretin channel presumably blocks the release of periplasmic
(cid:889)(cid:882) proteins when the secretin channel is in its resting state, i.e., when proteins or phages are not
(cid:889)(cid:883) being secreted and pili or needles are not assembled. However, the secretin channel is not
(cid:889)(cid:884) necessarily tightly closed. Several studies investigated the ability of reconstituted (resting
(cid:889)(cid:885) state) secretins to form pores or channels in non-native lipid bilayers. Data from conductance
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(cid:889)(cid:886) measurements were sometimes difficult to interpret. High currents were measured upon w
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(cid:889)(cid:887) reconstitution of the Pseudomonas aeruginosa secretin XcpQ in artificial membranes (17). lo
a
d
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(cid:889)(cid:888) Large structural fluctuations in XcpQ were proposed to cause the observed non-uniform d

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(cid:889)(cid:889) conductance, which was very high compared to porins, did not increase linearly with the o
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(cid:889)(cid:890) applied potential and even persisted when the applied potentials were high. The Yersinia t
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(cid:889)(cid:891) enterocolitica secretin YscC formed stable conductance channels when reconstituted in vitro, //
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(cid:890)(cid:882) but did not facilitate uptake of β-lactam antibiotics in vivo, indicative of a narrow molecular s
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(cid:890)(cid:883) weight cut-off (18). In contrast, PulD reconstituted into artificial bilayers only showed o
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(cid:890)(cid:884) fluctuating conductance when a high voltage was applied across the lipid bilayer (19), o
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(cid:890)(cid:885) suggesting that it is normally in a tightly closed conformation. However, while PulD
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(cid:890)(cid:886) produced in vivo in the presence of its chaperone PulS inserts in the outer membrane without m
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(cid:890)(cid:887) dramatically modifying its permeability, in the absence of PulS, it mislocalizes into the inner r

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(cid:890)(cid:888) membrane and induces a phage shock response (16). This phenomenon suggests that PulD ,

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(cid:890)(cid:889) forms a proton-permeable pore in the inner membrane. Electrophysiology studies also 1
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(cid:890)(cid:890) suggested that the pIV secretin channel is tightly closed in vitro, although it allows an y

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(cid:890)(cid:891) Escherichia coli strain lacking maltoporin (LamB) to grow on small maltodextrins (20). The e
s
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(cid:891)(cid:882) permeability of the pIV channel was increased by changes in the pIV sequence that allowed a
(cid:891)(cid:883) LamB-deficient strain to regain its ability to import maltopentose (21). These sequence
(cid:891)(cid:884) changes clustered mainly in two regions that were named Gates 1 and 2 because of their
(cid:891)(cid:885) proposed role in channel gating (21). To simplify the terminology, we will use the term
(cid:891)(cid:886)
(cid:887)
secretin channel to define the wide-open conduit that permits protein or phage egress from the
(cid:891)(cid:887) cell, and the term secretin pore to define the (hypothetical) partially closed conduit. The
(cid:891)(cid:888) existence and properties of a pore in a resting state secretin complex are the subjects of this
(cid:891)(cid:889) report.
(cid:891)(cid:890)
Many membrane proteins, including PulD and some other secretins, can be efficiently D
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(cid:891)(cid:891) synthesized and assembled into liposomes in a cell-free system (22-24). Here, we investigated n
lo
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(cid:883)(cid:882)(cid:882) the permeability properties of PulD, XcpQ, OutD and pIV inserted into liposomes preloaded d
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(cid:883)(cid:882)(cid:883) with small reporter molecules. A miniaturized version of the assay was used to screen a
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(cid:883)(cid:882)(cid:884) previously characterized PulD mutant library to identify residues influencing pore size (24,

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(cid:883)(cid:882)(cid:885) 25). tp
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(cid:883)(cid:882)(cid:886) jb
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(cid:883)(cid:882)(cid:887) .
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Materials and Methods
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Bacterial strains and growth conditions D
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E. coli K-12 PAP105 [Δ(lac-pro) F' (lacIq1 ΔlacZM15 proAB+ Tn10)], used for plasmid b
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(cid:883)(cid:882)(cid:890) manipulations and E. coli PAP7447 [MC4100 (F' lacIq pro+ Tn10)], with pulS, pulA, and 2
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(cid:883)(cid:882)(cid:891) pulC-O integrated into malPp and with a large deletion in pulD, were grown in LB medium at 2
0
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(cid:883)(cid:883)(cid:882) 30°C. E. coli strain NB190 (26) lacking cyanocobalamin receptor BtuB (proC22, trpE23, 8

b
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(cid:883)(cid:883)(cid:883) metE70, lysA23, rpoB308, thi-1, lacZ36, xyl-5, mtl-1, rpsL109, cyc-19, tsx-67, supE44, (cid:507)btuB g
u
e
(cid:883)(cid:883)(cid:884) arg F’ (lacIq1 ΔlacZM15 proAB Tn10)) was maintained in LB medium at 30°C. s
+ t
(cid:883)(cid:883)(cid:885) Cyanocobalamin permeability assays with this strain were performed in 0.2% glucose M63B1
(cid:883)(cid:883)(cid:886) minimal medium (0.4 % maltose in experiments with pCHAP1226) supplemented with all
(cid:883)(cid:883)(cid:887) amino acids except methionine and cysteine. Ampicillin (100 (cid:541)g/ml), chloramphenicol (25
(cid:883)(cid:883)(cid:888) (cid:541)g/ ml) and kanamycin (50 (cid:541)g/ ml) were used when appropriate. 5 (cid:541)g/ml of tetracycline was
(cid:883)(cid:883)(cid:889)
(cid:888)
used to maintain the F’ in NB190, PAP105 and PAP7447. Staphylococcus aureus RN4220
(cid:883)(cid:883)(cid:890) was grown in LB medium (27) at 37°C. Cloning experiments were all done in PAP105 at 30
(cid:883)(cid:883)(cid:891) °C.
(cid:883)(cid:884)(cid:882)
Gene constructions and plasmids
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Plasmids and primers used in this study are listed in Table S1 and S2, respectively. w
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(cid:883)(cid:884)(cid:884) pCHAP3674 and pCHAP3675 carrying wild type fhuA and fhuAΔ322-355, respectively, were a
d
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(cid:883)(cid:884)(cid:885) obtained by excision of the genes from pHK763 (28) and pHK226 (29), respectively, using d

f
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(cid:883)(cid:884)(cid:886) EcoRI and HindIII and inserted into pBGS19 digested with the same enzymes. m

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(cid:883)(cid:884)(cid:887) pCHAP3909, encoding α-hemolysin with a C-terminal His-tag, was obtained in a two-step tt
p
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(cid:883)(cid:884)(cid:888) cloning reaction. pCHAP3908 was first obtained by ligation of a BlpI and BglII fragment /jb
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a
(cid:883)(cid:884)(cid:889) from pT7-HL (30) into pIVEX2.3MCS digested with the same enzymes. A hexahistidine tag s
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(cid:883)(cid:884)(cid:890) was then introduced using primers ING163 and ING164 on pCHAP3908. The resulting r
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(cid:883)(cid:884)(cid:891) fragment was cleaved using NdeI and BamHI and ligated into pIVEX2.3MCS cleaved with o
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(cid:883)(cid:885)(cid:882) the same endonucleases. e
c
e
(cid:883)(cid:885)(cid:883) Erwinia carotovora outD was amplified from pCPP2242 (31) with primers ING206 and m
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(cid:883)(cid:885)(cid:884) ING207, digested with NdeI and BamHI and ligated into pIVEX2.3MCS cleaved by the same r

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(cid:883)(cid:885)(cid:885) enzymes to give pCHAP9718. ,

2
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(cid:883)(cid:885)(cid:886) The linker deletion in Dickeya dadantii OutD was created stepwise. The outD fragments up- 1
8

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(cid:883)(cid:885)(cid:887) and down-stream of residues 297-353 were amplified by PCR from pCHAP3794 using y

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(cid:883)(cid:885)(cid:888) primers ING86 and DIS26 and DIS27 and ING91, in which DIS26 and 27 have overlapping e
s
t
(cid:883)(cid:885)(cid:889) termini. Both halves were assembled together by a second PCR reaction using ING86 and
(cid:883)(cid:885)(cid:890) ING91. The resulting amplicon was cloned into pIVEX2.3MCS after digestion with NdeI and
(cid:883)(cid:885)(cid:891) SacI. Additional mutations introduced by the primers DIS26 and DIS27 were corrected by
(cid:883)(cid:886)(cid:882) site directed mutagenesis.
(cid:883)(cid:886)(cid:883)
(cid:889)
pCHAP9724 containing a P252L substitution in the filamentous phage secretin pIV was
(cid:883)(cid:886)(cid:884) obtained by mutagenesis using pCHAP9147 (24) and primers DIS43 and DIS44.
(cid:883)(cid:886)(cid:885)
PulD28-42/259-660ΔGate2 (pCHAP9715) was constructed from pCHAP3716 in two PCR steps.
(cid:883)(cid:886)(cid:886) First, two fragments were generated using primers ING59 and DIS22 and primers ING62 and
(cid:883)(cid:886)(cid:887) DIS23. In a second step, both amplified fragments were used as templates in a third reaction D
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(cid:883)(cid:886)(cid:888) with primers ING59 and ING62. The resulting amplicon was inserted in the plasmid pIVEX n
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(cid:883)(cid:886)(cid:889) 2.3MCS digested with NdeI and BamHI. PulD28-42/259-660ΔGate1 (pCHAP9722) was d
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(cid:883)(cid:886)(cid:890) constructed by using the same procedure as the one described for PulD28-42/259-660ΔGate2 using f
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(cid:883)(cid:886)(cid:891) primers DIS39 and DIS40 instead of DIS22 and DIS23.
h
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(cid:883)(cid:887)(cid:882) p
:
Sac7d was amplified by PCR from pQUANTagen sac7d-phoA (32) using primers ING218 //
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(cid:883)(cid:887)(cid:883) and ING219. The amplicon and pASK2C (IBA), encoding for an OmpA signal peptide and a a
s
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(cid:883)(cid:887)(cid:884) Strep-tag at opposite sides of the multiple cloning site, were digested with EcoRI and PstI and .o
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(cid:883)(cid:887)(cid:885) ligated together to form pCHAP3978. The fragment containing the sequence for Sac7d with
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(cid:883)(cid:887)(cid:886) an N-terminal OmpA signal peptide and a C-terminal Strep tag was then subcloned in D
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(cid:883)(cid:887)(cid:887) pASK12 (IBA) using the XbaI/HindIII restriction sites to give pCHAP9725. e
m
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(cid:883)(cid:887)(cid:888) All constructions were verified by DNA sequencing e
r

2
(cid:883)(cid:887)(cid:889) 5
,
Preparation of liposomes 2
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(cid:883)(cid:887)(cid:890) 8

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC; Avanti Polar Lipids) dissolved in b
y

g
(cid:883)(cid:887)(cid:891) chloroform was dried under a stream of nitrogen. The resulting thin lipid film was hydrated u
e
s
t
(cid:883)(cid:888)(cid:882) with different buffered solutions (50 mM TrisCl pH 7.4) containing 50 mM disodium calcein,
(cid:883)(cid:888)(cid:883) 10 mM carboxyfluorescein or 100 mg/ml of vancomycin. Carboxyfluorescein was added
(cid:883)(cid:888)(cid:884) from a stock solution of 0.4 M in dimethyl sulfoxide. Multilamellar liposomes were probe-
(cid:883)(cid:888)(cid:885) sonicated three times for 2 min (with 1 min intervals on ice) at 50% duty cycle and at an
(cid:883)(cid:888)(cid:886)
(cid:890)
output of 3 using a Sonics Vibracell sonicator. Liposomes were predominantly unilamellar
(cid:883)(cid:888)(cid:887) when examined by electron microscopy. Non-encapsulated compounds were removed by
(cid:883)(cid:888)(cid:888) dialysis (Spectra/Por membrane; MWCO 6-8 kDa) at 4°C against three changes of 2 L of 50
(cid:883)(cid:888)(cid:889) mM TrisCl (pH 7.4). Liposomes were collected by centrifugation at 35000 x g for 15 min and
(cid:883)(cid:888)(cid:890) resuspended in the same buffer to a final concentration of 25 mg/ml
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(cid:883)(cid:888)(cid:891) w
In vitro synthesis and purification of α-hemolysin n
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(cid:883)(cid:889)(cid:882) d
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His-tagged α-hemolysin was synthesized for 24h at 30°C by adding pCHAP3909 (10 µg) to d

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(cid:883)(cid:889)(cid:883) the RTS500 E. coli HY in vitro transcription/translation system (5 PRIME). The reaction was m

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(cid:883)(cid:889)(cid:884) diluted in 25 mM phosphate buffer, pH 8, and applied to a Ni-NTA column (1 ml; Qiagen) tt
p
:
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(cid:883)(cid:889)(cid:885) equilibrated with the same buffer. After washing with 10 column volumes of the same buffer, /jb
.
a
(cid:883)(cid:889)(cid:886) α-hemolysin was eluted with the same buffer containing 250 mM imidazole. Imidazole was s
m
.
o
(cid:883)(cid:889)(cid:887) removed by dialysis against the same buffer before the protein was concentrated on an r
g
/

(cid:883)(cid:889)(cid:888) Amicon ultra-4 centrifugal filter (Millipore). Protein purity was verified by SDS-PAGE. o
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(cid:883)(cid:889)(cid:889) e
c
Calcein release by purified α-hemolysin e
m
b
(cid:883)(cid:889)(cid:890)
e
Calcein fluorescence was excited at 490 nm and the emission was monitored at 520 nm in an r
2
5
(cid:883)(cid:889)(cid:891) Infinitive F200 Pro Tecan spectrofluorometer. To initiate leakage, 3 µg of α-hemolysin was , 2
0
1
(cid:883)(cid:890)(cid:882) added to 15 µl of 50 mM TrisCl buffer (pH 7.4) containing 0.5 M sucrose (to enable 8

b
y
(cid:883)(cid:890)(cid:883) comparison with MscL leakage) and 50 µg of calcein-loaded liposomes. The reaction was g
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(cid:883)(cid:890)(cid:884) performed in 384-well plates (black, flat bottom, Greiner) at 30°C with continuous shaking. s
t
(cid:883)(cid:890)(cid:885) The total calcein amount in the liposomes was indicated by the fluorescence observed upon
(cid:883)(cid:890)(cid:886) the addition of 0.1% Triton X-100. Fluorescence (determined every 90 seconds) was plotted
(cid:883)(cid:890)(cid:887) as the percentage of the total calcein present in the reaction.
(cid:883)(cid:890)(cid:888)
(cid:891)
Calcein and carboxyfluorescein release during in vitro synthesis of α-hemolysin or
(cid:883)(cid:890)(cid:889) secretins
(cid:883)(cid:890)(cid:890)
Plasmid DNA (150 ng) and calcein- or carboxyfluorescein-loaded liposomes (50 µg) were
(cid:883)(cid:890)(cid:891) added to 15 µl of the RTS100 E. coli HY reaction kit (5 PRIME). In vitro synthesis was
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(cid:883)(cid:891)(cid:882) performed in 384-well plates (black, flat bottom, Greiner), and incubated in a o
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(cid:883)(cid:891)(cid:883) spectrofluorometer as above at 30°C with continuous shaking. Calcein and lo
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(cid:883)(cid:891)(cid:884) carboxyfluorescein were excited at 490 nm and 485 nm and the fluorescence emission was e
d

f
(cid:883)(cid:891)(cid:885) monitored at 520 nm and 513 nm, respectively. The fluorescence signal was measured every r
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(cid:883)(cid:891)(cid:886) 15 min for 2 hours or every 90 s for 2 hours in initial experiments with α-hemolysin. The total h
t
t
p
(cid:883)(cid:891)(cid:887) amount of liposome-encapsulated dye was indicated by the fluorescence measured upon the :/
/
jb
(cid:883)(cid:891)(cid:888) addition of 0.1% Triton X-100 at the end of the reaction. Fluorescence was plotted as a .a
s
m
(cid:883)(cid:891)(cid:889) percentage of total unquenched fluorescence. .
o
r
g
(cid:883)(cid:891)(cid:890) /

Calcein release after in vitro synthesis of MscL on

D
(cid:883)(cid:891)(cid:891) e
c
Plasmid DNA (150 ng) encoding MscL (33) was added to 15 µl of the RTS100 E. coli HY e
m
b
(cid:884)(cid:882)(cid:882) reaction kit supplemented with 50 µg calcein-loaded liposomes. The reaction mixture was e
r

2
(cid:884)(cid:882)(cid:883) incubated for 5 h at 30°C. Under these conditions, MscL inserts in the liposome in a closed 5
,

2
(cid:884)(cid:882)(cid:884) conformation (iso-osmotic conditions) and no calcein leakage occurs. Following MscL 0
1
8

(cid:884)(cid:882)(cid:885) synthesis, 2 (cid:541)l aliquots of the reaction mixture were diluted into 990 (cid:541)l of 50 mM TrisCl (pH b
y

g
(cid:884)(cid:882)(cid:886) 7.4) with or without 0.5 M sucrose. Calcein release is instantaneous and was measured u
e
s
(cid:884)(cid:882)(cid:887) immediately after mixing. The samples were excited at 490 nm and emission scans were t
(cid:884)(cid:882)(cid:888) monitored from 500 to 600 nm in a cuvette with path length of 1 cm in a Varian Cary Eclipse
(cid:884)(cid:882)(cid:889) fluorescence spectrophotometer. Buffer spectra were subtracted.
(cid:884)(cid:882)(cid:890)
Vancomycin release
(cid:884)(cid:882)(cid:891)
(cid:883)(cid:882)