the translocation domain in trimeric autotransporter adhesins … · the translocation domain in...

The Translocation Domain in Trimeric Autotransporter Adhesins IsNecessary and Sufficient for Trimerization and Autotransportation

Kornelia M. Mikula,a,b Jack C. Leo,a* Andrzej Łyskowski,a* Sylwia Kedracka-Krok,b Artur Pirog,b and Adrian Goldmana

Molecular X-Ray Crystallography Group, Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland,a and Department of PhysicalBiochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Polandb

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mecha-nism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hair-pin inside the �-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N ter-minus through the lumen of the �-barrel. We further investigated the phenomenon of autotransportation and the rules thatgovern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similarTAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing twomore distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to thecells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one sothat the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the �-barrel,but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.

The type V secretion system is the most widespread mechanismof protein secretion in disease-causing Gram-negative bacteria

(6). It is a Sec-dependent system consisting of three distinct path-ways: the classical (monomeric) autotransporter pathway (typeVa), two-partner secretion (type Vb), and the trimeric autotrans-porter pathway (type Vc).

Classical autotransporters are single-chain proteins comprisedof a signal peptide, a passenger domain that is exposed to theextracellular space and is in many cases cleaved after transporta-tion, and a C-terminal translocation domain. Passenger domainscontain the specific activity of the protein (14) and usually have acentral �-helical core (9, 11, 31) though there are exceptions (44).The C-terminal translocation domain forms a 12-stranded (30)�-barrel that is responsible for insertion in the outer membrane(OM) and translocation of the passenger (32). Trimeric auto-transporter adhesins (TAAs), which comprise the type Vc secre-tion pathway (26), are built of three identical polypeptide chainscarrying a signal peptide that directs them to the Sec-machinery.As for classical autotransporters, the structure of TAAs can bedivided into distinct regions: the N-terminal passenger domaintypically contains a trimeric lollipop-like head, a neck, and acoiled-coil stalk domain (15), followed by a C-terminal transloca-tion domain that is, as for classical autotransporters, believed to beresponsible for insertion and translocation of the passenger do-main to the outside of the cell (Fig. 1E). The translocation domainis a 12-stranded �-barrel in which four strands come from onemonomeric polypeptide (27).

In autotransporter systems, proteins are transported across theinner and outer membranes in two distinct steps. The first step forall types is translocation across the inner membrane in a Sec-dependent manner (6), directly followed by passage through theperiplasm. During this step, an important role is attributed to thesignal peptide, which acts as a transient membrane tether main-taining the passenger domain in an unfolded state and thus allow-ing passage of the protein through the periplasm (42).

The second step, insertion into the outer membrane and trans-location to the outside of the cell, is different for both pathways.

However, the assistance of the Bam complex (previously known asOmp85 or YaeT) is required in both (16, 20, 22, 25, 40). Strainswith deletion of Omp85 were not able to transport IgA protease, aclassical autotransporter. Its passenger domain, which is cleavedafter transportation on the cell surface, accumulated in a full-length form in the periplasm (46). Recently, Hagan et al. (13)showed in an ex vivo experiment that the Bam complex assembles�-barrel proteins into the OM.

Much work has been done to elucidate the mechanism of au-totransportation and folding in classical autotransporters (18, 19,29, 30, 34, 39, 45). Of the proposed models to explain autotrans-portation, two are still considered viable, the single-chain modeland the Bam complex-assisted model. In both, the C-terminaldomain folds first into the OM with the assistance of the Bamcomplex.

The single-chain model posits that the passenger domain istransported through the pore of the barrel to the extracellularspace without the assistance of other proteins, ending with anN-out topology, where the N terminus is distal to the OM and theC terminus is on the inside (2). The way in which the passengerdomain is transported remains under debate. Does the N termi-nus of the passenger domain thread through the pore of the barrel,or do the C terminus of the passenger domain and the N terminusof the barrel form a hairpin structure, leading to transport of thepassenger domain (30)? Considerable evidence supports the hair-

Received 17 May 2011 Accepted 30 November 2011

Published ahead of print 9 December 2011

Address correspondence to Adrian Goldman, [email protected].

* Present address: Jack C. Leo, Department of Protein Evolution, Max PlanckInstitute for Developmental Biology, Tuebingen, Germany; Andrzej Łyskowski, IMBInstitute of Molecular Biosciences, University of Graz, Graz, Austria.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.05322-11

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pin model: the presence of folding promoting regions in BrkA(29); mutagenesis of the linking region, which affects passengertranslocation but not barrel insertion (21); and the fact thatC-terminal disulfide bridges in the Prn passenger domain preventsurface exposure, whereas N-terminal ones do not (17).

In the second model, the Bam complex is posited to stabilize anopen conformation of the �-barrel that could allow partiallyfolded passenger domain passage through the pore (30). Recentresults conducted with OmpT and purified Bam complex in pro-teoliposomes indicate that insertion and folding of outer mem-brane proteins occurs without any energy source; i.e., the processis driven by the �G of protein folding (13). Moreover, it has alsobeen postulated by Ieva and Bernstein (16) that the Bam complexis involved in passenger assembly. Recent results published bySauri et al. showed that the �-barrel is necessary for both insertioninto the OM and translocation of the passenger domain and that itcooperates with BamA complex (39). The topology of the trans-port through the Bam complex-assisted model and the single-chain model can be the same; passage through the pore stabilizedby the Bam complex may still occur either from the N terminus tothe C terminus or from the C terminus to the N terminus.

The mechanism of autotransportation of TAAs has been lesswell-studied but is generally assumed to be similar to the classicalautotransporters (4) (Fig. 1). The most probable mechanism ofautotransportation is analogous to the single-chain model, but itis still unclear whether the N or the C terminus of the passenger

domain is first transported through the pore of the �-barrel. Theexperimental data support the hairpin model, where three hair-pins are formed from the three passenger domains (Fig. 1C). First,the entire passenger domain is not required for transportation(27, 35). Second, trimerization of the passenger domain seems tooccur after trimerization and incorporation of the translocationdomain into the outer membrane (5). Third, some TAA passengerdomains are over 3,000 residues in length (43), making threadingof the three N termini unlikely. On the other hand, the spaceavailable inside the �-barrel in its final conformation is limited, soeven though three �-hairpins (with each �-strand approximately4 Å in width) could fit into the barrel, the space would be very tight(27). Another possibility would therefore be that the C-terminalpart of the hairpin—possibly as an already folded helix as in theNalP structure (30)—is already in the pore, while the N-terminalpart is outside, surrounded by the Bam complex (Fig. 1D). This isnot consistent with recent results from Leyton et al. (25) on mo-nomeric ATs, suggesting that the role of the Bam complex is tokeep the transporter domain in a transportation-competent statebut that there are still very strict constraints on what can passthrough the barrel. It is also hard to reconcile this structure withthe absolute requirement for Gly in the center of the barrel (12).

Our study addresses the mechanism of translocation. We usedtwo different types of TAAs, YadA expressed by enteropathogenicYersinia strains and Eib proteins (EibA, EibC, and EibD) fromEscherichia coli reference strains, to study the role of the translo-

FIG 1 Models of passenger domain translocation across the OM. (A) Protein with folded �-barrel and inserted into the OM, before translocation and foldingof the passenger domain. Proposed models for passenger translocation are also shown: threading model (a submodel of the single-chain model), in which the Nterminus is translocated first and then the rest of the protein is translocated with an N-to-C polarity (B); hairpin model (second submodel of the single-chainmodels), where the C terminus of the passenger domain is first inserted into the pore of the �-barrel so that a hairpin structure is created and the rest of the proteinis translocated C to N (C); and a Bam complex-assisted model in which the Bam complex is involved not only in �-barrel insertion into the OM but also inpassenger domain translocation (D). Panel E presents the folded structure of EibD, with protein domains marked on the right-hand side (24).

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cation domain in transport and folding. We showed that two dif-ferent Eibs can form heterotrimers but that YadA does not formheterotrimers with EibA. Moreover, we showed that, thoughYadA/EibA heterotrimer does not form, it will when the barrel ofYadA is replaced by the EibA �-barrel or vice versa. Taken to-gether, our work shows that a functional �-barrel is necessary andsufficient for trimerization and autotransportation.

MATERIALS AND METHODSCloning of YadA and Eibs for cross-trimerization experiments. We firstconstructed a novel vector for coexpression of two different membraneproteins on the cell surface so that expression of the two proteins is in-duced by isopropyl-�-D-thiogalactopyranoside (IPTG), with each havinga unique signal sequence and tag. We used different signal sequences toincrease the genetic stability of the plasmid in expression strains by avoid-ing repeated sequences. We modified the expression vector pETDuet-1(Novagen), which allows expression of two inserts from separate T7 pro-moters, to include signal sequences for periplasmic targeting. We ampli-fied the signal sequence of EibD by PCR and inserted this between theNdeI and BglII sites of the second polylinker region of pETDuet-1 (primersequences in are given in Table S1 in the supplemental material). Theprimers were designed to include an N-terminal StrepII tag, located di-rectly after the cleavage site for the signal peptide. We then isolated thePelB signal sequence from pET22b (Novagen) by PCR (using T7 and T7terminator primers) and cloned the resulting product between the XbaIand NcoI sites in the first polylinker region of pETDuet-1. This resulted inthe first multiple cloning site that included a hexahistidine tag directlyafter the PelB cleavage site. Thus, this vector can be used to express twoinserts with signal peptides; the first will have an N-terminal His tag, andthe second will have an N-terminal StrepII tag. We called this vectorpETDuet-S (for secretion), in which the products of both inserts are tar-geted to the periplasm by the Sec machinery.

For the cloning of YadA and of EibA, EibC, and EibD, we amplified thesequences of the passenger domains and translocation units of the corre-sponding genes, omitting the native signal sequences (see Table S1 in thesupplemental material). The products were then cloned into the NcoI/HindIII sites (EibD), BamHI/HindIII sites (YadA), or BglII/AatII sites(EibA and EibC), either alone or in combination (Table 1).The correct-ness of all insertions was verified by sequencing.

We produced the YadA-EibA fusions by a two-step protocol. We sep-arately amplified the sequences corresponding to the YadA and EibA pas-senger domains and translocation units. The forward primers of the trans-location units were designed to contain a 5= overhang complementary tothe 3= end of the passenger domain to be fused (see Table S1). We thenannealed the products of these reactions (YadA passenger domain to theEibA barrel, forming YadA-EibA, and vice versa, forming EibA-YadA) andfilled in the fragments by PCR. The resulting inserts were then digestedwith BamHI and HindIII (YadA-EibA) or BglII and AatII (EibA-YadA).These were then cloned into pETDuet-S, either alone or in combinationwith wild-type YadA or EibA.

Expression and outer membrane protein purification. We trans-formed the plasmids for surface expression of full-length Eibs and YadAinto the strain BL21(DE3) Omp8, which is optimized for the expression ofOM proteins (33). These bacteria were grown overnight at 37°C in 5 ml ofLuria-Bertani (LB) medium supplemented with ampicillin at 100 �g/ml.The following morning the optical density (OD) was measured, and thebacteria were diluted to an OD of �0.3 in 20 ml of fresh LB supplementedwith 100 �g/ml ampicillin and allowed to grow to mid-log phase (OD at600 nm [OD600] of �0.5). Protein production was then induced by theaddition of 0.2 mM IPTG. After a further 2 h of incubation at 37°C, wediluted the cells to an OD600 of 0.6. Cells from 10 ml of this suspensionwere harvested by centrifugation for 10 min at 4,000 � g. We then washedthe cells by resuspending them in 1.5 ml of 10 mM HEPES (pH 7.4),centrifuged them again for 10 min at 4,000 � g, and resuspended the pelletin 2 ml of 10 mM HEPES (pH 7.4) for OM isolation. Cell lysates wereproduced by sonicating the resuspended cells twice for 30 s each time(MS73 probe [Bandelin Sonoplus]; 0.5-s duty cycle) and subjecting themto centrifugation for 2 min at 15,600 � g to remove cell debris. To isolatethe OM, we followed the protocol of Carlone et al. (3). Briefly, innermembranes were separated from Sarkosyl-insoluble OMs by solubiliza-tion with 400 �l of 1% N-lauroyl sarcosine. Pelleted, washed OMs wereresuspended in 50 �l of 10 mM HEPES (pH 7.4) and solubilized in SDS-containing sample buffer. They were then used immediately and run onSDS-PAGE gels or stored at �20°C.

Nondissociating SDS-PAGE. Electrophoresis was performed withsamples containing nonreducing SDS-PAGE sample buffer (41). Beforeloading, samples were heated at 50°C for 10 min and then loaded on theready-made 4 to 12% gradient gel (Bio-Rad Mini-Protean Precast Gels),

TABLE 1 Plasmids and constructs used in our studies with description of tags used to detect heterotrimers

Plasmid Insert(s)a (cloning site[s]) Comment

pETDuet-S Derivative of pETDuet-1; contains signal sequencesfor periplasmic targeting of both inserts

pETDuetS-EibA eibA29-392 (BglII, AatII) Contains an N-terminal Strep II tagpETDuetS-EibAD eibA29-392 (BglII, AatII), eibD27-511 (NcoI, HindIII) EibA contains an N-terminal Strep II tag; EibD

contains an N-terminal His tagpETDuetS-EibD eibD27-511 (NcoI, HindIII) Contains an N-terminal His tagpETDuetS-EibC eibC26-504 (BglII, AatII) Contains an N-terminal Strep II tagpETDuetS-EibCD eibC26-392 (BglII, AatII), eibD27-511 (NcoI, HindIII) EibC contains an N-terminal Strep II tag; EibD

contains an N-terminal His tagpETDuetS-YadA yadA26-455 (BamHI, HindIII) Contains an N-terminal His tagpETDuetS-EibA-YadA eibA29-298-yadA368-455 (BglII, AatII) Contains an N-terminal Strep II tagpETDuetS-YadA/EibA-YadA yadA26-455 (BamHI, HindIII), eibA29-298-yadA368-455 (BglII, AatII) YadA contains an N-terminal His tag; the fusion of

the EibA passenger and YadA translocatordomains contains an N-terminal Strep II tag

pETDuetS-Yada-EibA yadA26-367-eibA299-392 (BamHI, HindIII) Contains N-terminal His tagpETDuetS-EibA/YadA-EibA yadA26-367-eibA299-392 (BamHI, HindIII), eibA29-392 (BglII, AatII) Fusion of the YadA passenger and EibA

translocator domains contains an N-terminalHis tag; EibA contains an N-terminal Strep II tag

pETDuetS-YadA/EibA eibA29-392 (BglII, AatII), yadA26-455 (BamHI, HindIII) EibA contains an N-terminal Strep II tag; YadAcontains an N-terminal His tag

a The subscript after the gene name refers to the amino acid residues of the corresponding protein encoded by the cloned sequence.

TAA C-Terminal Domain Suffices for Autotransportation

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and electrophoresis was performed at 160 V. These conditions prevent thevery stable TAA trimers from dissociating.

Proteinase K treatment of TAA-expressing cells. Bacteria were cul-tured and induced as above. Cultures after induction were diluted to anOD600 of 0.6 in a volume of 20 ml and collected by centrifugation for 10min at 4,000 � g. The pellet was resuspended in 4 ml of phosphate-buffered saline (PBS) with the addition of 25 �g/ml of chloramphenicol toinhibit protein synthesis. After 5 min of incubation at room temperature(RT), the samples were split in half. To one half we added proteinase K (toa final concentration of 40 U/ml) and incubated the samples for 15 min atRT, while the other half were used as controls and treated in all respects thesame in the subsequent steps. Proteolysis was stopped by addition ofphenylmethylsulfonyl fluoride (PMSF) to a final concentration of 0.2 mMto both proteinase K-treated and control cells. The cells were pelleted bycentrifugation for 10 min at 4,000 � g, resuspended in 1.5 ml of cold (4°C)10 mM HEPES, pH 7.4 (containing protease inhibitor as above), recen-trifuged for 10 min at 4°C at 4,000 � g, and resuspended in 2 ml of cold 10mM HEPES, pH 7.2 (containing protease inhibitor as above). From thatpoint samples were either treated as in the protocol for outer membranepurification (above) or stored at �80°C for subsequent use.

Western blotting. After SDS-PAGE (described above), the proteinsamples were blotted onto nitrocellulose filters (Hybond C extra; GEHealthcare) with a Bio-Rad semidry apparatus, according to the manu-facturer’s instructions using standard transfer buffer. Blocking and incu-bations with antibodies differ for the various blots and are described be-low. The final step, detection of the tagged protein, was again the same forall steps. Proteins were detected by enhanced chemiluminescence (ECLPlus kit; GE Healthcare) according to the manufacturer’s instructions.

Detection of StrepII-tagged proteins using StrepTactin conjugatedto HRP. The membrane was blocked for 1 h at RT in 1% fat-free milkpowder in 20 ml of PBS with 0.1% (vol/vol) Tween. Then, we washedmembrane three times for 5 min in 20 ml of PBS– 0.1% Tween, and thisstep was followed by incubation in 10 ml of PBS– 0.1% Tween with 5 �l ofStrepTactin conjugated to horseradish peroxidase ([HRP] diluted beforeuse according to the manufacturer’s protocol). The membrane was thenwashed two times in 20 ml of PBS– 0.1% Tween for 5 min each and twotimes in 20 ml of PBS for 5 min each. Finally, the Strep-tagged protein wasdetected (see above).

Detection of His-tagged proteins using a Ni-NTA conjugate. Thesteps after the Western blotting procedure were done according to themanufacturer’s protocol (HisDetector Nickel-HRP, catalog number 24-01-01; KPL). Briefly, membrane was blocked in 20 ml of 1% bovine serumalbumin (BSA) at RT for 1 h, and then a Ni-nitrilotriacetic acid (NTA)-HRP conjugate was added at a 1:20,000 dilution. After incubation for 1 hat RT, the membrane was washed three times for 5 min each time in 20 mlof 1� Tris-buffered saline– 0.1% Tween (TBST), and proteins were de-tected (see above).

IgG Fc binding assay. After the membrane was blotted, it was blockedfor 1 h in 20 ml of PBS–1% fat free milk powder at RT. Human IgGFc-HRP (Jackson ImmunoResearch) diluted 1:8,000 was added to theblocking solution, and the sample was again incubated for 1 h in RT,washed in 20 ml of PBS three times for 5 min each time, and then detected(see above).

Collagen far-Western blotting. After the blotting step, the membranewas quickly rinsed with 20 ml of 1� PBS– 0.1% Tween and then blockedfor 1 h at RT in 20 ml of 1� PBS supplemented with 2% fat-free milkpowder. Collagen (bovine collagen type I; Sigma) was bound by incuba-tion for 1 h at RT in 20 ml of 1� PBS– 0.1% Tween containing 2% milkpowder and 10 �g ml�1 collagen. After two washes (10 min in 20 ml of 1�PBS– 0.1% Tween), the primary antibody was added (monoclonal ColE1[Sigma], diluted 1:2,000 in 20 ml of 1� PBS–2% milk powder) and incu-bated for 1 h at RT or overnight at 4°C. After two washes (10 min each in20 ml of 1� PBS), HRP-conjugated secondary (anti-mouse) antibody(Santa Cruz Biotechnology) was added at a dilution of 1:5,000 in 20 ml of

1� PBS–2% milk powder. The secondary antibody was detected (seeabove).

MS. To identify the protein in bands cut from SDS-PAGE, gel pieceswere destained at 37°C by several washes in 25% and 50% acetonitrile(ACN), reduced at 56°C for 45 min in 50 mM dithiothreitol (DTT), andalkylated for 2 h at RT in the dark using 55 mM iodoacetamide. Residualreagents were washed out with 50% acetonitrile in 25 mM ammoniumbicarbonate buffer (NH4HCO3). Gel pieces were dehydrated in 100%ACN and dried in a SpeedVac for 15 min. The gel pieces were reswollenwith 15 �l of trypsin solution (10 ng/�l in 25 mM NH4HCO3, pH 8.0) for15 min, and after that 20 �l of 25 mM NH4HCO3 was added. Digestionwas carried out at 37°C overnight. Peptides were extracted by sonicationand dried with 100% ACN. The extracts were evaporated to dryness andresuspended in 2% ACN with 0.05% trifluoroacetic acid (TFA). Peptideswere analyzed using an UltiMate 3000RS LCnanoSystem (Dionex) cou-pled with a MicrOTOF-Q II mass spectrometer (MS; Bruker) using anApollo Source ESI nano-sprayer equipped with a low-flow nebulizer.

The peptides were injected on a C18 precolumn (Acclaim PepMapNano Trap column) using 2% ACN with 0.05% TFA as a mobile phaseand further separated on a 15-cm by 75-�m reverse-phase (RP) column(Acclaim PepMap Nano Series column; 100-�m particle size; 100-Å poresize) using a 2 to 40% ACN gradient in 0.05% TFA for 60 min. The MS wasoperated in standard data-dependent acquisition (DDA) tandem MS(MS/MS) mode with fragmentation of the most abundant precursor ions.

Mascot Generic format (.mgf) peak lists were generated using Data-Analysis, version 4.0, software and further searched against the NCBIdatabase or a custom database containing chimeric protein sequences andcommon contaminants (adapted from the common Repository of Ad-ventitious Proteins [cRAP] database [http://www.thegpm.org/crap/index.html]) using our in-house Mascot server (version 3.0; Matrix Science,London, United Kingdom).

Because of the high sequence similarity of the proteins of interest aswell as of their chimeric forms, the identification of species was made onthe basis of unique peptides. The number of identified MS/MS spectra ofunique peptides can also be used for estimation of the protein amount,enabling semiquantitative analysis. Spectral count normalization was ap-plied to the estimation of relative protein levels or their constituents (pas-senger or �-barrel) in the studied samples. Normalized spectral abun-dance factors (NSAFs) were used to compare the amount of the particularprotein (k) between samples:

�NSAF�k �(SpC ⁄ Length)k

�i � 1

N

(SpC ⁄ Length)i

where the total number of tandem MS spectra counts (SpC) matched toprotein k was divided by the length of protein k; this value was thendivided by the sum of the quotients SpC/length for all N proteins occur-ring in the sample (10), where i is the ith element of the sum. NSAF valuesrange from 0 to 1; a value closer 1 indicates a higher protein level.

Maltose-binding protein (MBP) detection. (i) Preparation of mini-mal medium supplemented with maltose. Minimal medium supple-mented with maltose was prepared according to the protocol by Elbingand Brent (7). Briefly, for 1 liter of a 5� concentration of medium, 30 g ofNa2HPO4, 15 g of KH2PO4, 5 g of NH4Cl, 2.5 g of NaCl, and 15 mg ofCaCl2 were resuspended in MilliQ water. Then, the medium was auto-claved and diluted to a 1� concentration, and the following were added: 1ml of 1 M MgSO4, 10 ml of 20% maltose, 4 ml (25 mg/ml) of methionine,and 10 ml of (10 mg/ml) amino acids mix.

(ii) Protein expression in minimal medium supplemented withmaltose. BL21(DE3) Omp8 strains containing our constructs were grownovernight in minimal medium supplemented with maltose and 100 �g/mlampicillin in 5-ml cultures at 30°C (36). The following morning, the ODwas measured, and the bacteria were diluted to an OD of �0.4 in 40 ml offresh minimal medium supplemented with 20% maltose and 100 �g/mlampicillin and allowed to grow at 30°C to mid-log phase (OD600 of �0.5).

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Protein production was then induced by the addition of 0.2 mM IPTG.After a further 2 h of incubation at 30°C, we diluted cells to an OD600 of 1in a volume of 40 ml, and protein expression was stopped with the addi-tion of 25 �g/ml of chloramphenicol. The samples were split in two,proteinase K was added to a final concentration of 0.01 mg/ml to one ofthem (17), and the samples were incubated for 15 min. Proteolysis wasstopped by addition of phenylmethylsulfonyl fluoride (PMSF) to a finalconcentration of 0.2 mM. PMSF was added to the control samples as well.All samples were centrifuged for 10 min at 4,000 � g and resuspended in2 ml of cold 10 mM HEPES, pH 7.2. The cells were broken down bysonication twice for 30 s each time (MS73 probe [Bandelin Sonoplus];0.5-s duty cycle). Samples were taken, 2� nonreducing SDS-PAGE sam-ple buffer was added, and SDS-PAGE and Western blot transfer wereperformed.

(iii) Detection of maltose binding protein. After a blotting step, themembrane was blocked for 1 h at RT in 20 ml of 1� PBS supplementedwith 5% fat-free milk powder. Then, the membrane was washed threetimes for 5 min in 20 ml of 1� PBS– 0.05% Tween, and anti-MBP (Sigma-Aldrich) was bound by incubating the membrane for 2 h at RT in 20 ml of1� PBS–1% BSA. The membrane was then washed three times for 5 mineach time in 20 ml of 1� PBS– 0.05% Tween. After this, we incubated themembranes for 1 h at RT with HRP-conjugated secondary (anti-mouse)antibody (Santa Cruz Biotechnology) at a dilution of 1:5,000 in 20 ml of1� PBS– 0.05% Tween. The secondary antibody was detected (see above).

RESULTSHeterotrimers can be formed among closely related/highly sim-ilar TAAs. Our main goal has been to understand the transportmechanism of TAAs. We therefore first investigated whether het-erotrimers could form among members of the Eib group of TAAs.We cloned Eib proteins (Table 1) into the pETDuet-S vector, in-troduced them into the E. coli BL21 Omp8 strain, isolated OMfractions containing the expressed proteins, and separated themusing nondenaturing SDS-PAGE so that we could see trimericTAAs (Fig. 2A). In addition to the expected homotrimers (EibA,121.5 kDa; EibC, 156 kDa; EibD, 156.9 kDa), heterotrimersformed between both EibA and EibD and between EibC and EibD.The expression levels of the EibA and EibC homotrimers wereequal, whereas EibD was expressed at a lower level. For the EibADheterotrimer (from pETDuet-S encoding both EibA and EibD),we observed the following four bands, in order of size (from top tobottom on Fig. 2A): EibD3 (hardly visible), EibA/EibD2, EibA2/EibD, and EibA3. EibD3 is hardly visible because EibD expresses ata lower level than EibA, as also for homotrimers. Most of the EibDis, then, in the heterotrimers, so very little of the EibD homotrimeris present. The relative molecular masses of EibC and EibD differby only 0.9 kDa, making it impossible to distinguish them on a gel;as a result, the EibCD heterotrimers (from pETDuet-S-encodedEibC and EibD) ran as a broad band (Fig. 2A). This presumablymeant that the band contained both homo- and heterotrimers.

We used Western blotting to make sure our interpretation wascorrect. EibA and EibC have a StrepII tag and can be detected withStrepTactin (Fig. 2B), while EibD has a His tag and can be detectedusing a Ni-NTA conjugate (Fig. 2C). In the EibAD experiment(Fig. 2D), StrepTactin bound well to bands III and IV but boundonly slightly to band II, while Ni-NTA bound only bands II andIII. In other words, EibA3 and EibA2/EibD bound StrepTactinwell, while EibA/EibD2 bound it poorly, as expected with the Stre-pII tag on EibA. Conversely, only EibA2/EibD and EibA/EibD2

bound the Ni-NTA conjugate. (EibD3 was present in such smallamounts that binding was not observed.) As expected, the unre-solved EibCD band (Fig. 2A) bound both StrepTactin (Fig. 2B)

and Ni-NTA (Fig. 2C), consistent with the presence of hetero-trimers (Fig. 2E).

We confirmed these results by excising the bands from the geland examining them by mass spectrometry (Table 2). This notonly confirmed the formation of heterotrimers but also showedthe ratio of monomers within one band. To compare the amountsof monomers in one band, the NSAF was calculated (see Materialsand Methods). For samples 4, 5, and 6 we can see that abundanceof EibA increases, with the amount in sample 4 less than that insample 5, which is less than the amount in sample 6, and that thereis a concomitant decrease of EibD abundance. This is consistentwith the data from SDS-PAGE (Fig. 2A) and Western blotting(Fig. 2B and C). The expected NSAF ratio for EibA/EibD2 (sample4) is 1:2, and for EibA2/EibD (sample 5) it is 2:1. The observedNSAFs are 1:1.5 and 1.8:1, respectively. The results are consistent,given that NSAFs are semiquantitative. Moreover, in samples 1, 2,and 6, the StrepII tag was one of detected peptides.

The data above, however, do not prove that the heterotrimericpassenger domain is on the cell surface and generates a proteinwith the correct topology. To prove this, we used a proteinase Kassay on Eib-expressing cells. Proteinase K treatment of wholecells reduced the sizes of both homotrimeric and heterotrimericEibs (Fig. 2F). The relative molecular masses of EibC, EibD, andEibCD all decreased in the same way, as expected if the entirepassenger domain is cleaved off. In the absence of proteinase Ktreatment, there was a significant amount of cleaved protein (i.e.,barrel alone) in the EibCD heterotrimer lane and just a smallamount in EibC lane (Fig. 2F). This again indicates that the EibCDcoexpression generates genuine heterotrimers, which are then lessstable, rather than a stoichiometric mixture of homotrimers.

Finally, we tested whether the heterotrimers are able to bindIgG Fc nonimmunologically (23). We performed standard West-ern blotting with Fc(IgG)-HRP and developed the blot with thesubstrate for HRP (Fig. 2G). We could observe strong binding ofFc to homotrimers but weaker binding to EibA2/EibD and to theEibCD mixture. We did not observe binding to EibA/EibD2, pre-sumably due to the low level of protein expression or the misfold-ing of the heterotrimeric passenger domain.

Heterotrimers cannot be formed by distantly related TAAs.For the full-length sequence, there was 49.5% identity betweenEibA and EibD, 84.6% between EibC and EibD, and 48.3% be-tween EibA and EibC (data not shown). Alignment of transloca-tion domains of EibA with EibD and of EibA with EibC showed97.9% identity, while EibC and EibD are identical in this region(see Fig. S1 in the supplemental material). Since the highly con-served Eibs can form heterotrimers, we decided to test if this is alsopossible for more distantly related TAAs. We therefore coex-pressed YadA and EibA, where the full-length proteins are 32.5%identical (data not shown) and the translocation domains are42.9% identical (see Fig. S1). After induction, purification of OMproteins, and SDS-PAGE, we observed YadA alone, EibA alone, orno protein at all (Fig. 3). This was true for 10 different colonies(data not shown). To make sure that this was not due to degrada-tion of proteins that were stuck in the outer membrane or theperiplasm, unable to pass to the outside of the cell, we transformedthe plasmid into a �degP strain, a BL21(DE3) Omp8 derivative(12) which lacks the periplasmic protease DegP that clears aggre-gated or denatured proteins from the periplasmic space in E. coli(28). Again, cells were induced with IPTG, and the OM proteins




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were purified and separated by SDS-PAGE. The results were thesame as for the BL21 Omp8 strain (data not shown).

The results were puzzling as we had of course sequenced theplasmids before transforming them into the E. coli expressionstrains. These transformants did initially grow very slowly on theagar-ampicillin plates after transformation, producing only verysmall colonies after overnight growth. We therefore purified plas-mids from four liquid cultures after induction (two expressing

YadA and two expressing nothing at all) and resequenced them.When there was no EibA expression, a point mutation (35 Leu tostop) had occurred at the beginning of the eibA open readingframe. Plasmids from two cultures that expressed neither proteingave different and very short fragments after sequencing, indicat-ing a large deletion in both the yadA and eibA genes. This was alsotrue for two other transformants that expressed neither protein.Coexpression of YadA and EibA seems lethal for the cells.

FIG 2 Eib proteins form heterotrimers. Expression and outer membrane purification show formation of heterotrimers between EibA and EibD and betweenEibC and EibD. As a negative control, OM proteins purified from cultures transformed with empty pETDuet-S vector were used (marked �), and, as positivecontrols, strains expressing only EibA, EibC, or EibD were used. M, molecular mass marker (kDa). (A) Coomassie-stained SDS-PAGE. (B) Western blots withStrepTactin that binds EibA and EibC.(C) Ni-NTA conjugates that bind EibD. For better comparison of gel and blot results, two figure sets were composed forheterotrimers EibAD (D) and heterotrimers EibCD (E). In panels D and E, the letters above the lanes (a to i) show which lanes from the original gel and blotsshown in panels A to C were used; in panel D numbers I to IV mark heterotrimers, as follows: IV, EibA3; III, EibA2/EibD; II, EibA/EibD2; I, EibD3. Surfaceexpression of heterotrimers was confirmed by proteinase K assay (F). Arrows mark bands corresponding to full-length proteins, and arrowheads mark bandscorresponding to translocation domains of the proteins. �, untreated samples; �,samples treated with proteinase K. (G) A functional assay with Fc IgG bindingwas also made. For further confirmation of our interpretation, bands marked with numbers 1 to 7 in panel A were cut from the gel and sent for mass spectrometrymeasurements (Table 2).

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Heterotrimerization depends on the translocation domain.As YadA and EibA, with low percentage identity in the transloca-tion domain, do not form heterotrimers while the almost identicalEibA and EibD do, we decided to test if changing the translocationdomain in YadA to that of EibA and vice versa would yield viableheterotrimers. We expected that the translocation domain wouldbe more important for trimerization and transport because earlierresults (5) indicated that defects in passenger domain trimeriza-tion do not prevent TAA expression.

We created passenger-translocation chimeric TAAs EibA-YadA and YadA-EibA, where the translocation domains containthe �-barrels and linker helices. These were cloned with the TAAcorresponding to the translocation domain into the pETDuet-Svector (Table 1). Constructs were expressed, OM proteins werepurified, and the proteins were separated by nondenaturing SDS-PAGE so that we could visualize homo- and heterotrimers (Fig.4A). The majority of EibA proteins remained as trimers (121.5kDa), and only a small amount dissociated into monomers (40.5kDa) (Fig. 4A, lane 1). YadA, on the other hand, migrated as amixture with trimeric (138.3 kDa), dimeric (92.2 kDa), and mo-nomeric (46.1 kDa) proteins all present in similar amounts (Fig.4A, lane 2). This is consistent with previous results (47) that YadAtrimers dissociate into dimers and monomers under SDS-PAGEtreatment, but the results do not imply that this occurs in themembrane. In addition, the control expression of the EibA-YadA(trimer of 119.4 kDa) and the YadA-EibA (trimer of 140.4) chi-meras also produced some levels of trimers at the expected relativemolecular masses (Fig. 4A, lanes 3 and 4). Interestingly, the EibA-YadA chimera migrated primarily as a monomer (39.8 kDa), whileonly a small amount of YadA-EibA migrated as a monomer (46.8kDa). The lack of stability of YadA under these experimental con-ditions resides in the barrel.

The SDS-PAGE experiment also showed that the expectedheterotrimers were created. For the EibA/YadA-EibA constructwe obtained three bands, corresponding to EibA/(YadA-EibA)2, EibA2/YadA-EibA, and EibA3 (Fig. 4A, lane 5). For theYadA/EibA-YadA construct, we obtained YadA2/EibA-YadA,YadA/(EibA-YadA)2, and (EibA-YadA)3 (Fig. 4A, lane 6). Inaddition, we also observed monomeric YadA-EibA, which is

not surprising based on our YadA and EibA-YadA control ex-periments (above). Differences in expression levels among het-erotrimers within one construct presumably result from thedifferential barrel stability of each of the possible species, and,as for the control homotrimers, heterotrimers containing moreEibA are more stable.

To confirm this interpretation, we used Western blotting(with StrepTactin and Ni-NTA conjugate) as the two proteinswere differentially tagged (EibA with a StrepII tag and YadAwith a His tag). As expected, StrepTactin bound to both mo-nomeric and trimeric EibA and to homotrimers and monomersof EibA-YadA (Fig. 4B, lanes 1 and 3). For heterotrimers (Fig.4B, lanes 5 and 6) binding occurred only for EibA2/YadA-EibAand YadA/(EibA-YadA)2 and not for EibA/(YadA-EibA)2 andYadA2/EibA-YadA. Ni-NTA conjugate bound to all YadA andYadA-EibA forms (Fig. 4C) but only to the monomeric formof YadA when it was coexpressed with EibA-YadA (Fig. 4C, lane6). We could not observe binding to the heterotrimeric pro-teins. One possible reason for this is steric hindrance, whichcould be created due to mismatches in heterotrimeric passen-ger domains during folding.

We further confirmed these results and our interpretation byexcising the bands from the gel and examining them by mass spec-trometry (Table 3 and Fig. 5). For samples 1, 2, 3, 8, and 13, theStrepII tag was detected, and for sample 4 the His tag and thelinker sequence that contains amino acids of the YadA passengerdomain and EibA translocation domain were detected. For theseresults, comparison of the NSAFs is difficult as the �-barrels ofproteins ionize poorly and so were not detected by MS. However,the EibA passenger abundance increases in the heterotrimericsamples such that the amount in sample 6 is less than that insample 7 which is less than that in sample 8, and the YadA passen-ger abundance decreases from sample 9 to sample 10 and thenagain to sample 11, which is in strong agreement with our inter-pretation.

Proteinase K treatment on intact cells followed by inactivationof the enzyme and SDS-PAGE showed that all of the chimeraswere surface exposed (Fig. 4D); for the majority of the constructs[EibA, EibA-YadA, EibA/(YadA-EibA) and YadA/(EibA-YadA)],

FIG 3 YadA and EibA do not heterotrimerize. Based on coexpression inpETDuet-S vector of distinct TAAs, EibA and YadA showed no heterotrimerformation. The figure shows a Coomassie-stained SDS-PAGE gel after OMprotein purification from three cultures with different protein expression lev-els (EibA/YadA lanes 1, 2, and 3). As a negative control OM proteins purifiedfrom cultures transformed with empty vector pETDuet-S (marked �) wereused, and, as a positive control, homotrimers of EibA and YadA were used. M,molecular mass marker (kDa). Protein expression was done at 37°C, whereYadA expression is higher than expression of EibA.

TABLE 2 Mass spectrometry measurements of Eib homo-and heterotrimersa

Sampleno.

No. ofuniquepeptides

No. ofsequencesforuniquepeptides

No. ofsequencedpeptides

Total no. ofMS spectra(SpC) Protein NSAF

1 16 166 19b 172 EibA 1.002 8 33 21b 210 EibC 1.003 10 57 45 119 EibD 1.004 8 32 15 56 EibD 0.594 7 20 9 29 EibA 0.415 9 31 14 56 EibD 0.355 15 70 16 80 EibA 0.656 18 215 18b 224 EibA 1.007 9 180 18 240 EibC 0.757 9 29 19 80 EibD 0.25a Bands cut for mass spectrometry measurements are marked with numbers 1 to 7 onFig. 2A.b The StrepTactin sequence (QWSHPQFEK) is present in the sequenced peptide.




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the band corresponding to the trimeric TAA (Fig. 4D, arrow) dis-appears and is replaced by a band corresponding to the transloca-tion domain trimer. For the YadA homotrimer and the YadA-EibA chimeric homotrimer, there was a significant decrease in theamount of full-length trimer, and a band corresponding to the sizeof the translocation domain appeared. Moreover, the disappear-ance of YadA dimers and monomers and the formation of justtrimeric YadA translocation domain bands further shows thatthose forms are created during SDS-PAGE; the results imply that,in SDS, the full-length protein is less stable than the YadA trans-location domain alone. To prove the integrity of the OM and showthat disappearance of the bands corresponding to full-length pro-teins does not result from leaking of proteinase K into theperiplasm, we used Western blotting to detect MBP (Fig. 6). It isclear that the outer membrane is intact as the signals from treated(Fig. 6, � lanes) and untreated (Fig. 6, � lanes) cells are the same.In addition, we observed nonspecific binding of the secondaryantibody by the EibA passenger domain (EibA, EibA-YadA, EibA/YadA-EibA, and YadA/EibA-YadA) and its digestion by protei-nase K, as shown in Fig. 4. This proves that constructs that weexpressed are expressed on the cell surface.

Finally, we showed that the chimeric heterotrimers possessthe activity of both passenger domains. Membranes wereprobed with IgG Fc or collagen, followed by an anti-collagenantibody. IgG Fc bound the EibA passenger domain in bothmonomeric and trimeric forms (Fig. 4E). However, as withbinding the StrepTactin, not all trimers containing the EibApassenger domain bound Fc. Fc bound to EibA2/YadA-EibAand EibA3 (Fig. 4E, lane 5), the YadA/(EibA-YadA)2 hetero-trimer, (EibA-YadA)3, and apparently to the EibA-YadAmonomer (Fig. 4E, lane 6). Collagen bound to trimeric and,surprisingly, to dimeric and monomeric forms of YadA (Fig.4F, lane 2) and to trimers and monomers of the YadA-EibAchimera (Fig. 4F, lane 4). Binding to trimeric YadA and YadA-EibA is much more efficient than to other forms of YadA andmonomeric YadA-EibA, as expected. Binding to dimers andmonomers of YadA can be explained by refolding of YadA onthe nitrocellulose membrane (8). Lack of binding to hetero-trimers (Fig. 4F, lanes 5 and 6) is according to our predictionsbecause there is no band containing a trimeric form of theYadA passenger domain. In the collagen blot the nonspecificbinding to the YadA/(EibA-YadA) (lane 6) is most probably

FIG 4 The translocation domain is responsible for trimerization and translocation. Coexpression and OM purification of chimeric proteins with EibA orYadA prove sufficiency of �-barrel for trimerization and translocation of passenger domain. For all panels, lanes are as follows: lane �, control sample OMproteins from expression of empty vector pETDuet-S; lane 1, EibA; lane 2, YadA; lane 3, EibA-YadA; lane 4, YadA-EibA; lanes 5 and 6, investigatedsamples EibA/YadA-EibA and YadA/EibA-YadA, respectively, that form heterotrimers; lane M molecular mass marker (kDa). (A) Coomassie-stainedSDS-PAGE gel. Western blotting with StrepTactin, which binds EibA (B), and with Ni-NTA conjugate, which binds YadA (C) showed heterotrimerformation. (D) Surface expression of heterotrimers was confirmed with proteinase K assay. Arrows mark bands corresponding to full-length proteins, andarrowheads mark bands corresponding to translocation domains of the proteins.�, untreated samples; �, samples treated with proteinase K. Functionalassays with Fc IgG binding (E) and with collagen (F) were performed showing that heterotrimers still posses the activity of comprised proteins. Finally,for further confirmation of our interpretation, bands marked with numbers 1 to 13 in panel A were cut from the gel and sent for mass spectrometrymeasurements (Table 3).

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due to nonimmune interactions between EibA and the anti-bodies. This could be seen by comparing the strength of thesignals in lanes 2 and 4 with that in lane 6.

DISCUSSIONFolding and autotransport: implications for mechanism. If thesignal for transport of the TAA passenger domain to the cell sur-face is not in the passenger domain, it should logically be in thetranslocation (barrel plus linker) domain. This is consistent withstudies on the structure and pore opening of the monomeric au-totransporter NalP (30), where the helical linker region wasshown to move in and out of the barrel domain. In YadA, dele-tions of different regions of the passenger domain did not disturbsurface expression (35). Translocation occurred even when one ormore of the head, neck, or stalk domains were deleted. The linkerand �-barrel domain were, however, always required. Similarly,Cotter et al. (5) showed that null passenger domains still led tofolded �-barrels in the outer membrane. In addition, we haverecently shown that the EibD passenger domain by itself under-goes a trimer-dimer equilibrium during gel filtration (24). On the

other hand, Ieva and Bernstein (16) showed that, during translo-cation by the monomeric autotransporters, BamA interacts notjust with the translocation domain but also with the passengerdomain. However, they still support the hairpin model as stallingof autotransportation resulted in only the C terminus of the pas-senger domain being degraded by proteinase K.

Studying the heterotrimeric barrels would, we thought, giveinsight into the rules of barrel folding and transport. Theyformed heterotrimers if the barrels were of the Eib group (iden-tity � 97%) (Fig. 2), but YadA and EibA (barrel sequence iden-tity of 43%) did not form hetero-barrels at all. To our surprisethe combination was clearly toxic to the cell, with either one orboth genes eliminated shortly after transformation (Fig. 3).YadA and Hia were reported in a previous study to be coex-pressed (5), but the authors used two plasmids, which may haveled to one of the two plasmids being eliminated stochastically,and the observed expression of both proteins could be the re-sult of a heterologous population of cells within the cultures, someexpressing only Hia and some only YadA. Alternatively, YadA andHia maybe divergent enough (21.8% identity of translocation do-

TABLE 3 Mass spectrometry measurements of YadA and EibA heterotrimersa

Sampleno.

No. of uniquepeptides

No. of sequences forunique peptides

No. of sequencedpeptides

Total no. of MSspectra (SpC) Protein (part of protein) NSAF

1 21 172 21b 172 EibA (passenger) 0.601 6 28 6 28 YadA (barrel) 0.402 22 285 22b 285 EibA (passenger) 0.562 7 54 7 54 YadA (barrel) 0.463 26 197 26b 197 EibA (passenger) 0.483 6 52 6 52 YadA (barrel) 0.524 22 229 22c 229 YadA (passenger) 0.954 0 0 1 2 EibA (barrel) 0.034 1 3 1 3 EibA (passenger) 0.02d

5 15 117 15 117 YadA (passenger) 0.675 1 6 2 15 EibA (barrel) 0.336 6 40 6 40 EibA (passenger) 0.376 10 86 10 86 YadA (passenger) 0.637 14 97 14 97 EibA (passenger) 0.547 14 106 14 106 YadA (passenger) 0.468 19 178 19b 178 EibA (passenger) 1.009 9 61 9 61 YadA (passenger) 0.459 7 54 7 54 EibA (passenger) 0.519 0 0 1 1 YadA (barrel) 0.0410 13 83 13 83 YadA (passenger) 0.3010 11 83 11 83 EibA (passenger) 0.3810 0 0 3 17 YadA (barrel) 0.3211 1 1 1 1 YadA (passenger) 0.004d

11 14 111 14 111 EibA (passenger) 0.5911 0 0 4 18 YadA (barrel) 0.4012 1 3 1 3 YadA (passenger) 0.01d

12 21 174 21 174 EibA (passenger) 0.4912 0 0 6 42 YadA (barrel) 0.5013 2 2 2 2 YadA (passenger) 0.004d

13 20 168 20b 168 EibA (passenger) 0.4313 0 0 6 52 YadA (barrel) 0.56a Bands cut for MS measurements are marked with numbers 1 to 13 on Fig. 4A.b The StrepTactin sequence (QWSHPQFEK) is present.c The His tag sequence (MGSSHHHHHHSQDPDDYDGIPNLTAVQISPNADPALGLEYPVRPPVPGAGGLNASAK) and the linker sequence containing the C terminus of the YadApassenger and the N terminus of the EibA barrel are present.d The abundance of the YadA passenger domain in samples 11, 12, and 13 is insignificant relative to the peak area (Fig. 5A and B) and appears to be due to sample contaminationthat occurred when the bands were excised. It is then a baseline for other measurements. The EibA passenger domain in sample 4 is also due to contamination, as this sample wasfrom YadA-EibA, and so no EibA passenger domain could be present.




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main) that heterotrimerization is not even attempted. On theother hand, as long as the translocation domain was not heterol-ogous (i.e., for YadA, either YadA or YadA/EibA-YadA; for EibA,either EibA or EibA/YadA-EibA), a heterotrimeric proteinformed, and the now heterotrimeric passenger domain was trans-ported to the cell surface (Fig. 4D).

Consequently, we suggest that trimerization of the transloca-tion domain, which seems to occur during or prior to membraneinsertion, is required for translocation of the passenger domain tothe cell surface. Passenger trimerization, on the other hand, is notrequired for membrane insertion and so presumably happenslater. What, then, is the mechanism of passenger trimerizationand transport? We start from the assumption that the three do-mains must be transported together because individually they donot form folded entities, and so the protein folding would notdrive transport in a ratchet-like manner. Given this, one possiblemechanism is through the translocation domain, with three pas-senger domains passing through the barrel domain as hairpins atthe same time (Fig. 1C). This is based on results of Grosskinskyand coworkers (12), who showed that the conserved Gly389(YadA numbering) in the center of the TAA barrel is required forfolding but not structure. Even the G389A mutation let to reduced

expression (12). Furthermore, our results indicate that foldingand transport are intrinsic to the translocation domain; the YadA/EibA heterotrimer formed only when the barrels were the same(Fig. 4D). On the other hand, space limitations in the �-barrellumen indicate that the hairpin may form the other way: one halfof the hairpin would be inside the �-barrel lumen while the otherhalf would be between the �-barrel and the Bam complex (Fig.1D), as suggested by Ieva and Bernstein (16).

Overall, we prefer the internal hairpin model (Fig. 1C) for thefollowing reasons: (i) this model does not require BamA for trans-location of the passenger domain but explains its role in barrelassembly and makes its role the same for all �-barrels insertinginto the OM (13); (ii) the model leads to the greatest level of stericcrowding around Gly389 during translocation; and (iii) the modelexplains the requirement for both linker and �-barrel in translo-cation.

Biological implications. Are the mixed barrels we see in vitro(above) relevant in vivo? In the highly homologous Eib family of IgFc-binding proteins, such mixed trimers may be physiologicallyrelevant. We demonstrated that EibAD and EibCD heterotrimersoccur. As EibC and EibD are almost identical (97% between EibAand EibD), it is highly likely that EibAC heterotrimers also occur.This finding is very interesting as multiple Eib genes occur natu-rally in E. coli strains (37). Their natural coexpression can result inthe binding of different subsets of Igs as different Eibs bind withvarious affinities or not at all to different Ig subtypes (23, 37, 38).There are two possibilities: either bacteria have a mechanism thatprevents heterotrimerization, or heterotrimers form and are atleast not disadvantageous. If it is the former, one possibility wouldbe transcription regulation so that only one eib gene is transcribedat a time. The latter model seems more likely because Eibs natu-rally form heterotrimers, and this may confer a selection advan-tage. First, it would increase the levels of expression of the Eibs bya gene dose effect, and, second, the heterotrimers, which still bindtheir ligands (Fig. 2G and 4E and F), would provide various affin-ities and specificities in comparison with homotrimers. This couldbe advantageous during infection as a way of increasing surfaceheterogeneity and thus immune evasion.

FIG 6 The outer membrane is intact. Expression of chimeric proteins withEibA or YadA in minimal medium supplemented with maltose followed byproteinase K treatment and MBP detection proves integrity of OM. MBP levelsare shown for the empty pETDuet-S vector (�; positive control for MBPlevel),wild-type proteins EibA and YadA, chimeric EibA-YadA and YadA-EibA, and coexpressed EibA/YadA-EibA (E/Y-E) and YadA/EibA-YadA (Y/E-Y). Lane M, molecular mass marker (kDa); �, untreated sample; � sampletreated with proteinase K. The MBP band is boxed. Results were similar for theEibA, -C, and -D heterotrimers (data not shown).

FIG 5 The comparison of precursor MS spectra: baseline creation. Spectra oftwo sequences, TTLETAEEHANSVAR (A) and SSSVLGIANNYTDSK (B), theonly unique sequences found for the YadA passenger domain, are shown.Spectra for samples containing full-length YadA protein (bold dashed line,sample 9; bold solid line, sample 10) are compared with samples that shouldnot contain a YadA passenger domain unless contaminated (unbolded line,samples 11, 12, and 13).

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In the YadA-EibA coexpression studies, coexpression of dis-tant homologues appears to be deleterious. We suggest that, insuch cases, the genes will be tightly regulated to ensure that con-current expression does not occur. This suggestion is consistentwith the existence of allelic TAAs in Haemophilus influenzae, Hiaand Hsf (1), that are very different in length (1,096 and 2,413residues, respectively) but have very similar translocation do-mains. Even though their �-barrels would allow heterotrimeriza-tion, it is highly unlikely that an active passenger domain couldform due to the 700-residue difference in length. Allelic expres-sion is certainly consistent with our model. Elucidating the foldingrules for TAAs and what happens in vivo is clearly an importantgoal.

ACKNOWLEDGMENTS

We thank Ralf Koebnik for BL21 Omp8 and Dirk Linke for BL21 Omp�DegP. We also thank Mikael Skurnik for the template containing theyadA gene from Y. enterocolitica serotype O:3.

This work was funded by a Socrates/Erasmus Fellowship (to K.M.M.), bythe Sigrid Juselius foundation (to A.G.), and by the EU FP7 Marie CurieTrimBAT project (to A.Ł). The MS research was done with the equipmentpurchased thanks to the European Regional Development Fund in the frame-work of the Polish Innovation Economy Operational Program (contractnumber POIG.02.01.00-12-167/08, project Małopolska Center of Biotech-nology) and supported by grant number 2023/B/P01/2010/39 (K/PBW/000676) from the Polish Ministry of Science and Higher Education.

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