Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 16, Issue of April 22, pp. 12233-12239, 1994 Printed in U.S.A.

Stable Fiber-forming and Nonfiber-forming Chaperone-Subunit Complexes in Pilus Biogenesis*

(Received for publication, January 24, 1994)

Robert Striker$$, Frangoise Jacob-Dubuisson$n, Carl Friedenll, Scott J. Hultgren$** From the $Department of Molecular Microbiology and the lpepartment of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin. P pilus assembly depends upon the periplasmic chaperone PapD. The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly. Pap& the major subunit of the pilus rod, formed two periplasmic complexes (DA, and DA) with PapD. PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK). Only “fiber forming“ or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm. Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions. Apulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

Gram-negative bacteria have evolved filamentous polymeric structures called pili to mediate their specific adherence to host cells and subsequent colonization (1-3). For example, most uropathogenic Escherichia coli express P pili that contain an adhesin, PapG, that mediates specific binding to Gala(1-4)Gal present in the globoseries of glycolipids on epithelial cells lining the urinary tract (4). P pili are heteropolymeric structures consisting of a rod joined to an adhesive tip fibrillum (5). The rod is composed of the repeating major pilin subunit PapA that forms a right-handed helix of 3.3 subunitslturn (61, while the bulk of the tip fibrillum is composed of PapE subunits polymerized into a linear fiber (5). The PapG adhesin is joined to the distal end of the tip fibrillum via the PapF adaptor subunit, and the tip fibrillum is joined to the pilus rod via the PapK adaptor subunit (PapG, F, and K are present in single or low copy number in each pilus). Thus, the ordered assembly of pili depends upon the complementary surfaces of each subunit type (7). In addition, an outer membrane assembly protein called PapC serves as a molecular doorkeeper to selectively incorporate tip fibrillar subunits into the pilus across the outer membrane and has been termed an usher (8). Virtually nothing is

* This work was supported by Support Grant lROlAI29549 (to S. J. H.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This

with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked “aduertisernent” in accordance

8 Member of the medical scientist training program and recipient of support from National Institutes of Health Grant lROlAI29549.

11 Recipient of a long term postdoctoral fellowship from the European Molecular Biology Organization (91-93) and is now a Keck Foundation Fellow.

** To whom correspondence should be addressed. Tel.: 314-362-6772; Fax: 314-362-1998.

known about the elongation phase of either PapE polymerization in the tip or PapA polymerization in the rod.

The formation of P pili and other pili (with the exception of the type IV class of pili) has been shown to depend on periplasmic chaperones. Twelve periplasmic chaperones, homologous to PapD, have been identified in diverse Gram-negative bacteria and are essential for the assembly of a number of different extracellular virulence structures, most of which are pili (9,101. The three-dimensional structure of PapD has been solved to 2.0 A. It consists of two immunoglobin-like domains oriented to form a cleft which is conserved among the members of the PapD family of chaperones (111.’ Site-directed mutations of arginine 8, an invariant residue protruding into the cleft, have been shown to abolish subunit binding. The highly conserved chaperone cleft has therefore been proposed to be the subunit binding site (12). In P pili biogenesis, subunits are bound by the chaperone, PapD, after secretion across the cytoplasmic membrane, and presented to the outer membrane usher, PapC, in an assembly competent state. A stable chaperone adhesin complex PapD-PapG (DG) has been purified previously, and PapG in the complex was shown to bind the Gala(1-4)Gal moiety suggesting that even in the preassembly complex it was folded (13, 14). It has been suggested that the function of the chaperone is to cap interactive surfaces on subunits to prevent their nonproductive aggregation (13, 15). In the absence of the chaperone, the ag- gregated subunits are degraded by proteases. It has therefore been difficult to define the role of the chaperone beyond pro- tection from proteolytic degradation. Possible other chaperone functions include (i) correctly orienting quaternary interactions between two subunits; (ii) mediating subunit folding; (iii) im- porting subunits into the periplasm; or (iv) retarding subunit tertiary structure formation (similar to a cytoplasmic chaperone) or quaternary structure before secretion across the outer membrane.

Pilus subunits have considerable homology to each other, but have different structural roles in the pilus, and different func- tional roles in assembly (3) (see model in Fig. 1). PapA and PapK are 30% identical at the amino acid level, yet PapA forms a homopolymeric thick helical rod while PapK does not. In this work we report for the first time the purification of stable complexes formed between both the major subunit PapA and PapD and also between PapK and PapD. We show that PapK binds to PapD only as a monomer, but that PapA is complexed to PapD both as a monomer and a homodimer. The ability to form multimers in the periplasm was found to be restricted to subunits that are homopolymers in the pilus (PapA and PapE but not PapK or PapG). Further a complex between PapD and PapA was shown to be a true assembly intermediate in vivo. This work represents a significant step toward an in vitro assembly system.

D. Ogg, manuscript in preparation.

12233

12234 Chaperone-assisted Pilus Biogenesis

4 Adhesin

HO

Gala(1-4)Gal

OCH, i ) } Repeating Subunits of Pap€

lnltlator of Rod assembly Tip-Rod Adaptor

Pilus Shaft Repeating Subunits of PapA

J - Anchor: H

1. Targeting 2. Chaperone Uncapping 3. Polymerization

Membrane Outer

\

Periplasm

Cytoplasmic Membrane

with each subunit after the subunit is translocated across the membrane in an unfolded state to form preassembly complexes. The preassembly FIG. 1. Model of chaperone-assisted P pili assembly. Details of the model are discussed in the text. Briefly the PapD chaperone interacts

complexes are then targeted to PapC at the outer membrane where the chaperone is dissociated and the subunits are incorporated in an ordered fashion in the growing pilus. In the absence of the chaperone, subunits aggregate and are degraded by the DegP protease. Only the PapG and PapE-chaperone complexes were proven to exist prior to this report. Only PapE and PapA subunits self-polymerize into fibers; PapA subunits form the pilus rod and PapE subunits form the tip fibrillum.

MATERIALS AND METHODS Strains and Plasmids-All experiments except the pulse chase were

carried out in E. coli HB101. The pulse-chase analysis was done with E. coli W3110 cells to ensure strong induction of the ara promoter. pRS2A was created by cloning papA from P P A P A Y ~ ~ ~ ~ into pTRC99A (Phar- macia LKB Biotechnology, Inc.) behind the trc promoter. pFJ29 contains pupAHCDJKEFG under the D c promoter. It was obtained by replacing the EcoRI-NsiI fragment of pFJ3 (7) by a fragment containing la@ and the trc promoter that was obtained by polymerase chain reaction am- plification of the corresponding region of the pTRC99A plasmid. pFJ2O was constructed by cloning papC from pKDlOl (7) into pMON6235 (courtesy of Mark Obukowitz, Monsanto Corp., St. Louis) downstream of the ara promoter. pFJ22 encodes papDJKEFGA under the tac promoter. It was constructed by cloning papA from pRS2A downstream of pupG in pPAP58 (14).

Protein Purification-HB101 carrying the plasmid pLSlOl (papD (12)) and either pRS2A (PUPA), pFJ l l (papK(7)), pPAP63 (pa@ (16)), or pJPl (papDG (17)) were used to purify DA and D 4 DK, DE, and DG complexes, respectively. Three liters of cells were typically grown in Luna broth to an optical density of OD, = 0.8 before induction with 0.5 nm isopropyl-P-D-thiogalactoside and preparation of periplasmic extracts as previously described (12).

The PapD- and PapA-containing extracts were equilibrated in 1 M ammonium sulfate, 50 nm phosphate buffer, pH 7.0, and fractionated on a fast protein liquid chromatography phenyl superose (10/10 Pharma- cia) hydrophobic interaction column (HIC)3 by eluting with a linear gradient to 0 M ammonium sulfate at 1 mVmin over 30 min. The earliest eluting two-thirds of the fractions that contained both PapD and PapA

M. J. Wick, and S. N. Normark, manuscript submitted. The abbreviations used are: HIC, hydrophobic interaction chroma-

tography; PAGE, polyacrylamide gel electrophoresis.

had the least impurities and so were pooled, raised to 0.8 M ammonium sulfate, and further chromatographed on a propyl HIC column (J. T. Baker Chemical Co., HPLC). This first two-thirds contained only the 5.8 species while the last third contained both the 5.8 and 6.8 PI species. Bound protein was eluted from the propyl HIC column with a gradient from 0.8 to 0 M ammonium sulfate at 1 mumin over 20 min. Two main peaks were eluted, free PapD and the DA, complex.

Free PapA and the DA complex were obtained by use of a C-18 reverse-phase (Beckman, Ultrasphere-ODS 4.6 x 150 mm) column. Pu- rified DA, was injected onto the column in 0.05% trifluoroacetic acid/ H,O. Free PapA and the DA complex eluted with a linear gradient of 95% acetonitrile, 0.05% trifluoroacetic acid at 1 mVmin over 30 min.

Periplasm containing PapD-PapK was dialyzed into 35 m~ diethanol- amine, pH 8.5, and chromatographed on an anion-exchange column (Hi Load MonoQ, Pharmacia). Both free PapD and DK complex flowed through while a majority of lower PI contaminants were retained. Flow- through was dialyzed in 0.8 M ammonium sulfate and was further fractionated using the same HIC steps as described above for PapD- PapA. The DK complex eluted as a separate peak from free PapD, which eluted first.

The PapD-PapE complex was partially purified from periplasmic extracts using the same chromatographic steps as those described for DK purification. DG was purified as described previously (14).

Pili were purified as described previously (12). To obtain the pattern of different oligomers of PapA on a SDS-PAGE gel, pili were suspended in 8 M urea, the urea was dialyzed away, and the preparation was then diluted into SDS sample buffer and incubated a t 24 "C for 5 min. To obtain the monomeric PapA, the sample was incubated at 95 "C instead of 24 "C for 5 min before SDS-PAGE.

Amino Acid Composition and Sequncing--Amino acid composition and sequencing was done by Washington University Protein Chemistry Facility. For amino acid composition analysis, proteins were hydrolyzed

Chaperone-assisted Pilus Biogenesis 12235

A B I 2 3 4 5 6 6.8 5.8 - PapD .ll -*r - " "-

~. .- - - PapD

s -PapA -6.8

- - 5.8

C 1 2

b - 6.8

HRlOl cells expressing: both PapD and PapA under the lac promoter (lane 2 ) ; only PapA under the lac promoter ( fnnr .7 I ; 81 wrll charactcrizrd FIG. 2. A, two complrxes hrtwern Pap11 and I'apA exist in the periplasm. Silver-stained isoelectric focusing gel of pcmpl:lsmlc extracts from

mutant of PapD (RHG PapD) that does not hind subunits coexpressed with PapA. both genes expressed from the tnc promoter (lanr 4 1; PapD coexpressed from tar and PapA under its own promoter (lane 5 ); the entire pap operon expressed from the tar promoter whrrr induction confws pilus production (lane 6 ) . Lane I shows isoelectric focusing standards. R , SDS-PAGE shows purified 6.8 and 5.8 species containing Pap11 and PapA. The 6.8 and 5.8 PapD-PapAcomplexes were purified and analyzed on Coomassie Blue-stained SDS-PAGE grls. Thr identity of the hands as Pap11 and PapA was confirmed by amino-terminal sequencing and Western blot with anti-PapAantisera. C, low pH and acrtonitrilr convert9 purified I ) h 2 complex to DAcomplex. Silver-stained isoelectric focusinggel of purified D 4 complex prior to the low pH of the rrvrrse-phnsr column rlnnr I ). Afirr the low pH reverse-phase column, the D 4 complex was converted into a DA complex with a pl of 6.8 (Ianr 2 j . Not shown is somr frrr PapA.

using 6 N HCI vapor a t 110 "C for 16 h in vacuo. Amino acid analysis was performed on a Reckman model 6300 amino acid analyzer using nin- hydrin with monitoring a t 570 nm for amino acids and 440 nm for imino acids. Proteins were sequenced on an Applied Biosystems model 470A automated protein sequencer using gas-phase chemistry. An Applied Riosystems model 12A"on-line" phenylthiohydantoin Analyzer with UV detection was used to identify the phenylthiohydantoin amino acids a t each cycle. The yield of histidine (unique to PapK) was taken as the concentration of PapK. There are no amino acid residues unique to PapD. Therefore, the ratio of PapK to PapD that would account for the ohserved amount of Glx (Glu and Gln) and Asx (Asp and Asn) in the hydrolyzed complex was calculated based on the known amount of PapK (histidine). Residues to compare in the composition and sequencing analysis were chosen on the hasis of ohtaining similarly quantita- tive yields (18).

Electrophoresis: Isorlectric Focusing, Native Gels, and Western Blors-Isoelectric focusing was done with precast PI 5 4 gels from Phar- macia on a Pharmacia Phast. The gels were then silver stained as per manufacturer's instructions. Acidic native gel electrophoreses were done with precast homogenous 20% gels from Pharmacia on a Pharma- cia Phast with a reverse polarity electrode as per Application File No. 300 (Pharmacia). The pH of the buffer was 4.0. These gels were either Coomassie Blue stained or blotted to polyvinylidene difluoride with acidic transfer huffer (20% methanol, 0.16 M p-alanine, pH 4. with acetic acid). The blots were developed with either anti-PapD, anti-tip fihrillum (7). or anti-PapA rahhit antisera as the primary antihody and goat anti-rabbit I g G antisera conjugated to alkaline phosphatase as the sec- ondary antisera (Sigma). SDS-PAGE and non-native Western blots were done as in Ref. 12 with the above antisera.

Pulse-chase Laheling of PapA to Follow Pilus Assemhly"W31101 pFJ20,pFJ22 cells were grown in minimal A medium containing 0.2% glycerol, 1 mM MgSO,, and 50 mdml of each amino acid (except for Met and Cys) and grown to an OD, 0.8 a t 600 nm. Isopropyl-p-n-thiogalactoside was added to 5 mM to induce expression of the genes behind the tar promoter for 5 min prior to labeling. Trans""S-label (Amersham) was then added to 100 pCiIml of culture (0.1 pw Met. 0.02 p~ Cys). After a 45-s pulse, the label was chased by the addition of cold methionine and cysteine to 5 mM each, andpapC was induced at the same time using 5T arahinose. After 2 more min, chloramphenicol was added to 100 pg/ml to inhihit further protein synthesis. Aliquots were taken a t various time points after the chase. quickly mixed with the same volume of ice-cold 0.1 M sodium phosphate 0.15 M NaCI. pH 8. incubated for a few minutes a t -15 "C to stop assembly. and removed prior to freezing. Periplasmic extracts were prepared from half of each aliquot as described above and analyzed hy isoelectric focusing and autoradiography. Pili were heat extracted from the other half and immunoprecipitated using an anti- PapA monoclonal antihody. Immunoprecipitations were analyzed by SDS-PAGE and autoradiography using an intensifier (Entensify, New England Nuclear). Laheled protein hands were quantitated by densitometry (Digital Imaging Systems IS-1000).

RESULTS

PapD Forms Stahle Complrxm with PnpA in thr Periplasm-We investigated PapD-PapA interactions by exam- ining periplasmic extracts from cells co-expressing PapA and PapD (HBlOl/pRS2A + pLSI01) on silver-shined isoelectric focusing gels. PapA is proteolytically deqaded when expressed in the absence of PapD (12). Two hands were ohserved after isoelectric focusing of the periplasmic extracts from cells coexpressing PapA and PapD (Fig. 2 . 4 , lane 2 ) that were absent in cells expressing only PapA or only PapD (Fig. 2 . 4 , lane .? ). These two bands had isoelectric points of 6.8 and 5.8 which are he- tween the isoelectric points of PapA (PI = 4.6) and PapD (PI = 9.4). These species were also formed when PapA was cwx- pressed from its own promoter (pPAP43 ( 19)) with PapD (Fig. 2 4 , lane i i ) . Small amounts of both bands were present in the periplasm of a strain producing pili, HRIOl/pFJ29, which contains an inducible pap operon (Fig. 2.4, lanr 6 ), suggesting that they may represent preassemhly intermediates. The two puta- tive PapD-PapA species were not present in the periplasm (Fig. 2 A , lane 4 ) when PapA was coexpressed with a well characterized point mutant of PapD (R8G PapD) in HR101/pPAP43 + pR8G consistent with the observation that this mutation a h l - ishes the ability of PapD to bind and stahilize PapA ( 12).

Purification and Stoichiometp of Fi.8 a n d 6.8 PapD-PapA Complexes-The majority of the 5.8 PI species was separated from the 6.8 species and partially purified from the periplasm of HBlOl/pLSlOl+ pRS2A(papD+ papA 1 by phenyl-Superose HIC. This was then applied to a propyl HIC column, and a single PapA-containing peak was identified in the chromato- graph which consisted entirely of PapD and PapA (Fig. 28 1.

Isoelectric focusing gels of the purified material from the propyl HIC revealed the presence of a distinct moiety that miqated with a PI of 5.8 (Fig. 2C, lanr 1 ) with a minor contaminant of a PI of 6.8. Amino acid analysis of the purified 5.8 PapD-PapA complex revealed that the histidine peak (2 per PapA), which is unique to PapA, was one-third the concentration of the a r6ninr peak which is unique to PapD ( 13 per PapD). This armes for a 2:l ratio for the PapA-PapD complex having a PI of 5.8. In addition, amino-terminal sequencing also revealed that there was twice as much proline (PapA) as valine (PapD) in the second cycle (both proteins have alanine as their first residue). This evidence also suggests that there is twicp as much PapAas


FIG. 3. A, native gel showing DK complex. Coomassie Blue-stained acidic native gel showing periplasmic extracts from HRlOl cells expressing: Papl) only tlnnr I ); PapK only (lane 2 ) ; PapD and PapK uninduced (lone 3 I: PapD and PapK induced ( h e 4 ); purified PapD and IIK complex tlnnr 5 ) (PapD and PapK are under the tnc promoter). R, purified IIK complex contains PapD and PapK. Analysis of the purified DK complex on a Coomassie Blue-stained gel confirmed identity by amino-terminal sequencing and Westem hlot analysis using anti- PapK antisera.

PapD in the 5.8 complex. This complex will be referred to as the DA, complex.

The DA, complex was applied to a reverse-phase C-18 column at a low pH (pH = 3.0). Two species eluted off the column with increasing acetonitrile that were identified as PapA only (data not shown) and a PapD-PapA complex (Fig. 2R ). Isoelec- tric focusing of the PapD-PapA complex revealed that it now had a PI of 6.8 identical to the species in the periplasm (Fig. 2C, lane 2) . The elution of both free PapA and a PapD-PapA complex with a PI of 6.8 suggested that PapA was being removed from the DA, complex to convert it into a DA complex by reverse-phase chromatography at low pH. The loss of PapA from the D 4 complex was accompanied by a shift in isoelectric point from 5.8 to 6.8 which is approximately the average of the PI values of PapD (9.4) and PapA (4.6). again suggesting the 6.8 complex is a 1:l species. Although clearly present in the crude periplasmic extract (Fig. 2.4 ), this was the best procedure found for separating DA from D 4 .

Identification, Purification, a n d Stoichiometry of the DK Complex-PapD-PapK interactions were analyzed using a n acidic native PAGE. Analysis of periplasmic extracts from cells induced to express both PapD and PapK (HB101/ pLSlOl+pFJl l , ( 7 ) ) revealed the presence of free PapD and a unique band (Fig. 3A, lane 4 ) that was not present in the periplasm of cells induced to express PapD only (HBlOl/ pLS101, Fig. 3 A , lane I ) or PapK only (HBlOl/pFJll, Fig. 3 A , lane 2) . PapK is proteolytically degraded in the absence of a n interaction with PapD (12). SDS-PAGE demonstrated that the unique band was comprised of PapD and PapK, and their identity was confirmed by Western blotting and amino-terminal sequencing (Fig. 3R ).

A peak containing both PapD and PapK was purified by ion-exchange and HIC from periplasmic extracts of HB101/ pFJll+pLS101 (papKandpapD)(Fig. 3A). In addition, a separate peak containing only PapD was obtained. Native PAGE analysis of the peak containing both PapD and PapK revealed two distinct bands (Fig. 3A, lane -5 ). The slower migrating band was confirmed as the DK complex while the faster migrating band was free PapD as determined by Western blotting of the native gel by anti-PapD and anti-PapK antisera (data not shown). Thus, HIC did not completely separate the DK complex from free PapD probably since they have such similar chromatographic properties.

The stoichiometry of the DK complex was investigated by amino acid analysis and amino-terminal sequencing in a similar manner as t h e D 4 . Amino acid analysis suggested a ratio of PapK to PapD of 0.83 (see "Materials and Methods"). Amino- terminal sequencing of the two bands blotted onto polyvinylidene difluoride paper from a SDS-PAGE gave a ratio of PapK to

A

A2-

1 2 3 4 5 6 7 8

D A l -"D - "K

+ - + - + - + - 9 5 o c Pili DA, DA DK

8 , 2 3 4

40 - - A 4 - A 3

30- - - A 2

-

- - r - - A

15-

+ - + - 9 5 o c DA2 DA

FIG. 4. A, DA, complex and not DA contains PapA-PapA dimrr. Pili and PapD-subunit complexes were incubated at 95 (: I + I nr 24 C ( - 1 in 1'T SIX loading huffrr. suhjected to SI)S-PA(;E, and s tninrd hy Cno- massie Rlur. Pili hrcome complrtrly monnmrnc only aRrr inruhntion at 95 'C tlnnr I ) , hut producr n multimrric Iaddrr nt 24 ( ' Ilonr 2 I. Altrr 95 "C preincubation the DA, complrx was dlssorintcd Into monomc.ric PapD and monomeric I'apA clnnr .7 I. hu t aftrr 24 C: prrlncuhation t h r DA, was dissociated into monomrric PnplI and prrdomlnantly d lmrnc I'apA tlnnr 4 I. I'apA dissociated from thr I)A complrx was monomrric aftrr hoth 95 "C clnnr .51 and 24 C incuhatlon Ilonr f i ~ . I'npK ~ I S W C I -

atcd from thr DK complex was also monomrric aRrr 95 and 24 incubation rlnnr 7 and H I . R. Wrstrm hlot wlth nntl-l'npA an t iwnrm confirms tha t the DAL complex IS compowd of monnmrnc PapA aRrr 95 C incuhation Ilnnr I I, hut a t 24 (' lncuhation contninrd pnncipnlly dimeric and somr higher oligomrrs of PnpA llnnr 2 I. T h r I)A complrx IS primarily monomeric hnth a t 95 ' C llnnv .'t I and 24 (' lncuhntion llnnr 4 I.

PapD of 1.3 by comparing similar residues in the fourth cycle (alanine for PapK and leucine for PapD). Given that the native PAGE gel shows some free PapD these data suggest that PapD and PapK are in a discrete 1:l complex.

Dissociation of thr PapD Chapmonr from Fibrr- and Nnnfi- her-forming Subunits-Subunit-suhunit interactions within a pilus fiber are resistant to denaturants and also to SDS f lr? ) a t 24 "C (20). This fact was used to investigate suhunit interactions in the complexes by SDS-PAGE after incuhation in SDS a t 95 or 24 "C. At 95 "C the DA, complex was dissociated into a 19 kDa PapA monomeric hand and the 28 kDa PapD hand (Fig. 4A, lane 3 ). Interestingly the DA, complex a t 24 'C dissociated into two components that migrated as 38- and 28-kDa species as detected on Coomassie Blue-stained SDS-PACE gels (Fig. 4A, lane 4 ). Western blotting using anti-PapA and anti-PapD antisera revealed that the 38 kDa band was comprised only of PapA (Fig. 4R, lane 2 ) whereas the 28 kDa hand was PapD (data not shown). The 38 kDa PapA hand presumahly represents a PapA dimer which is dissociated into monomers a t 95 "C in SDS (compare Fig. 4 8 , Ianc. 2 to Ianr 1 ). Low amounts of trimeric and tetrameric polymers of PapA were dc1tectc.d hy the Western blot of DA, after incuhation at 24 "C hut not a t 95 "C.

The DA complex dissociated into a 19-kDa PapA monomer and 28-kDa PapD at 24 and 95 "C as determined by Coomassie Blue staining (Fig. 4A, Ianrs -5 and 6 ) and Western blotting using anti-PapA antisera (Fig. 4B. 1anc.s .? a n d 4 1. The small amount of oligomeric species present in the DA at 24 "C was probably due to a slight contamination of DA,. The monomeric and dimeric PapA bands were also dctmted in crude periplns- mic extracts incubated at 24 "C in SDS using anti-PapA anti-

Chaperone-assisted Pilus Biogenesis 12237

46. G -

3 0

21,

14, - K

-E

+ - + - + - + - 9 5 O C

DG OK DE

B 1 2 3 4 5 6

DA2 L

DA D

=DG

c DE cDK ' D

FIG. 5. Only fiber-forming subunits form multisubunit complexes.A, Western blot of DG, DK, and partially purified DE complexes with anti-tip fibrillum antiserum (reacts against PapKEFG) shows only PapE form multimeric complexes. PapG from the DG complex was monomeric after 95 "C (lane 1 ) or 24 "C incubation (lane 2). PapK from the DK complex was also monomeric after 95 "C (lane 3) or 24 "C incubation (lane 4). Partially purified DE complex contained multimers of PapE after 24 "C but not 95 "C incubation (compare lanes 6 and 5) . This was also true for periplasmic extracts from cells expressing both PapD and PapE (compare lanes 8 and 7). B, anti-PapD Western blot of a native gel of different periplasmic extracts from HBlOl cells expressing: both PapD and PapA(1ane 1 ), a mutant of PapD (R8G) that does not bind PapA and PapA (lane Z) , PapD and PapE (lane 3 ), PapD and PapK (lane 4 ) , PapD and PapG (lane 5). Lane 6 shows where purified uncom- plexed PapD migrates. Note that PapD forms two complexes with PapA but only one with any other subunit.

serum in a Western blot (data not shown). Since the DA, complex is only dissociated into dimers of PapA and PapD under these conditions, the PapA monomers and dimers observed in periplasmic extract were presumably derived from the DA and D 4 complexes, respectively. Similarly, the DK complex dissociated into a 19-kDa PapK monomer and the 28-kDa PapD (Fig. 4A, lanes 7 and 8) at both 24 and 95 "C.

In order to compare the aggregative state of the fiber-forming subunits (PapA and PapE) to nonfiber-forming subunits (PapK and PapG), partially purified DE complex and purified DK and DG complexes were also incubated at 24 and 95 "C in SDS sample buffer, analyzed by SDS-PAGE, and Western blotted with anti-tip fibrillum antiserum (Fig. 5A). All five complexes dissociated into monomeric PapD and monomeric subunits at 95 "C (Figs. 4B and 5A). Interestingly, incubation at 24 "C dif- ferentiated fiber-forming from nonfiber-forming subunits. PapG and PapK were dissociated from the DG and DK complexes as monomeric PapK and PapG species at 24 "C (Fig. 5A, lanes 2 and 41, and unlike DA, no oligomeric species of either subunit protein was present. In contrast, the partially purified DE complex produced monomers, dimers, and higher order species when incubated at 24 "C in SDS sample buffer (Fig. 5 A , lane 6). This behavior was also observed in periplasmic extracts from HBlOWpPAP63 + pLSlOl coexpressing PapD and PapE (Fig. 5A, lane 8). No PapD was present in the higher order PapE or PapA oligomers as determined by Western blotting using anti-PapD antiserum (data not shown). These results argue that the resistance of subunit-subunit interactions

n l "0 10 20 30

Time (minutes)

2 5 IO 25 Time

--- polymerized PapA

FIG. 6 . A , DAcomplex is a true intermediate in pilus assembly. W3110 cells expressing papDJKEFGA under the tac promoter were induced, pulsed with [3sSlmethionine for 45 s, and chased with cold methionine at the time of papC induction by arabinose which synchronizes pilus assembly. At zero time ( 2 min into the chase), chloramphenicol was added to stop protein synthesis. Pilus assembly was monitored by ra- dioimmunoprecipitation of the pili at various time points with anti- PapA monoclonal antibody, and the PapA band was quantitated by densitometry after SDS-PAGE and autoradiography. Concurrent periplasmic extractions were analyzed by isoelectric focusing for quan- titation of the DA, and DA complexes. The results were averaged from three experiments and graphed as relative percent of pili, D h , and DA complexes. The time courses for the appearance of pili and the disap- pearance of DA can both be fitted to first-order reactions by the rate constants 7.4 2.6 x min" and 4.7 f 1.5 x lo-* min", respectively, which are comparable within experimental error. B, typical autoradio- grams show the depletion of DA as pili are made and the fluctuation in the low level of D 4 .

to SDS at 24 "C is specific to subunits that polymerize to themselves.

The DA, D 4 , DK, DE, and DG complexes were also detected in periplasmic extracts from cells coexpressing the respective subunit, with PapD, as determined by acidic native PAGE fol- lowed by Western blotting using anti-PapD antisera (Fig. 5B 1. The use of anti-PapD antiserum ensured that only PapD containing complexes were detected. The same DA, and the DA species were also detected using anti-pilus antiserum as were the DK, DG, and DE complexes using anti-tip fibrillum antiserum (data not shown). The ladder of PapE oligomers seen in the SDS gels at 24 "C was also present on native polyacrylamide gels of periplasmic extracts isolated from cells expressing both PapD and PapE as detected with anti-tip-fibrillum antiserum (data not shown). Only subunits that polymerize to themselves were found in multisubunit complexes in native gels despite considerable primary sequence homology between the subunits.

The DA Complex Is a Due Pilus Assembly Intermediate- PapDJKEFGA were induced in W311OlpFJ22, pFJ20 (papD- JKEFGA, papC ) cells and pulse labeled for 45 s with [35Slme- thionine. PapC was induced with arabinose at the time of the chase to synchronize pilus assembly, and 2 min later chloramphenicol was added to prevent further protein synthesis. Under these conditions the labeled periplasmic PapD-PapA complexes had already accumulated, and thus only the conversion of these complexes into pili was measured. At various time points after


the addition of chloramphenicol, aliquots of cells were collected, and both pili and periplasm were extracted. The radiolabeled pili were immunoprecipitated with anti-PapAantisera and sub- jected to SDS-PAGE, while the periplasmic extracts were sub- jected to isoelectric focusing. The pili (polymerized PapA), D 4 , and DA bands were quantitated by autoradiography, and the complexes were normalized to a periplasmic band that was constant throughout the experiment. The time courses are shown graphically in Fig. 6A with representative autoradio- grams (Fig. 6B ). Labeled PapA subunits were rapidly incorporated into pili, and the DA complex was simultaneously depleted with both time courses virtually over in 10 min. The DA, complex was present in low levels with a slight rise and fall during the time course. The low level of D 4 may be due to the location ofpupA at the most distal location from the tuc promoter in construct pFJ22. Therefore, it is inconclusive whether the D 4 complex is a true intermediate. In addition, a portion of both the DA and D 4 complexes may be assembly incompetent since they were not completely depleted from the periplasm (Fig. 6B). This is, however, the first direct evidence of a chaperone complex (DA) as a true intermediate in pilus assembly.

DISCUSSION

PapD-PapA and PapD-PapK complexes were identified, purified, and characterized. PapD has already been shown to form stable complexes with the adhesin PapG (14) and another mi- nor subunit PapE (16). While PapD complexes with PapA and PapK have long been suggested by pilus assembly models this is the first proof that they exist and are stable for long periods. The purification and investigation of the stability and oligomeric state of these complexes is the first step in future studies of in vitro assembly.

Virtually all pilin subunits assembled by the PapD family of chaperones share some amino- and carboxyl-terminal homology. They also share an intramolecular disulfide bond whose spacing and position is conserved between subunits (21). PapA and PapK in particular are 30% identical and -50% similar at the amino acid level. PapA must fit into the tight packing of the pilus rod, and PapK must adapt that rod to the tip of the pilus so it is reasonable to assume they share the same global fold. Despite these similarities, the use of that global fold by a major subunit (PapA) or adaptor protein (PapK) is obviously different. Here we show that PapK and PapA also differ in their ability to form various stoichiometric complexes with the chaperone.

Several different methods were used to determine that PapA forms both a D 4 and a DA complex while PapK forms only a DK complex. The DA and D 4 complexes could be stripped of PapD by SDS a t 24 "C yielding either a PapA monomer or a PapA dimer, respectively. The dimer was dissociated into monomers by heating to 95 "C in SDS. Similarly, dissociation of P pili into monomeric subunits requires boiling in 4 M urea and SDS. The D 4 complex could also be stripped of a PapA subunit by reverse-phase chromatography to yield a homogeneous DA complex having a PI of 6.8 between that of PapD (PI = 9.4) and of PapA (PI = 4.6). The migration of the purified DK, D&, and DA complexes on native or isoelectric gels was identical to their migration when present in crude periplasmic extracts showing that the purification process did not affect the ratio of subunits to chaperone. SDS-PAGE has been used to investigate the multimeric state of other subunits that form polymers like the influenza hemagglutinin (22). The resistance of intersubunit interactions to SDS at 24 "C is specific to PapA and PapE which form homopolymers in the pilus. In contrast PapK and PapG which are not homopolymers in the pilus also do not form SDS-resistant multisubunit complexes in the periplasm.

Electron microscopy and fiber diffraction studies on pili have led Bullitt et aL4 to propose a model of the pilus where each PapA has a complementary surface not only for the PapA on either side (n f l), but also for those three ahead and behind (n f 3). We suggest that the subunit-subunit interaction in the DA, complex reflects one of these quaternary interactions that is formed in the pilus. I t is stable, resistant to SDS unless boiled, and specific to subunits that polymerize. By identifying intersubunit interactions in the periplasm, we are now able to separate the process of making subunit contacts away from the more complex process of making pili.

If the D 4 is a true intermediate, it suggests PapD may play a role in orienting subunit-subunit contacts such that the thick PapA helix is formed. If the DA, is not a true intermediate, then it might suggest that the PapC outer membrane usher protein can only incorporate one subunit at a time. Alternatively, since PapA does make four contacts in the pilus, DA, could represent an assembly incompetent n 3 PapA interaction and therefore cannot be incorporated into the pilus. The inability of PapK to form multisubunit interactions must be due to its not having the proper interactive surface to do so. PapK is a considerably more basic protein (PI = 8.85) than PapA (PI = 4.6), and it is possible that overall charge plays some role. This is unlikely since PapE (PI = 8.1) can form subunit multimers in the periplasm. The fact that both the DA and the D 4 complexes can be detected in the periplasm of a strain producing pili is circumstantial evidence that both may be true intermediates in pilus assembly. The more definitive pulse-chase experiment clearly shows that the DA complex decreases as pili are assembled suggesting that the conversion of DA to pili can be simply modeled as a first-order process. This is the first direct evidence that a chaperone complex is a true pilus assembly intermediate.

Pilus assembly is a complex process. After secretion across the inner membrane, the subunits must be protected from degradation and aggregation, form tertiary and possibly some quaternary structure, dissociate from the chaperone, and be se- creted across the outer membrane. Little is known structurally about the extremely stable subunit interactions that the assembly process produces. Mutational studies can be difflcult to interpret since some interactions are transient, and subunits in nonproductive pathways can be destroyed by proteases. Analy- ses of mutations in PapA will allow us to differentiate between residues necessary for binding PapD and residues necessary for dimeric subunit interactions from those critical for other interactions. By studying stable blocks in the pathway, additional insight can be gained into the assembly of these important virulence structures.

of pPapAYl62 from which pRS2A was constructed, Meta Kuehn for Acknowledgments-We are grateful to Mary J o Wick for construction

critical reading of the manuscript, and Staffan Normark for helpful advice.

REFERENCES 1. Beachey, E. H. (1981) J. Infect. Dis. 143,325-345 2. Hultgren, S. J., Normark, S. J.. and Abraham, S. N. (1991) Annu. Reu. Micro-

biol. 45, 383415 3. Hultgren, S. J., Abraham, S., Caparon, M., Falk, P., St. &me, J., 111, and

Normark, S. (1993) Cell 73,887-901 4. Bock, K., Breimer, M. E., Brignole, A., Hansson, G. C., Karlsson, K.-A., Larson,

G., Leffler, H., Samuelsson, B. E., Stromberg, N., Svanlmrg-Eden, C., and

5. Kuehn, M. J., Heuser, J., Normark, S., and Hultgren, S . J. (1992) Nature 356, Thurin, J. (1985) J . B i d . Chem. 260,85454551

6. Gong, M., and Makowski, L. (1992) J. Mol. Biof. 228,735742 252-255

7. Jacob-Dubuisson, F., Heuser, J. , Dodson, K., Normark, S., and Hultgren, S. J.

8. Dodson, K. W., Jacob-Dubuisson, E , Striker, R. T., and Hultgren, S. J. (1993) (1993) EMBO J . 12,837-847

Proc. Natl. Acad. Sci. U. S. A. 90, 3670-3674

E. Bullitt, and L. Makowski, manuscript in preparation.

Chaperone-assisted Pilus Biogenesis 12239 9. Holmgren, A,, Kuehn, M. J., Brandh, C.-I., and Hultgren, S. J. (1992) EMBO

10. Jacob-Dubuisson, F., Kuebn, M., and Hultgren, S. J. (1993) 'Den& Microbial.

11. Holmgren, A,, and Brandh, C . (1989) Nature 342,248-251 12. Slonim, L. N., Pinkner, J. S., Branden, C. I., and Hultgren, S. J. (1992) EMBO

13. Kuehn, M. J., Normark, S., and Hultgren, S. J. (1991) Proc. Natl. Acad. Sci.

14. Hultgren, S. J., Lindberg, F., Magnusson, G., Kihlberg, J., Tennent, J. M., and

15. Bakker, D., Vader, C. E., Roosendaal, B., Mooi, F. R., Oudega, B., and De Graaf,

J. 11, 1617-1622

1,5C-58

J . 11,47414756

U. S. A . 88, 10586-10590

Normark, S. (1989) Proc. Natl. Acad. Sci. U. S. A 66,43574361

F. K. (1991) Mol. Microbiol. 5, 875-886

16. Lindberg, F., Tennent, J. M., Hultgren, S. J., Lund, B., and Normark, S. (1989)

17. Jones, C. H., Pinkner, J., Nicholes, A., Slonim, L., Abraham, S. N., and Hult-

18. Grant, G. A. (1992) in Synthetic Peptides-A Users' Guide: UWBC Biotechnical

19. Lund, B., Lindberg, F. P., Baga, M., and Normark, S. (1985) J. Bacterid. 162,

20. Eshdat, Y., Silverblatt, F. J., and Sharon, N. (1981) J . Bacterid. 148,308-314 21. Simons, B. L., Rathman, P., Mblij, C. R., Oudega, B., and de Graaf, F. K. (1990)

22. Boulay, F., Dams, R. W., Webster, R. G., and Helenius, A. (1988) J. Cell B i d .

J . Bacteriol. 171, 60524058

gren, S. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8397-8401

Series (Burgess, R., ed) p. 185, W. H. Freeman & Co., New York

1293-1301

FEMS Microbiol. Lett. 67, 107-112

106,629-639

Documents

Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis