Proc. Nall. Acad. Sci. USA Vol. 89, pp. 6210-6214, July 1992 Microbiology

Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae JOEL A. PEEK AND RONALD K. TAYLOR Department of Microbiology and Immunology, University of Tennessee, Memphis, TN 38163

Communicated by Stanley Falkow, March 26, 1992

A number of ToxR-regulated genes that enABSTRACT code products required for the biogenesis or function of the toxin-coregulated colonization pilus (TCP) of Vibrio cholrae have been identified previously by TnphoA fusions. In this study we have examined the role of the product of one of these genes, tcpG, to which a fusion results in a piliated cell lacking all ofthe in vivo and in vitro functions associated with TCP. Our results show that TcpG is not an ancillary pilus adhesin component as suggested by the mutant phenotype but instead is a 24-kDa periplasmic protein that shares active-site homology with several different bacterial thioredoxins and protein disulfide isomerase, as well as overall homology with the disulfide bond-forming DsbA periplasmic oxidoreductase protein of E. coli. Corresponding activity can be demonstrated in vitro for TcpG-enriched fractions from a wild-type strain but is absent in a similarly fractionated tcpG-phoA mutant. The phenotype conferred by a tcpG mutation was found to be pleiotropic in nature, also affecting the extracellular secretion of cholera toxin A subunit and a major protease. This suggests a general role for TcpG in allowing a group of virulenceassociated (and perhaps other) proteins that contain diulfide bonds to assume a secretion or functionally competent state.

the same extent as a tcpA mutant in infant mouse studies (4, 6). This phenotype and the identification of tcpG by phoA fusion suggested that the tcpG gene product might function as a secreted pilus-associated adhesin molecule, similar to that described for other pilus systems such as type 1 fimbriae or P pili (for review, see ref. 7). The findings presented here contradict the notion that TcpG is an adhesin molecule and instead characterize it as a periplasmically localized protein capable of mediating thioldisulfide interchange reactions. The tcpG mutation was also found to be pleiotropic, affecting the secretion of at least one other ToxR-regulated protein, cholera toxin itself. Sequence* homology with another oxidoreductase molecule, DsbA, which affects disulfide bond formation, suggests that TcpG may be a member of a family of periplasmic proteins that assist in the conformational maturation of secreted proteins containing disulfide bonds.

MATERIALS AND METHODS

Cloing the tcpG-phoA Fusion and Intact Gene. Chromosomal DNA isolated from strain KP8-96 was digested with BamHI, ligated into similarly digested pBR322, and transformed into E. coli strain MC1061 (8-10). Transformants were selected on agar containing kanamycin (45 ,g/ml), ampicillin (100 ,.g/ml), and the alkaline phosphatase chromogenic substrate X-P (5-bromo-4-chloro-3-indolyl phosphate; 40 Itg/ml). Two antibiotic-resistant blue colonies resulted. Both recombinant plasmids were shown by restriction analysis to contain a 6.7-kilobase (kb) BamHI fusion fragment, of which 1.7 kb was Vibrio DNA and the remainder was from TnphoA. The plasmid used throughout this study is referred to as p8-96.1. The intact gene was cloned by inserting an antibiotic cartridge adjacent to the tcpG coding region on the Vibrio chromosome. The antibiotic cartridge was utilized as a cloning marker resulting in the tcpG clone pATG1 (unpublished data). DNA Sequence Determination. The BamHI fragment of p8-96.1 and the BamHI/Pst I fragment of pATG1 were subcloned into the appropriate restriction sites of M13mpl8 and transformed into JM103 (11) derivative strain JF626 (J. Felton). Additional subclones generated in both M13mpl8 and M13mp19 were used to determine the DNA sequence from both strands by the dideoxynucleotide chaintermination method with the universal lac, phoA, and additional 20-base-pair (bp) synthetically generated primers. Sequence analyses were performed utilizing Wisconsin Genetics Computer Group Algorithms (12). Antibodies Dircted Apinst TcpG. Kyte and Doolittle analysis (13) indicated a strong hydrophilic peak corresponding to residues 108-130 of the predicted amino acid sequence of the tcpG open reading frame (see Fig. 4B). A 24-amino acid peptide, peptide 1, corresponding to this region plus a C-ter-

The gram-negative bacterium Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that can lead to death through severe dehydration with resultant ion imbalance and hypoglycemia. These disease manifestations are the result of the organism's ability to colonize the epithelial surface of the small bowel and elaborate a potent exotoxin that mediates the ADP-ribosylation of the guanine nucleotide stimulatory (G.) subunit of host adenylate cyclase (1). Expression of cholera toxin and at least one of the colonization factors, the toxin coregulated pilus (TCP), is coordinately regulated within the ToxR virulence regulon (2, 3); ToxR is a transmembrane protein that activates transcription of the cholera toxin operon. TCP is a polymer composed of a 20.5-kDa major pilin subunit encoded by the tcpA gene. A series of TnphoA insertion mutations has defined a number of genes in addition to tcpA that are required for pilus assembly and function (4, 5); TnphoA is a derivative of TnS used to create fusions between target genes and phoA, the Escherichia coli gene for alkaline phosphatase. So far, all of these genes are coordinately regulated by ToxR, and all but one (tcpG) are closely linked and oriented in the same direction, perhaps forming an operon. A TnphoA insertion in tcpG results in a strain that elaborates pili that appear morphologically normal and yet are incapable of mediating any TCPattributable functions. As such, 0395-derived tcpG mutant strain KP8-% no longer autoagglutinates when grown under TCP-expressing conditions in culture, fails to mediate fucoseresistant hemagglutination, and is colonization defective to

Abbreviations: TCP, toxin-coregulated pilus; X-P, 5-bromo-4chloro-3-indolyl phosphate; PDI, protein disulfide isomerase. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M93713).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Microbiology: Peek and Taylor minal cysteine residue to facilitate keyhole limpet hemocyanin (KLH) coupling, was synthesized on an Applied Biosystems peptide synthesizer. The KLH-coupled TcpG peptide was resuspended in phosphate-buffered saline (PBS; 0.154 M NaCl/0.67 M KCl/1.9 mM Na2HPO4/0.1 mM KH2PO4) and emulsified at a 1:1 ratio with Freund's complete adjuvant or with Ribi's adjuvant according to reconstitution instructions provided by Ribi Immunochem. Rabbits were bled for preimmune sera and then immunized with 150-200 ,Ag of the antigen. After a routine immunization protocol, the rabbits were bled and then administered a booster with 75-150 ,ug of antigen solubilized in PBS (for Freund's rabbit) or Ribi's adjuvant (for Ribi's rabbit) (14). Either antiserum was used to detect TcpG by Western immunoblot (15), and both are collectively referred to as "anti-TcpG antiserum." Purification of TcpG. Cultures of 0395 were grown in LB medium (pH 6.5) at 30'C (TCP-expressing conditions) to an optical density of 1.7-1.9 at 600 nm as described (2). These cells were chilled on ice and pelleted at 10,000 x g for 10 min. Cells were then resuspended in cold PBS at a 20-fold concentration. A stock solution of polymyxin B sulfate at 10 mg/ml in PBS was added to the cells to a final concentration of 2 mg/ml. This mixture was gently stirred in an ice bath for 10-12 min. Polymyxin B-treated cells were removed by centrifugation at 10,000 x g for 10 min. The supernatant was then dialyzed against 10 mM Tris.HCl/1 mM EDTA, pH 6.8, overnight at 4°C and concentrated about 4-fold by ultrafiltration using an Amicon PM10 membrane at 50 psi (1 psi = 6.89 kPa) of N2 at 4°C. The retentate was applied to a column of DEAE (DE 52) cellulose (2.6 x 15 cm) that had been equilibrated with 10 mM Tris-HCl/1 mM EDTA, pH 6.8 at 4°C. These ionic conditions were such that TcpG was eluted with the flow-through of the DEAE column. Flow-through fractions were analyzed by Western blot, and TcpG-containing fractions were pooled and concentrated as above. The concentrated TcpG fractions were applied to a column of G-100 Sephadex (1.6 x 96 cm) that had been equilibrated with 50 mM Tris HCl/1 mM EDTA, pH 6.8. Fractions of 1 ml each were collected at a flow rate of 12 ml/hr. TcpG-containing fractions were identified by Western blot and by insulin assay. Insulin Assay. The catalyzed reduction of insulin in the presence of dithiothreitol was measured turbidimetrically at 600 nm as described (16). Reaction mixtures contained 500 ,ul ofinsulin at 1 mg/ml in 0.1 M potassium phosphate buffer, pH 7.0/2 mM EDTA and 3-20 ug of sample. Water was added to a final volume of 1 ml. The reaction was started with the addition of 1-5 ,ul of 100 mM dithiothreitol. Measurements were taken at 60-sec intervals for 60-80 min. Measurements were taken again at -24 hr to assure that equivalent reducing potential was contained in all of the samples. Slopes of the dithiothreitol control and the reaction samples were determined, and activities were calculated as described (16). The assay parameters were first worked out by utilizing lyophilized E. coli thioredoxin (Sigma) that had been resuspended in 0.1 M potassium phosphate buffer, pH 7.0/2 mM EDTA. Once optimized, the assay indicated a thioredoxin activity of 2.9 OD/min per mg of protein slightly below the activity reported by Sigma of 3.0-5.0 OD/min per mg.

RESULTS Identification of TcpG. Antiserum prepared against synthetic TcpG peptide 1 was used to identify TcpG in total-cell protein extracts. Samples prepared from strains RT110.21 (tcpA-phoA), iB1 (toxR-), 0395 (wild type), and KP8-% (tcpG-phoA) were separated by SDS/PAGE and analyzed by Western immunoblot (Fig. 1). A 24-kDa protein recognized by the TcpG antibodies was detected in RT110.21 and 0395 protein extracts and to a lesser extent in iB1. This apparent regulation by ToxR is consistent with the manner in which the tcpG gene was originally identified (4). The KP8-% lane

A

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6211

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FIG. 1. Immunoblot identification of TcpG. Whole-cell extracts SDS/PAGE and probed with anti-peptide 1 TcpG antiserum. Extracts in lanes are from the following strains: A, RT110.21 (tcpA); B, iB1 (toxR); C, KP8-96 (tcpG-phoA); and D, 0395 (wild type): Lane E shows molecular mass markers in kDa.

were resolved by

lacked the 24-kDa protein and instead expressed a 64-kDa protein that cross-reacted with both anti-TcpG and antialkaline phosphatase antisera (data not shown). Subcellular Locliation of TcpG. Since the anti-peptide antibody appeared to be specific for TcpG, it was utilized to localize the TcpG protein. If TcpG were to function as an adhesin molecule as was initially expected, it should be exposed on the cell surface. Repeated attempts utilizing whole-cell ELISA, immunofluorescence, and immunoelectron microscopy of intact bacteria failed to detect TcpG on the exterior of the bacteria (data not shown). This suggested that TcpG is not surface-exposed or that the native epitopes are inaccessible or unrecognizable by the peptide-generated antibodies. Since phoA fusion data indicated that the TcpG molecule was exported beyond the cytoplasm, a fractionation technique with polymyxin B sulfate was used to localize TcpG to either the periplasm or the membrane fraction (4, 17). Cells were washed and then treated with polymyxin B, allowing the periplasmic contents to be solubilized. Polymyxin B-treated cells were pelleted by centrifugation, leaving the periplasmic contents in the supernatant. The pellet and supernatant were then examined for the presence of TcpG. A Western blot of polymyxin B-fractionated 0395 probed with anti-TcpG antibodies showed that all of the detectable TcpG was released from the periplasm and contained in the supernatant fraction (Fig. 2). Fig. 2 also shows a duplicate blot probed with anti-TcpA antibodies that shows TcpA in the pellet fraction. Thus, TcpA and TcpG are separated in different fractions, further suggesting that TcpG does not function as an adhesin molecule but rather works by modulating TCP function, perhaps at a step during pilus assembly. The periplasmic location of TcpG was also demonstrated by AntiTcpA

msp

AntiTcpG

m

I FIG. 2. Immunoblot of polymyxin B-fractionated 0395. Washed cells were treated with polymyxin B and then centrifuged at 16,000 x g for 10 min to separate the periplasmic contents (supernatant) from the treated cells (pellet).S Proteins from corresponding fractions (lanes and P) were resolved by SDS/PAGE and separately probed with anti-TcpA (Left) or antiTcpG (Right) antisera. Immunoblots were overdeveloped to enhance detection of trace amounts of antigen.

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Proc. Natl. Acad. Sci. USA 89 (1992)

immunoelectron microscopy of Lowicryl thin sections of strain 0395 and Western analysis after EDTA-lysozyme fractionation (data not shown). Purification of TcpG. To investigate potential activities of TcpG by in vitro assays, the protein was purified from the periplasmic space as described in Materials and Methods. Absorbance profiles from the G-100 elution of KP8-% and 0395 samples were similar, with peak material consistently being eluted at characteristic molecular weights. Only the amplitude of these peaks varied from run to run, possibly because of slightly differing culture conditions. While the SDS/PAGE protein profiles of corresponding peaks from both strains appeared to be identical (Fig. 3 Left), a duplicate gel run simultaneously and analyzed by Western blot with anti-TcpG antibodies showed a 24-kDa band for 0395 (lanes M-Q in Fig. 3 Right) but no 24-kDa cross-reactive band in the corresponding lanes for KP8-96. Interestingly, while TcpG was thus apparently absent in KP8-96, other fractions from this strain contained either a 20-kDa or 29-kDa cross-reactive protein (Fig. 3 Right, bands a and b). These bands have been noted previously in whole-cell samples of KP8-96, where the predominate cross-reactive band is always the 64-kDa hybrid protein, with bands of lesser intensity from 20-35 kDa, suggesting that these smaller bands are degradation products of the hybrid protein since they are only seen in KP8-% and never in 0395. TcpG Thiol-Disulfide Interchange Activity. Comparison of the predicted amino acid sequence of TcpG to entries in the Swiss protein data base using the TFASTA algorithm (18) revealed homology to the reactive redox site of protein disulfide isomerase (PDI) (19) and several different bacterial thioredoxins (20) (Fig. 4A), suggesting that a similar activity might be attributable to TcpG. The most widely used method for monitoring the activity of E. coli thioredoxin during isolation is an insulin assay developed by Holmgren (16) that spectrophotometrically records the precipitation of the insoluble B chain that is produced when the interchain disulfide bonds of insulin are reduced. For strains KP8-% and 0395, G-100 elution profiles were used to choose samples from each of the characteristic elution peaks to be assayed for redox activity. Due to time-dependent loss of activity of the samples, they were assayed as they were eluted from the G-100 column or shortly thereafter. Subsequent to the assay, the elution peaks from both strains appeared to contain identical protein profiles with the exception of TcpG being absent in the KP8-% profile (Fig. 3). Based on the difference in the slope of the reaction sample to that of the dithiothreitol control, samples taken from the area ofthe 0395 elution profile that contained protein in the 24-kDa range (corresponding to lane P in Fig. 3) showed an activity of 2.7 OD/min per mg of protein (Fig. 5). Multiple A B C D E F

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1 MEKIWLALAGLVLAFSASAAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCP 50 51 HCNTFEPII... AQLKQQLPEGAKFQKNHVSFMGGNMGQAMSKAYATMIA 97 51 HCYQFEEVLHISDNVKKKLPEGVEOTKYHVNFMGGDLGKDLTQAWAVAMA 100 98 LEVEDKMVPVMFNRIHTLRKPPKDEQELRQIFLDEGIDAAKFDAAYNGFA 147

101 LGVEDKVTVPLFEGVQK. TQTIRSASDIRDVTINAGIKGEEYDAAWNSFV 149 148 VDSMVRRFDKQFQDSGLTGVPAVVVNNRYLVQGQSVKS....,.LDEYFD 191 150 VKSLVAQQEKAADVQLRGVPAMFVNGKYQLNPQGMDTSN)VFVQQYAD 199 192 LVNYLLTLK 200

200 TVKYLSEKK 208

FIG. 4. (A) Active site homologies. TcpG residues 36-58 are aligned with the region surrounding the catalytic site (boxed) of rat liver PDI (19), thioredoxin from Corynebacterium nephridii (20), and the highly related E. coli DsbA protein (21). (B) Overall homology between TcpG (top line) and DsbA was determined by the algorithm of Needleman and Wunsch (22). Vertical lines show identity; comparison value 2 0.50; ., comparison value 2 0.10.

0395 purification analyses yielded thio-disulfide interchange activities from 2.7 to 4.2 OD/min per mg. This activity remained constant with a doubling ofthe sample concentration (data not shown). Thus, the "thioredoxin-like" reactive site of TcpG can function as a thiol-disulfide interchange site. Samples taken from analogous areas of the KP8-96 protein profile had no activity (Fig. 5). It was noted that one KP8-96 sample, corresponding to lane C in Fig. 3, had increased performance (0.64 OD/min per mg) over similar nonactive 0395 fractions. Subsequent Western analysis revealed that the assayed sample contained the probable degradation product of the fusion protein described above (Fig. 3 Right, band a). Since the fusion protein contains the redox site from TcpG, the activity of this sample may be attributable to this degradation product or to a compensatory overexpression of another redox molecule or redox reaction intermediary. Plelotpic Nature of the tcpG-phoA Fusion Mutation. The thiol-disulfide interchange activity of TcpG led us to investigate whether another ToxR-regulated disulfide-containing molecule, cholera toxin itself (23), was affected by a tcpG

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FIG. 3. 0395 and KP8-96 G-100 gel filtration protein profiles. Samples were resolved by SDS/PAGE and stained with Coomassie blue (Left) or were transferred to nitrocellulose and probed with anti-TcpG (Right). Lanes: A, a polymyxin B supernatant from 0395; B-J, KP8-96 elution

fractions; K-S, corresponding 0395 elution fractions.

Proc. Natl. Acad. Sci. USA 89 (1992)

Microbiology: Peek and Taylor

0

0.2 0

0.1

0

20

10

30 40 Time, min

50

60

FIG. 5. Oxidoreductase activity assay of 0395 and KP8-% gel filtration samples. Each reaction mixture contains 500 /4I of insulin at 1 mg/ml to which -3 itg of protein sample was added, and the volume was then adjusted to 1 ml. The reactions were started with the addition of 1 A.l of 100 mM dithiothreitol to the reaction cuvette, and the OD was recorded at 600 nm for 60 min. o, DTT control; *, 0395 sample corresponding to lane P of Fig. 3; and *, corresponding KP8-96 sample.

mutation. To assess the effects of TcpG on toxin, cultures of KP8-96 and 0395 were grown to an equivalent OD at 600 nm under toxin-expressing conditions. Both whole-cell and supernatant samples were resolved by SDS/PAGE and analyzed by Western blot (Fig. 6) with polyclonal anti-holotoxin antiserum (provided by J. Mekalanos). Most notable was the finding that the toxin A subunit profiles were markedly different between the two strains. The A subunit of 0395 was found in the unnicked A form in the whole-cell extracts, and both the unnicked A and Al forms in the supernatant. KP8-96, on the other hand, showed elevated levels ofunnicked A in the cellular extracts, but reduced levels of secreted unnicked A and virtually no Al form in the supernatant. In contrast the secreted B subunit appeared to be at wild-type levels or above for strain KP8-96. Toxin secretion remains defective in a tcpG null mutant as well (unpublished data), indicating that the toxin A subunit secretion defect seen in the tcpG-phoA mutant is not a

b

c

d

FIG. 6. Comparison of KP8-96 and 0395 toxin profiles. KP8-% and 0395 cultures were centrifuged at 10,000 x g for 10 min to pellet cells. The culture supernatants were concentrated -20-fold by using an Amicon PM10 membrane at 50 psi at 40C. Proteins from the supernatant and from whole-cell samples were resolved by SDS/ PAGE and immunoblotted with anti-whole toxin antibodies. Lanes: a, 0395 whole-cell sample; b, 0395 culture medium; c, KP8-% whole-cell sample; d, KP8-96 culture medium.

6213

due to a general secretion defect caused by the improper localization of the TcpG-PhoA hybrid protein. The tcpG mutant was also found to be defective in protease secretion. This was evident from growth of the wild-type and mutant strains on casein agar, where the parent caused a substantial clearing around the colonies, that was barely evident around equally sized colonies of KP8-96 or a tcpG deletion/insertion mutant strain (data not shown). Homology Between TcpG and DsbA. Recently, a periplasmic protein named DsbA has been identified in E. coli that also shares primary structural homology with the active site of disulfide oxidoreductases. Furthermore, this protein has been shown to be required for the efficient disulfide bond formation of several periplasmic and outer membrane proteins in vivo. Like TcpG, DsbA can also catalyze the reduction of insulin in vitro (21). Comparison of the amino acid sequences of TcpG and DsbA using the Needleman and Wunsch program (22) revealed 63% similarity, with a striking 40%o identity, between the two proteins (Fig. 4B). Neither of these proteins shares significant homology with the other oxidoreductases beyond the region surrounding the active site, suggesting that DsbA and TcpG form a distinct class of enzymes involved in the formation of disulfide bonds within the periplasm.

DISCUSSION The tcpG mutant strain KP8-96 elaborates TCP colonization pili that appear morphologically normal by transmission electron microscopy of negatively stained samples, yet fail to mediate colonization in vivo, and are functionally deficient by several in vitro assays (4, 6). This phenotype suggested that tcpG would encode a surface or pilus-associated adhesin molecule, or possibly a product that mediated pilus function in some other manner. The findings presented here are consistent with the latter interpretation. By using a variety of techniques, no detectable TcpG was found on the exterior of the cell or associated with assembled TCP pili. Instead, TcpG appears to be localized exclusively to the periplasm. The predicted amino acid sequence of TcpG initially revealed homology to both thioredoxin and PDI, centering around the thiol-disulfide interchange sites of these molecules. We were able to show that the homologous redox site in TcpG can function in vitro with a level of activity comparable to that of E. coli thioredoxin. Thus, TcpG might interact with target proteins that contain disulfide bonds. Consistent with this hypothesis, the major TcpA pilin subunit contains an intrachain disulfide bond (unpublished data; ref. 24). The amino acid sequence encompassed by this disulfide bond is hydrophobic in nature, and it is likely that this domain is externalized, leading to the very hydrophobic characteristics of the pilus (2). This domain has been implicated in the function of TCP-mediated colonization based on its recognition by monoclonal antibodies that provide passive immunity to experimental cholera (24). A similar carboxyl cystine loop in the type 4 pilin of Pseudomonas aeruginosa PAK has also been proposed to be an exposed adhesive domain (25, 26). Preliminary findings have indicated that KP8-96 pili are severalfold less hydrophobic than wild-type pili, yet the pilin in these defective structures retains its disulfide bond (unpublished data). The presence of the disulfide-bonded pilin in the tcpG mutant may not be surprising, since atmospheric oxidation would still be expected to occur. Thus, the TCP defect in the tcpG mutant may be attributable to the domain between the disulfide bond misfolding, resulting in the hydrophobic area in this region being internalized. The properties of TcpG, coupled with the ToxR regulation of its expression, prompted us to examine the effect of the tcpG-phoA fusion on other disulfide-containing, ToxRregulated molecules such as cholera exotoxin, where both the toxin A and B subunits contain disulfide bonds (21, 27). Western analysis of whole cholera toxin revealed a defect in

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the secretion of A subunit, while the B subunit was unaffected or present in greater than wild-type levels. Since the A subunit must associate with the B pentamer to be exported (28), it seems likely that the conformations required for this association are not efficiently achieved in the tcpG mutant. The pleiotropic nature of a tcpG mutation was also seen to include a significant decrease in the amount of active secreted protease by such mutants. A likely protease to be affected is the soluble hemagglutinin/protease that contains four cysteine residues and shares extensive homology with elastase of P. aeruginosa (29) where these residues have been shown to be disulfide-bonded (30). While the exact roles and mechanisms of TcpG function are not yet defined, insight may be provided from the functions attributable to related thiol-disulfide interchange molecules. For example, a protein with a homologous redox site, PDI, is a soluble multifunctional homodimer (2 x 57,000) found on the luminal side of the endoplasmic reticulum membrane (19). PDI has been shown to catalyze thioldisulfide interchange reactions in protein substrates and has also been closely correlated to the process of secretory protein synthesis and secretion (31, 32). Thus, proteins involved with disulfide bond formation may be important in mediating protein conformation during the export process. The idea that protein conformation is dictated solely by amino acid sequence has long been considered the fundamental principle behind protein folding (33). This model was initially reinforced by in vitro refolding studies of small proteins following denaturation and may be analogous to what usually occurs in the cytoplasm of cells. However, spontaneous protein folding based on primary structure fails to take into account the multiple physiochemical environments encountered by translocated polypeptides or the catalysis of disulfide bonds at a physiological rate. In vitro folding assays with proteins that contain disulfide bonds often give low yields (34). Thus, while bacterial disulfide-bonded proteins may spontaneously oxidize once they are secreted beyond the cytoplasm, it may not occur at a physiological rate without catalysis by an oxido-reductase protein such as TcpG. Given the properties of the TcpA pilin in the tcpG mutant, TcpG may have two roles in polypeptide maturation, one as a thiol oxidant and possibly also as an isomerase/ chaperone similar to BiP (binding protein) (35), where TcpG may act to locate and guide portions of polypeptide chains into a state whereby complex surfaces can form. These surfaces, such as the externalized hydrophobic domain of TcpA, might otherwise be energetically unfavorable and would rarely form under physiological conditions. This analysis suggests that the successful maturation of many translocated polypeptides would require the assistance of oxido-reductase enzymes on the distal side of membranes. The presence of a global class of periplasmic thiol-disulfide interchange proteins is supported by the independent findings of Bardwell et al., who have isolated the protein DsbA from the periplasm of E. coli that participates in disulfide-bond formation of several periplasmic and outer membrane proteins (21). As shown in Fig. 4, comparison of the E. coli dsbA gene product to TcpG reveals 40%o identity throughout the entire sequence, extending beyond the active site. This conservation of primary structure and function suggests the presence of a unique class of bacterial periplasmic proteins necessary for the functional maturation of some secreted proteins and illuminates another step in the secretion pathway of bacterial proteins. Consistent with this function the tcpG gene product has a higher basal level than most ToxRregulated gene products and is further induced under ToxRexpressing conditions (4). This response may reflect a cellular mechanism for maintaining the functional conformation of exported proteins before and during times of high expression

of translocated polypeptides, which in this case represent a coregulated set of secreted virulence factors. The authors thank L. Hatmaker, P. Hoffman, J. Katze, M. Kaufman, J. Mekalanos, and K. Peterson for contributing strains, reagents, equipment, and technical advice and for participating in insightful discussions. We also thank J. Bardwell and J. Beckwith for discussing results prior to publication. This work was supported by U.S. Public Health Service Grant AI-25096 to R.K.T.; J.A.P. is the recipient of National Institutes of Health Predoctoral Training Grant

AI-07238.

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Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae.

A number of ToxR-regulated genes that encode products required for the biogenesis or function of the toxin-coregulated colonization pilus (TCP) of Vib...
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