In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC*

Caterina Guzmán-VerriDagger §, Esteban Chaves-Olarte||, Fernando García||, Staffan ArvidsonDagger , and Edgardo Moreno§

From the Dagger  Microbiology & Tumorbiology Center, Box 280, Karolinska Institute, S-171-77 Stockholm, Sweden, § Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Aptdo 304-3000 Heredia, Costa Rica, and || Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, 1000, San José, Costa Rica

Received for publication, October 18, 2000, and in revised form, February 13, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Escherichia coli hemolysin (HlyA)1 is the major representative of a family of proteins secreted by proteobacteria known as "repeats in toxin" (RTX). Other members of this family are leukotoxins from Pasteurella hemolytica and Actinobacillus pleuropneumoniae, the adenylate cyclase-hemolysin from Bordetella pertussis, the cytotoxin RtxA from Vibrio cholerae, and antigenically related exoproteins from Neisseria meningitidis and Rhizobium leguminosarum (1-12). Most of these proteins are pore-forming toxins proposed to play a role in the pathogenicity of these microorganisms. E. coli hemolysin (HlyA) is synthesized as an inactive precursor, which is activated through fatty acid acylation of residues Lys564 and Lys690 (13, 14) when both the HlyC protein and the acylated form of acyl-carrier protein are present (13, 15, 16).

HlyC is a small protein of 170 amino acid residues (19.8 kDa) homologous only to other known activator C proteins (17-20). Primary structure comparison of 13 activator C proteins from different bacterial species showed a high degree of similarity, suggesting a common function (17). At present, only HlyC from E. coli and CyaC from B. pertussis have been shown to be involved in the fatty acylation of their corresponding RTX toxin (13, 21). Recently, a novel type of fatty acyltransferase activity, that catalyzes the formation of an internal protein amide bond has been described for HlyC (22, 23), despite the fact that HlyC does not show any significant similarity to known acyl transferases (13, 19), the protein seems to acylate HlyA in an equimolar fashion, and it appears to be functionally consumed during the reaction (16, 22). In this respect, it is also notable that HlyC and HlyA are encoded in the same mRNA and therefore synthesized at nearly equimolar amounts (24, 25). The unique acylation reaction catalyzed by HlyC does not involve covalent attachment of fatty acid molecules to the acyltransferase but a direct interaction between acylACP-HlyC and pro-HlyA through a non-covalent ternary complex (22). A HlyC binding domain in pro-HlyA has been proposed (26, 27), and some highly conserved amino acid residues in the carboxyl and amino termini of HlyC essential for the acylation process have been identified (17, 28-30).

Understanding the mechanism of activation and processing of RTX systems is important for therapeutic applications and vaccine production. In this respect, promising results have been obtained using the A. pleuropneumoniae Apx toxins to elicit a protective immune response to prevent porcine pleuropneumonia (3). Here we present evidence that HlyC is degraded in vivo by different protease systems when HlyA is present.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains, Plasmids, and Culture Media-- The recombinant plasmids (Table I) were propagated in E. coli 5K (SmrlacYI tonA21 thr-1 supE44 thi rk-mk+) or XL1Blue (Stratagene, La Jolla, California). When two plasmids were introduced in the same host, their integrity was corroborated by restriction enzyme digestion. The protease system-deficient strains used are listed in Table I. Bacteria were grown in 2-fold YT broth or LB broth (31) supplemented with ampicillin (50 µg/ml) and/or chloramphenicol (10 µg/ml).

Construction of Recombinant DNA, Expression and Purification of GST-HlyC-- HlyC was expressed as a GST tag fusion protein using vector pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden). Cloning and purification of the GST fusion protein was done according to the manufacturer's instructions (Amersham Pharmacia Biotech). Briefly, a PCR product from plasmid pFG1a encoding hlyC was obtained by using the following pair of primers: 5'-ACGTGAATTCTGAATATAAACAAACCATTAG-3' and 5'-TATCGAATTCTTTAATTACCTCTTAACCAG-3', containing EcoRI restriction site. Vector and PCR product were digested, ligated, and transformed into E. coli BL21-competent cells for propagation of the generated plasmid, pGEX2T-HlyC. GST-HlyC was functionally equivalent to wild type HlyC as judged by hemolytic activity assays. Expression of GST-HlyC was achieved by growing E. coli BL21 harboring pGEX2T-HlyC in 2-fold YT medium with aeration at 37 °C to mid exponential growth (A600 nm = 0.5). Cultures were then induced with 0.1 mM IPTG at 30 °C for 2 h. Bacteria were collected by centrifugation at 7700 × g, resuspended in phosphate-buffered saline and lysed by sonication. After centrifugation at 12,000 × g, the supernatant was incubated with glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 2 h at room temperature. Beads were washed twice with phosphate-buffered saline, and GST-HlyC was eluted with 10 mM glutathione (Sigma). The suspension was stored at -70 °C for further use.

Determination of Hemolytic Activity in Culture Supernatants-- E. coli strains were grown in 20 ml of 2-fold YT medium containing the appropriate antibiotics at 37 °C with shaking. Samples were taken at late logarithmic phase of growth and analyzed as described (32). Culture supernatant from E. coli 5K(pANN202-812B, pUC18) was used as a negative control, and sheep erythrocytes lysed with water were used as total lysis control. The results were expressed as percentage of hemolysis produced by strain 5K(pANN202-812B, pFG1a) ("wild type hemolysin").

Antiserum Production and Purification-- Rabbit anti-HlyC antibodies were produced by four intramuscularly applied boosts of GST-HlyC (250 µg) in complete (first boost) or incomplete (second to fourth boost) Freund adjuvant (Sigma). Antibodies from 2 ml of serum were adsorbed to GST-HlyC-Sepharose beads, eluted with 0.2 M glycine, pH 2.5, and collected in 1 M Tris, pH 9.0, according to Amersham Pharmacia Biotech recommendations. The antibodies were concentrated by ultrafiltration and stored at -20 °C in 50% glycerol.

Sample Preparation and Western Blotting-- Cell samples from the various strains were taken at different time points throughout cell growth and resuspended in water, 0.1 mM PMSF (Sigma) unless otherwise stated. Cell lysates obtained by sonication were centrifuged at 100,000 × g for 1 h, and supernatants were collected. Twenty µg of total protein were separated on a 12.5% SDS-PAGE according to Ref. 33, transferred to a polyvinylidene difluoride membrane (Roche Molecular Biochemicals), and probed with the rabbit polyclonal antiserum against HlyC or with a rabbit polyclonal antiserum against HlyA provided by Dr. Ivaylo Gentschev (University of Würzburg, Würzburg, Germany). Probing with a peroxidase-labeled secondary antibody and developing were carried out using a chemiluminescence Western blotting kit (Roche Molecular Biochemicals). Samples of culture supernatants were prepared as described below.

Northern Blot Analysis-- Total RNA from strains 5KpFG1a, 5K(pANN202-812B,pFG1a), and 5K(pACYC184,pUC18) at different time points was prepared using the RNeasy kit (Qiagen GmbH). For Northern blot analysis, 20 µg of total RNA were subjected to electrophoresis, transference to a Byodine A nylon membrane (Pall Ultrafine Filtration Corp.) and hybridization as described (34). A 450-base pair PCR product spanning nucleotides 726-1176 from Ref. 35, obtained from plasmid pFG1a was used as a probe to detect the 5' end of hlyC. The fragment was radiolabeled to a specific activity of 1-5 × 108 cpm µg-1 with [alpha -32P]dCTP (Amersham Pharmacia Biotech) using a random primer labeling kit (Roche Molecular Biochemicals). After hybridization, the filters were washed once for 1 h at 42 °C in 2-fold 0.15 M NaCl plus 0.015 M sodium citrate containing 0.1% SDS. After 2 h of exposure, Northern blot images were processed using the ImageQuant software.

HlyC Immunoprecipitation-- Cell samples from strains XL1BluepFG1a and XL1BluepUC18 were collected after IPTG induction according to Ref. 26. Seventy µg of total protein obtained after sonication of bacteria were mixed with 500 µl of immunoprecipitation buffer (150 mM NaCl, 1% Triton X-100, 10 mM Tris, pH 7.4, 1 mM EDTA, and 0.2 mM NaO3V) as described (36). Twenty µg of protein A-Sepharose (Amersham Pharmacia Biotech) were added to the immunoprecipitation mix for preclearing. After 2 h at room temperature, the samples were centrifuged at 1600 × g. The supernatants were incubated with 1.5 µg of purified anti-HlyC antibody and incubated for 2 h at room temperature, followed by addition of 20 µl of protein A-Sepharose and further incubation for 2 h at room temperature. The samples were centrifuged at 1600 × g for 5 min, and the pellet was resuspended in 1 ml of immunoprecipitation buffer. After three washing steps, samples were resuspended in 50 µl of SDS-PAGE sample buffer for further analysis on a 12.5% SDS-PAGE and silver staining as described (37).

Analysis of HlyA by Two-dimensional Polyacrylamide Gel Electrophoresis-- Supernatants corresponding to 5 × 108 cells at late logarithmic growth phase were precipitated with ice-cold trichloroacetic acid (final concentration 10%) for 1 h on ice. The pellet obtained after centrifugation was washed twice with ice-cold acetone and stored for no longer than 1 day at -20 °C. For analysis on two-dimensional polyacrylamide gel electrophoresis, the protocol described by Ludwig et al. (26) was followed with some modifications. Briefly, precipitated culture supernatants were resuspended in 20 µl of isoelectrofocusing sample buffer (62.5 mM Tris-HCl, pH 6.8, 0.5% SDS, and 10% glycerol) and 2 µl of 1 M Tris-HCl, pH 9.0. After heating at 56 °C for 15 min, 7.5 mg of urea/10 µl (ultra pure urea, Bio-Rad) were added, followed by two volumes of isoelectrofocusing lysis buffer: 9.5 M urea, 2% Nonidet P-40, 5% beta -mercaptoethanol and 5% ampholines, pH range 3-10 (Bio-Lyte 3/10 ampholyte, Bio-Rad). The lower electrode solution was 13.6% H3PO4 and the upper electrode solution, 20 mM NaOH. After isoelectric focusing separation in a miniature two-dimensional electrophoresis cell, the proteins were separated in the second dimension by SDS-PAGE and stained with Coomassie Brilliant Blue as described (33, 37).

Site-directed Mutagenesis-- Deletion of codons 10-18 in hlyC (pFG1a) was made by the ExSite PCR-based site-directed mutagenesis kit (Stratagene) with some modifications. Briefly, mutagenesis primers were designed according to Stratagene's recommendations and synthesized at Amersham Pharmacia Biotech. The sequence of the synthetic oligonucleotides is: 5'-AATCTCTAATGGTTTGTTTATATT and 5'-AGTTCTCCACTACACAGAAACT. One pmol of pFG1a prepared as described (31) was mixed with 15 pmol of each primer in 1% Me2SO, 25 µmol of dNTP mix, and 2.5 units of Taq DNA polymerase premixed with 2.5 units of Taq extender PCR additive. The PCR was performed in a PerkinElmer Life Sciences GeneAmp PCR system 9600, and the cycles were as follows: one cycle at 94 °C for 4 min, 55 °C for 2 min, and 72 °C for 3 min; eight cycles at 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min, followed by 10 min at 72 °C. The digestion of the original template and polishing of the PCR product was performed according to the Stratagene protocol. After verifying the integrity of the PCR product by agarose gel electrophoresis, 5 µl of the mutagenesis reaction were ligated for 1 h at 37 °C with 4 units of T4 ligase and 5 µmol of ATP in a 12-µl final volume, and used to transform competent XL1Blue cells. Mutants were identified by terminator cycle sequencing using fluorescence-labeled dideoxynucleotides (ABI Prism dye terminator cycle sequencing core kit, PerkinElmer Life Sciences) and a pair of primers designed for that purpose. The sequencing primers corresponded to nucleotides 726-743 and 1030-1049 of the reference nucleotide sequence (35). The reactions were analyzed on an ABI Prism 373A DNA sequencer. Further transference of the mutated plasmid to E. coli 5K was performed by transformation according to Ref. 31.

Identification of Proteins by MALDI-Mass Spectrometry and Peptide Mass Mapping-- Strains XL1BluepFG1a and XL1BluepUC18 were induced with IPTG as described (26). Cell lysates from both strains were obtained by resuspension in 2× SDS-PAGE sample buffer. Three hundred µg of total protein were precipitated with acetone for 20 min and resuspended in 60 µl of isoelectrofocusing lysis buffer. For two-dimensional separation, Immobiline DryStrip gels from Amersham Pharmacia Biotech were used and run according to manufacturer's instructions. After second dimension separation on SDS-PAGE and Coomassie staining, the proteins were localized by using reference spots from the same gel and from a Western blot run in parallel using the same sample. Identification of protein spots cut out from two-dimensional gels was carried out by MALDI-mass spectrometry and peptide mass mapping at the Protein Analysis Center, Karolinska Institute, Stockholm, Sweden. The tryptic peptide masses obtained were compared with the predicted masses of tryptic peptides from a protein sequence data base. All the analyzed protein spots generated peptides that matched the theoretical HlyC sequence.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HlyC Migrates as Two Protein Bands on SDS-PAGE-- To raise a polyclonal antibody against HlyC, a GST-HlyC fusion protein was constructed, expressed, and purified. Affinity-purified GST-HlyC migrates on SDS-PAGE as two discrete bands corresponding to the expected molecular weight for the fusion protein (Fig. 1, lane 5). Affinity-purified antibodies against GST-HlyC were obtained using this preparation linked to Sepharose beads. Their specificity was evaluated by Western blot analysis of cell lysates from an E. coli strain encoding only hlyC and from a hemolytic strain carrying hlyA and hlyC in trans. Two bands close to 20 kDa were detected in cell lysates when HlyC was expressed alone (Fig. 1, lanes 1 and 2). These bands were not detected in cell lysates when HlyC was co-expressed with HlyA, and rather a 10-kDa protein band was observed (Fig. 1, lane 4). These results indicate that the produced antibodies specifically recognize two forms of HlyC, in agreement with the two forms observed with purified GST-HlyC. In addition, reactivity of anti-HlyC antibodies with a lower molecular weight peptide in lysates where the full-length protein was not present, suggests proteolysis of the mature protein. This 10-kDa band was detected only when PMSF was added to the cell lysates (Fig. 1, lanes 3 and 4). The same molecular mass and mobility of the 20-kDa bands remained after treatment of lysates with and without beta -mercaptoethanol, electrophoretic re-running of the isolated SDS-PAGE bands, and immunoprecipitation excluding degradation of the proteins by manipulation (data not shown).


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Fig. 1.   Electrophoretic analysis of HlyC in E. coli extracts. Lane 1, 5KpFG1a Coomassie Blue-stained cell lysates containing HlyC; lane 2, Western blot of 5KpFG1a cell lysates using anti-HlyC antibodies; lane 3, Western blot of 5K(pANN202-812B,pFG1a) expressing hlyA and hlyC in trans in the absence of PMSF, using anti-HlyC antibodies; lane 4, same as lane 3 in the presence of PMSF; lane 5, affinity-purified GST-HlyC that was Coomassie Blue-stained.

The Presence of HlyA Correlates with HlyC Processing-- To further investigate a possible role of HlyA in HlyC processing, cell samples from different 5K E. coli strains were taken throughout growth and analyzed by Western blot. HlyC was detected at all time points tested in E. coli carrying only hlyC (Fig. 2A). Instead, in lysates from the hemolytic strain encoding hlyABD and hlyC in trans, only the 10-kDa band was evident throughout growth. A small amount of the upper 20-kDa HlyC band was detected in the 5-h sample, indicating incomplete degradation at this particular time (Fig. 2A). When HlyA and HlyC were expressed in cis, again HlyC was not found, regardless of the copy number of the plasmids used (Fig. 2B). The presence of HlyA throughout growth was analyzed by Western blot using anti-HlyA antibodies (Fig. 2C). Increasing amounts of this protein were found in cell samples from E. coli carrying hlyABD and hlyC in trans, showing that HlyA was normally translated. These data indicate that the disappearance of HlyC proceeds throughout growth and is related to the presence of HlyA, indifferently of whether hlyA is encoded in cis or in trans to hlyC or in a high or low copy number plasmid. A small amount of the 10-kDa peptide band was detected in cell lysates obtained at late hours of growth from E. coli carrying only hlyC, suggesting that incomplete proteolysis may happen even in the absence of HlyA at later times.


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Fig. 2.   Expression of HlyC and HlyA throughout growth. A, 20 µg of total protein from strain 5K(pANN202-812B,pFG1a) carrying hlyABD and hlyC in trans (a) or 5 µg of total protein from strain 5KpFG1a encoding only hlyC taken at different time points were analyzed by Western blot (b). Lane - contains a cell lysate from 5K(pANN202-812B,pUC18). B, Western blot analysis using anti-HlyC antibodies of a 5-h sample (20 µg) from strain 5K(pANN202-812) encoding hly in cis (a) is shown together with a positive control (b, 5KpFG1a cell lysate at 5 h). In the c lanes, cell lysates (20 µg) from strain 5K(pANN202-312*) taken at different time points and carrying hly in cis are shown, and in the d lanes, from strain 5KpFG1a encoding only hlyC. Samples from strain 5KpFG1a contain only 5 µg of total protein due to the high intensity signal. C, Western blot analysis of cell lysates taken at different time points from strain 5K(pANN202-812B,pFG1a) carrying hlyABD and hlyC in trans using anti-HlyA serum. Lane + contains a trichloroacetic acid-precipitated culture supernatant at 6 h from the same strain. Lane -, cell lysate from strain 5KpFG1a.

To determine if the lack of HlyC in the hlyCABD encoding strain was due to impaired transcription of hlyC, total RNA was obtained throughout cell growth and analyzed by Northern blot using as probe the 5' end sequence of hlyC. Equal amounts of hlyC RNA transcript were found at the time points tested in cells carrying hlyA and hlyC in trans or in bacteria carrying only hlyC (Fig. 3A). Detection of three different hlyC encoding mRNA (a, b, and c) may be due to different transcription start points upstream from hlyC as has been described elsewhere (38, 39). An indirect proof of HlyC expression is the presence of acylated HlyA, since it is known that HlyA acylation is an HlyC-dependent reaction (13, 15, 16). The acylation status of HlyA was therefore assessed in culture supernatants from different strains by two-dimensional polyacrylamide gel electrophoresis. Fig. 3B (panel a) shows that a single protein spot corresponding to fully acylated toxin was detected in E. coli encoding hlyABD and hlyC in trans, whereas in a strain lacking hlyC (panel b) nonacylated HlyA was detected. This protein spot was not detected at all in E. coli encoding only HlyC (panel c). Thus, in E. coli carrying hlyA and hlyC in trans, HlyC must have been produced. HlyC was not co-secreted with HlyA to the extracellular medium as demonstrated by Western blot analysis of concentrated culture supernatants (data not shown). Altogether, these data indicate that HlyC is processed in vivo following expression of HlyA.


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Fig. 3.   Northern blot analysis of hlyC mRNA and electrophoretic analysis of HlyA. A, Northen blot analysis of hlyC transcripts (a, b, and c) at different time points from strains 5K(pANN202-812B,pFG1a) and 5KpFG1a. The size of the hlyC transcript from pFG1a is unknown due to the lack of a transcription stop in this construct. The same pattern of transcription was detected for both strains. No hlyC transcript was detected in total RNA extracted from control strain 5K(pACYC184,pUC18). B, two-dimensional polyacrylamide gel electrophoresis of culture supernatant from strain 5K(pANN202-812B,pFG1a) (a) showed a single protein spot that corresponds to fully acylated toxin according to control experiments: culture supernatant from the pro-HlyA producing strain 5KpANN202-812B (b) and culture supernatant from the non producing HlyA strain 5KpFG1a (c).

Acylation of HlyA Is Not Required for HlyC Processing-- To confirm that the lack of HlyC is related to the presence of HlyA only and not to the toxin secretion machinery encoded by hlyB and hlyD, two hlyA mutant strains were used (Table I). When HlyC was co-expressed in cis or in trans to a derivative of HlyA lacking both acylation sites, HlyC was detected (Fig. 4, lanes b and e) as compared with control bacteria carrying wild type hlyA or only hlyC (Fig. 4, lanes d and c). Moreover, HlyC was detected in cell lysates from a strain encoding HlyC and another form of HlyA (HlyADelta 722-724, Table I), lacking part of a putative HlyC binding domain (26, 27) (Fig. 4, lane a). Thus, HlyC processing is directly related to the presence of intact HlyA, since subtle changes in its sequence induce HlyC stabilization.

                              
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Table I
Bacterial strains and plasmids used in this study


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Fig. 4.   Western blot analysis using anti-HlyC antibodies of cell lysates from 5K strains expressing HlyC and different HlyA derivatives. Cell lysate samples taken at late log growth phase from: lane a, strain 5K expressing a mutated form of HlyA (HlyADelta 722-724) in cis to hlyC (20 µg); lane b, 5K expressing a mutated form of HlyA lacking both acylation sites in cis to hlyC (20 µg); lane c, 5KpFG1a expressing only hlyC (5 µg); lane d, 5K(pANN202-812B,pFG1a) encoding hlyABD and hlyC in trans (20 µg);. lane e, 5K expressing a mutated form of HlyA lacking both acylation sites in trans to hlyC (20 µg).

From previous work it is known that to modify HlyA, HlyC (i) binds to and then (ii) acylates HlyA (22). To examine whether HlyA acylation is a requisite for HlyC degradation, HlyC mutants with impaired abilities to activate pro-HlyA were used. Western blot analysis of cell samples from strains encoding different mutations in hlyC demonstrated that the HlyC mutants were produced at similar levels as wild type HlyC when expressed alone (Table II). Only a deletion at the amino-terminal region of the protein affected its stability. When the different mutated hlyC were expressed in trans to hlyA, none of the HlyC mutants was detected by Western blot. Determination of hemolytic activity of culture supernatants from these strains was used as assessment of HlyC function. As shown in Table II, these strains have different abilities to activate pro-HlyA. None of the mutated HlyC forms was detected when HlyA was present, regardless of their ability to acylate pro-HlyA. It is therefore concluded that HlyC processing is independent of the HlyA acylation reaction and probably depends solely on binding to HlyA.

                              
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Table II
Features of the HlyC mutants analyzed

HlyC Is Degraded by Different ATP-dependent Proteases-- To determine if an E. coli protease system was involved in the processing of HlyC, a set of protease deficient strains transformed with a plasmid encoding the hly operon were tested by Western blot. The cell lysates taken at late logarithmic growth phase of Clp and FtsH mutants showed the two 20-kDa HlyC bands, without generating the 10-kDa product (Fig. 5). Thus, the Clp and FtsH protease systems are responsible of HlyC degradation. In contrast, only the 10-kDa product was detected in the lon-ompT mutant strain. This protein band was detected in large quantity as compared with the wild type situation (Fig. 5), suggesting that Lon and/or OmpT participate in additional degradation of this low molecular mass fragment. To determine if the 20-kDa bands detected in the protease deficient strains indeed correspond to HlyC, two-dimensional polyacrylamide gel electrophoresis in combination with Western blot were used. Interestingly, anti-HlyC antibodies recognized four HlyC spots in lysates from E. coli clpB carrying the hly determinant (Fig. 6b). The same protein pattern was observed in the control strain carrying only hlyC, indicating that the protein bands detected in the Clp and FtsH protease mutant strains are indeed HlyC (Fig. 6a). To further confirm this, the protein spots obtained from the control strain were cut out from a two-dimensional gel and sequenced. Each of the protein spots subjected to MALDI-mass spectrometry generated tryptic peptides with sequences corresponding to fragments of the HlyC protein, as indicated by peptide mass mapping. At present it is unknown what kind of modifications induce HlyC to migrate to more than one spot in two-dimensional gels.


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Fig. 5.   Immunoblot analysis of cell lysates from protease deficient strains transformed with plasmid pANN202-312*. Lane + is a cell lysate (5 µg of total protein) from strain 5KpFG1a, encoding only hlyC, used as a positive control, and lane - is a cell lysate (20 µg of total protein) from strain 5KpANN202-312* encoding hlyCABD used as a HlyC negative control. The rest of the lanes contain 20 µg of total protein from the respective mutant strain.


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Fig. 6.   Analysis of HlyC by two-dimensional electrophoresis and immunoblot. Cell samples from strain 5KpFG1a, encoding hlyC (a), and strain SG22100 (clpB), carrying the hly operon on plasmid pANN202-312* (b), were electrophoresed, blotted, and probed with anti-HlyC antibodies. A similar protein pattern was observed in both samples, despite the different absolute quantities among the spots. No HlyC spots were detected in cell samples from the control negative strain 5KpUC18 (c).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate that the HlyC bands and their degradation product were not the result of proteolysis during the separation procedures, but rather a physiological phenomenon related to the turnover of the HlyC protein. In general, HlyC has been estimated to be a single protein that participates in the acylation of HlyA hemolysin secreted by E. coli strains (23). Electrophoretic separation in combination with immunochemical detection, MALDI-mass spectrometry, and peptide mass mapping demonstrated that the four protein spots correspond to HlyC isoforms. Since the exact molecular size of each spot could not be determined by MALDI-mass spectrometry due to the low recovery of intact protein from a SDS gel, the molecular properties that generate the observed HlyC heterogeneity remain elusive. Based in our results and published data, it is possible to make some conjectures regarding the nature of the recorded differences. Stanley et al. (22) have shown that [3H]palmitoyl-HlyC generated two bands in SDS-PAGE. Although distinctive acylation may explain the differences in charge, as is the case of HlyA (17, 26), the contribution in mass of the linked acyl groups is not enough to account for the differences in molecular weight observed among the HlyC spots. Alternative possibilities to explain the heterogeneity of HlyC are as follows. 1) The isoforms may be synthesized from mRNA transcripts of variable size, 2) normal translation of HlyC may be interrupted in a particular region within its sequence, or 3) HlyC might be susceptible to limited proteolysis even in the absence of HlyA. Although hlyC encoding mRNAs of various sizes have been described (this work and Refs. 38 and 39), it is unlikely that different mRNA transcripts would be responsible for the two HlyC isoforms detected, since expression of HlyC as a GST-fusion protein under the control of the tac promoter generated two isoforms as well. Furthermore, the two HlyC isoforms were present at all times tested, despite the fact that a long hlyC mRNA was detected at early hours of growth but not at late hours, in the wild type and control strain. Traces of the larger HlyC isoform were found in some cases, particularly at late hours of growth even when HlyA was expressed (Fig. 2A, 5 h lane a; Fig. 4, lane d). This might be interpreted as the upper band being more stable than the lower band. The lower band could be the result of proteolysis that occurs independently of HlyA presence. Nevertheless, HlyC processing is enhanced when HlyA is expressed as demonstrated in our experiments.

The similar levels of hlyC transcription, throughout growth of bacteria carrying only hlyC and bacteria carrying hlyA and hlyC in trans, show that the lack of HlyC when HlyA is present, was not due to impaired transcription of hlyC. Moreover, since hlyA is transcribed after hlyC to generate hlyCA or hlyCABD mRNA (24, 25), detection of HlyA is an indirect proof that hlyC is indeed transcribed. Fully acylated HlyA was detected in culture supernatants of cells carrying hlyA and hlyC in trans or in cis (26), demonstrating that HlyC must have been produced. Failure to detect HlyC regardless of whether hlyC was supplied in cis or in trans to hlyABD or in a high or low copy number plasmid indicates that this was not the result of transcriptional effects, differential stability of different hlyC containing mRNA, copy number, or cis/trans effects. We therefore conclude that HlyC is processed in vivo when HlyA is present.

The proteolytic processing of HlyC in the presence of HlyA described here is supported by the results of Hardie et al. (16). These investigators demonstrated that the function of HlyA, after reaching a saturation level, could only be re-established upon addition of HlyC, suggesting that the later protein is inactivated or consumed during the reaction. The degradation of HlyC appears to be dependent on the presence of HlyA and/or HlyB and HlyD, since the lack of HlyC was evident only when the entire hly determinant was expressed in cis or in trans. It is likely that the processing of HlyC is linked to the expression of HlyA rather than to the secretion of the toxin, as no particular interaction has been described between HlyC and the Hly secretion machinery. This view receives support from the fact that HlyC is detected in cell extracts of E. coli strains expressing mutated forms of HlyA but which secretion machinery remains intact.

A working model considering our results and those of Stanley et al. (22) is presented in Fig. 7. It has been suggested that, during the activation of HlyA, two consecutive events take place (22). First, there is interaction between acylACP-HlyC and pro-HlyA; second, the acylation of pro-HlyA proceeds. Our results suggest that, although the interaction event is important for HlyC processing, the acylation is not required, as demonstrated by the lack of correlation between the hemolytic activity and the detection of HlyC (Table II). HlyC mutants, regardless of their ability to acylate or not pro-HlyA, are detected only when expressed out of the context of the hly determinant. This interpretation is also supported by the fact that a deletion of three amino acids in a putative HlyC binding site of HlyA (26) allows HlyC detection. The fact that HlyC was detected in a mutant where the acylation sites of HlyA were changed (26) might be due to the lack of the lysine residues per se, independently of its acylation status. Recently, Stanley et al. (22) proposed that HlyC generates a binary complex with acyl-ACP, before binding to pro-HlyA. HlyC is detected in the absence of HlyA, and under these conditions it has been shown that an acylated HlyC-ACP intermediate is formed, ruling out the possibility that this interaction results in HlyC processing. The interaction of HlyC-acyl-ACP complex with pro-HlyA should be the reason for HlyC degradation. Based on these results and our data, we postulate that a conformational change of HlyC occurs upon interaction with pro-HlyA, generating specific sites for different protease systems to process the active HlyC proteins. Considering that activation of one molecule of pro-HlyA seems to require one molecule of HlyC (16, 22, 23), it is likely that degradation of HlyC is necessary to prevent its intracellular accumulation as HlyA is continuously secreted throughout growth.


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Fig. 7.   Model for HlyC degradation. HlyC breakdown occurs when HlyC binds acyl-ACP in the presence of pro-HlyA (22) and the protease systems Clp and FtsH. A, B, binding of the binary complex acyl-HlyC-ACP to pro-HlyA induces a conformational change in HlyC, creating a recognition domain for interaction with proteases of the Clp and FtsH systems. The acylACP-HlyC-proHlyA ternary complex is generated. C, transference of the acyl chain from acyl-HlyC-ACP to pro-HlyA proceeds. D, the ternary complex is then dissociated. E, the Clp and FtsH protease systems perform the HlyC breakdown. F and G, further degradation of HlyC peptides is carried out by the Lon protease system.

Protein processing is a well known mechanism of turnover in bacteria (40-42). The protease Clp systems, namely ClpXP and ClpAP, and the FtsH system seem to be responsible for the limited proteolysis of HlyC. The ATP-dependent protease Lon might be involved in further degradation of the 10-kDa polypeptide, since in the lon mutant strain this peptide band accumulated, as compared with the wild type strain. Furthermore, the 10-kDa band was detected only when PMSF is added during the preparation of the lysates, suggesting that the enzyme involved in the degradation of the 10-kDa product is a serine protease, as is the case of Lon. ClpB also seems to be involved in HlyC processing. This protein has been recently associated to the DnaK machinery (43), which is involved in the degradation of proteins such as the RpoH factors from Bradyrhizobium japonicum (44). Redundancy in protein degradation has been described in other cases like the cell division inhibitor SulA, the positive regulator of capsule transcription RcsA, Xis of phage lambda , the heat shock sigma factor RpoH or sigma 32, and SsrA-tagged proteins (45-48). Overlapping specificity of the different proteases may provide an effective means to accelerate the degradation of critical common substrates (47, 48).

    ACKNOWLEDGEMENTS

We thank Dr. Werner Goebel's laboratory for providing wild type and HlyA mutants and Dr. Susan Gottesman for providing the protease mutants. We are grateful to Cristine Jacobs for stimulating discussions and Andrea Parra for technical help.

    FOOTNOTES

* This work was supported in part by Research Contract ICA4-CT-1999-10001 from the European Community, Research and Technological Development Project NOVELTARGETVACCINES, and Ministerio de Ciencia y Tecnología de Costa Rica/Consejo Nacional de Ciencia y Tecnología de Costa Rica, Costa Rica.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a grant from the Swedish International Development Agency, as part of the Karolinska International Research Training Program. To whom correspondence should be addressed. Tel.: 506-2380761; Fax: 506-2381298; E-mail: piet@ns.medvet.una.ac.cr.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M009514200

    ABBREVIATIONS

The abbreviations used are: HlyA, Escherichia coli hemolysin; RTX, repeats in toxins; GST, glutathione S-transferase; PCR, polymerase chain reaction; IPTG, isopropyl beta -D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption/ionization; HlyC, Escherichia coli HlyC acyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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