In Vivo Proteolytic Degradation of the
Escherichia coli Acyltransferase
HlyC*
Caterina
Guzmán-Verri
§¶,
Esteban
Chaves-Olarte
,
Fernando
García
,
Staffan
Arvidson
, and
Edgardo
Moreno§
From the
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 |
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 |
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 |
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
[
-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%
-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 |
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
-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.
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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.
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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).
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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
(HlyA
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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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 (HlyA 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).
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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.
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).
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DISCUSSION |
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
, the heat shock sigma
factor RpoH or
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
-D-thiogalactopyranoside;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
MALDI, matrix-assisted laser desorption/ionization;
HlyC, Escherichia coli HlyC acyltransferase.
 |
REFERENCES |
1.
|
Chang, Y. F.,
Ma, D. P.,
Shi, J.,
and Chengappa, M. M.
(1993)
Infect. Immun.
61,
2089-2095[Abstract]
|
2.
|
Chang, Y. F.,
Young, R.,
and Struck, D. K.
(1989)
DNA
8,
635-647[Medline]
[Order article via Infotrieve]
|
3.
|
Frey, J.
(1995)
Trends Microbiol.
3,
257-261[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Glaser, P.,
Sakamoto, H.,
Bellalou, J.,
Ullmann, A.,
and Danchin, A.
(1988)
EMBO J.
7,
3997-4004[Abstract]
|
5.
|
Jansen, R.,
Briaire, J.,
Smithe, H. E.,
Dom, P.,
Haesebrouck, F.,
Kamp, E. M.,
Gielkins, A. L.,
and Smits, M. A.
(1995)
Infect. Immun.
63,
27-37[Abstract]
|
6.
|
Koronakis, V.,
Cross, M.,
Senior, B.,
Koronakis, E.,
and Hughes, C.
(1987)
J. Bacteriol.
169,
1509-1515[Medline]
[Order article via Infotrieve]
|
7.
|
Kuhnert, P.,
Heyberger-Meyer, B.,
Nicolet, J.,
and Frey, J.
(2000)
Infect. Immun.
68,
6-12[Abstract/Free Full Text]
|
8.
|
Lin, W.,
Fullner, K. J.,
Clayton, R.,
Sexton, J. A.,
Rogers, M. B.,
Calia, K. E.,
Calderwood, S. B.,
Fraser, C.,
and Mekalanos, J. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1071-1076[Abstract/Free Full Text]
|
9.
|
Sutton, J. M.,
Lea, E. J.,
and Downie, J. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9990-9994[Abstract/Free Full Text]
|
10.
|
Thompson, S. A.,
Wang, L. L.,
West, A.,
and Sparling, P. F.
(1993)
J. Bacteriol.
175,
811-818[Abstract]
|
11.
|
Thompson, S. A.,
and Sparling, P. F.
(1993)
Infect. Immun.
61,
2906-2911[Abstract]
|
12.
|
Welch, R. A.,
Dellinger, E. P.,
Minshew, B.,
and Falkow, S.
(1981)
Nature
294,
665-667[Medline]
[Order article via Infotrieve]
|
13.
|
Issartel, J. P.,
Koronakis, V.,
and Hughes, C.
(1991)
Nature
351,
759-761[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Stanley, P.,
Packman, L. C.,
Koronakis, V.,
and Hughes, C.
(1994)
Science
266,
1992-1996[Medline]
[Order article via Infotrieve]
|
15.
|
Goebel, W.,
and Hedgpeth, J.
(1982)
J. Bacteriol.
151,
1290-1298[Medline]
[Order article via Infotrieve]
|
16.
|
Hardie, K. R.,
Issartel, J. P.,
Koronakis, E.,
Hughes, C.,
and Koronakis, V.
(1991)
Mol. Microbiol.
5,
1669-1679[Medline]
[Order article via Infotrieve]
|
17.
|
Guzmán-Verri, C.,
Garcia, F.,
and Arvidson, S.
(1997)
J. Bacteriol.
179,
5959-5962[Abstract]
|
18.
|
Hartlein, M.,
Schiell, S.,
Wagner, W.,
Rdest, U.,
Kreft, J.,
and Goebel, W.
(1983)
J. Cell. Biochem.
22,
87-97[Medline]
[Order article via Infotrieve]
|
19.
|
Hughes, C.,
Issartel, J. P.,
Hardie, K.,
Stanley, P.,
Koronakis, E.,
and Koronakis, V.
(1992)
FEMS Microbiol. Immunol.
105,
37-44
|
20.
|
Nicaud, J. M.,
Mackman, N.,
Gray, L.,
and Holland, I. B.
(1985)
FEBS Lett.
187,
339-344[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Hackett, M.,
Guo, L.,
Shabanowitz, J.,
Hunt, D. F.,
and Hewlett, E. L.
(1994)
Science
266,
433-435[Medline]
[Order article via Infotrieve]
|
22.
|
Stanley, P.,
Hyland, C.,
Koronakis, V.,
and Hughes, C.
(1999)
Mol. Microbiol.
34,
887-901[Medline]
[Order article via Infotrieve]
|
23.
|
Trent, M. S.,
Worsham, L. M.,
and Ernst-Fonberg, M. L.
(1998)
Biochemistry
37,
4644-4652[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Koronakis, V.,
Cross, M.,
and Hughes, C.
(1988)
Nucleic Acids Res.
16,
4789-4800[Abstract]
|
25.
|
Welch, R. A.,
and Pellet, S.
(1988)
J. Bacteriol.
170,
1622-1630[Medline]
[Order article via Infotrieve]
|
26.
|
Ludwig, A.,
García, F.,
Bauer, S.,
Jarchau, T.,
Benz, R.,
Hoppe, J.,
and Goebel, W.
(1996)
J. Bacteriol.
178,
5422-5430[Abstract]
|
27.
|
Stanley, P.,
Koronakis, V.,
Hardie, K.,
and Hughes, C.
(1996)
Mol. Microbiol.
20,
813-822[Medline]
[Order article via Infotrieve]
|
28.
|
Trent, M. S.,
Worsham, L. M.,
and Ernst-Fonberg, M. L.
(1999)
Biochemistry
38,
9541-9548[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Trent, M. S.,
Worsham, L. M.,
and Ernst-Fonberg, M. L.
(1999)
Biochemistry
38,
8831-8838[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Trent, M. S.,
Worsham, L. M.,
and Ernst-Fonberg, M. L.
(1999)
Biochemistry
38,
3433-3439[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
32.
|
Bauer, M. E.,
and Welch, R. A.
(1996)
Infect. Immun.
64,
167-175[Abstract]
|
33.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
34.
|
Janzon, L.,
Lofdahl, S.,
and Arvidson, S.
(1986)
FEMS Microbiol. Lett.
33,
193-198
|
35.
|
Hess, J.,
Wels, W.,
Vogel, M.,
and Goebel, W.
(1986)
FEMS Microbiol. Lett.
34,
1-11
|
36.
|
Harlow, E.,
and Lane, D.
(1999)
Using Antibodies: A Laboratory Manual
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
37.
|
Coligan, J. E.,
Dunn, B. M.,
Ploegh, H. L.,
Speicher, D. W.,
and Wingfield, P. T.
(1995)
in
Current Protocols in Protein Science
(Benson Chanda, V., ed)
, p. 10.5.1, John Wiley & Sons, Inc., New York
|
38.
|
Koronakis, V.,
and Hughes, C.
(1988)
Mol. Gen. Genet.
213,
99-104[Medline]
[Order article via Infotrieve]
|
39.
|
Koronakis, V.,
Cross, M.,
and Hughes, C.
(1989)
Mol. Microbiol.
3,
1397-1404[Medline]
[Order article via Infotrieve]
|
40.
|
Gottesman, S.,
Maurizi, M. R.,
and Wickner, S.
(1997)
Cell
91,
435-438[Medline]
[Order article via Infotrieve]
|
41.
|
Gottesman, S.
(1999)
Curr. Opin. Microbiol.
2,
142-147[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Porankiewicz, J.,
Wang, J.,
and Clarke, A. K.
(1999)
Mol. Microbiol.
32,
449-458[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Zolkiewski, M.
(1999)
J. Biol. Chem.
274,
28083-28086[Abstract/Free Full Text]
|
44.
|
Urech, C.,
Koby, S.,
Oppenheim, A. B.,
Münchbach, M.,
Hennecke, H.,
and Narberhaus, F.
(2000)
Eur. J. Biochem.
267,
4831-4839[Abstract/Free Full Text]
|
45.
|
Kanemori, M.,
Nishihara, K.,
Yanegi, H.,
and Yura, T.
(1997)
J. Bacteriol.
179,
7219-7225[Abstract]
|
46.
|
Leffers, G. G., Jr.,
and Gottesman, S.
(1998)
J. Bacteriol.
180,
1573-1577[Abstract/Free Full Text]
|
47.
|
Kanemori, M.,
Yanagi, H.,
and Yura, T.
(1999)
J. Bacteriol.
181,
3674-3680[Abstract/Free Full Text]
|
48.
|
Wu, W.-F.,
Zhou, Y.,
and Gottesman, S.
(1999)
J. Bacteriol.
181,
3681-3687[Abstract/Free Full Text]
|
49.
|
Gottesman, S.,
Clark, W. P.,
de Crecy-Lagard, V.,
and Maurizi, M. R.
(1993)
J. Biol. Chem.
268,
22618-22626[Abstract/Free Full Text]
|
50.
|
Squires, C.,
and Squires, C. L.
(1992)
J. Bacteriol.
174,
1081-1085[Medline]
[Order article via Infotrieve]
|
51.
|
Gottesman, S.,
Roche, E.,
Zhou, Y.,
and Sauer, R. T.
(1998)
Genes Dev.
12,
1338-1347[Abstract/Free Full Text]
|
52.
|
Bukhari, A. I.,
and Zipser, D.
(1973)
Nat. New Biol.
243,
238-241[Medline]
[Order article via Infotrieve]
|
53.
|
Grodberg, J.,
and Dunn, J. J.
(1988)
J. Bacteriol.
170,
1245-1253[Medline]
[Order article via Infotrieve]
|
54.
|
Gottesman, S.
(1989)
Annu. Rev. Genet.
23,
163-198[CrossRef][Medline]
[Order article via Infotrieve]
|
55.
|
Vogel, M.,
Hess, J.,
Then, I.,
Juarez, A.,
and Goebel, W.
(1988)
Mol. Gen. Genet.
212,
76-84[Medline]
[Order article via Infotrieve]
|
56.
|
Ludwig, A.,
Vogel, M.,
and Goebel, W.
(1987)
Mol. Gen. Genet.
206,
238-245[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.