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INTRODUCTION |
The adenylate cyclase toxin-hemolysin
(ACT,1 AC-Hly, or CyaA) is a
key virulence factor of the whooping cough agent Bordetella pertussis and a promising protective antigen candidate for
acellular pertussis vaccines (1-5). ACT belongs to the RTX
(repeats in toxin) protein family
(6) and has the capacity to form small cation-selective membrane
channels, which account for its weak hemolytic activity (8-11). The
major cytotoxic activity of the 1706-residue-long protein, however,
consists in its capacity to invade a variety of eukaryotic cells
directly across their cytoplasmic membrane (12-14) and to deliver into
cells a catalytic adenylate cyclase (AC) domain. This intoxicates cells
by unregulated conversion of ATP to cAMP (15-18) and causes impairment
of microbicidal functions of immune effector cells and apoptosis of
lung macrophages (19).
The capacity of ACT to penetrate into target cell membranes and to
intoxicate cells depends on a posttranslational activation by the
accessory protein, CyaC (20, 21). It was first established for the
Escherichia coli
-hemolysin (HlyA) that the activation of
RTX toxins consists in amide linked fatty-acylation (22), and Hackett
et al. (23) have demonstrated by mass spectrometric analysis
that native ACT produced by Bordetella (Bp-ACT)
is mono-acylated by a palmitoyl residue at the
-amino group of
lysine 983. In contrast, the HlyA from E. coli was found to
be acylated at two lysines, both in vitro and in
vivo (24, 25). Moreover, two highly conserved RTX acylation sites
(25), corresponding to lysine 983 (Lys983) and lysine 860 (Lys860) are also found in ACT, and for an unknown reason,
the CyaC-activated recombinant ACT produced in E. coli
(r-Ec-ACT) is palmitoylated also at Lys860, in
addition to acylation of Lys983 (26). When compared with
the native mono-acylated Bp-ACT, the doubly acylated
r-Ec-ACT exhibits about four times lower specific hemolytic
activity on sheep erythrocytes (20) and about 10 times lower specific
channel-forming activity in artificial planar lipid bilayers (10). At
the same time, however, the characteristics of the channels formed by
both proteins are identical, and both proteins have identical capacity
to insert into the target membranes and to deliver the invasive AC
domain into cells (5, 20, 26). In contrast to membrane insertion and AC
translocation into cells, the channel-forming activity of ACT exhibits
a cooperative concentration dependence curve, indicative of an
oligomerization step involved in ACT channel formation (5, 8, 26). On this basis, a hypothesis was formulated that the acylation of Lys860 might interfere selectively with the oligomerization
of ACT within target membranes (26).
Interestingly, the capacity of ACT to induce protective immune response
against Bordetella infection in mice also depends on the
CyaC-mediated acylation, and the doubly acylated r-Ec-ACT exhibits significantly lower protective antigenicity than the mono-acylated Bp-ACT (5). These differences in immunological properties of native and recombinant ACT also indicate that acylation at Lys860 may alter the conformation of the toxin.
We hypothesized that preventing acylation at Lys860 by
substitution of the lysine 860 residue might result in production of
native-like and fully active recombinant ACT in E. coli. In
this report we demonstrate, however, that lysine 860 is by itself
important for toxin action.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Growth Conditions, and Plasmids--
The
E. coli K12 strain XL1-Blue (Stratagene) was used throughout
this work for DNA manipulation and for expression of ACT. Bacteria were
grown at 37 °C in LB medium supplemented with 150 µg/ml of
ampicillin. pT7CACT1 is a construct derived from pCACT3 (5), allowing
enhanced production of recombinant CyaC-activated ACT in E. coli (r-Ec-ACT) under control of the IPTG-inducible lacZp promoter. To construct pT7CACT1, first the
cyaC open reading frame was amplified by PCR, using
purpose-designed primers (5'-GCGCGCGCCATATGCTTCCGTCCGCCCAAG and
5'-CCCGGGGGATCCTTAGGCGGTGCCCCGCGGTCG, respectively). The PCR product was fused to the translational enhancement and initiation signals of gene 10 from phage T7 by cloning into the NdeI
and BamHI sites of pT7-7 (27). Absence of undesired
mutations was verified by sequencing, and the XbaI fragment
containing the gene together with the expression signals was inserted
into a blunted HindIII site of pDLACT1 (5), to
substitute the cyaC allele in pCACT3, thereby placing it
under lacZp control and yielding pT7CACT1.
Site-directed Mutagenesis of cyaA--
The substitutions of the
codon for Lys860 were introduced into the cyaA
gene by PCR mutagenesis using three pairs of PCR primers, consisting of
one common forward primer 5'-GTGGTCCTCGACGTCGCCGCC and one
mutation-specific backward primer. These were
5'-TGTGGTGAATTCAGATCTGCCCGTTTTCGTGCG for the K860R substitution,
5'-TGTGGTGAATTCGCTCAGGCCTGTTTTCGTGCG for the K860L substitution, and
5'-TGTGGTGAATTCGCTGCAGCCCGTTTTCGTGCG, for the K860C substitution,
respectively. Using the proofreading Vent DNA polymerase (New England
Biolabs) and appropriate primer pairs, 144-base pair fragments of the
cyaA gene with the incorporated mutations were
PCR-amplified, purified on agarose gels, and cloned as
ClaI-EcoRI fragments into appropriately digested
pT7CACT1. The presence of the desired substitutions and the absence of
any unrelated mutations were verified by DNA sequencing of the cloned PCR product. In a second step, the complete mutant cyaA
alleles were assembled by addition of a 2617-base pair EcoRI
fragment encoding the COOH-terminal part of ACT, downstream to the
mutagenized residue 860.
Plasmids for expression of the truncated mutant ACT were obtained by
replacement of the XhoI-ScaI fragments of the
respective mutant pT7CACT1 derivatives by the
KpnI-ScaI fragment of pACT
C1322 (36). This
resulted in insertion of a TAA stop codon at the XhoI site,
leading to production of truncated ACT proteins (ACT
) with a
COOH-terminal Asp1008 residue.
The nonacylated variants of the intact and mutant proteins were
expressed from plasmids from which the cyaC gene was deleted as an NdeI-BamHI fragment.
Production and Purification of the CyaA-derived
Proteins--
The wild-type ACT and the different mutant derivatives
were produced in the presence, or in the absence of the activating protein CyaC, using the E. coli strain XL1-Blue (Stratagene)
bearing the appropriate plasmid derived from pT7CACT1. Exponential
500-ml cultures were induced with IPTG (1 mM), and the
extracts of insoluble cell debris after sonication were prepared in 8 M urea, 50 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, as described previously (20). The
proteins were further purified by ion-exchange chromatography on
DEAE-Sepharose and phenyl-Sepharose (Amersham Pharmacia Biotech) as
described previously (28). In the final step, the proteins were eluted
with 8 M urea, 50 mM Tris-HCl, pH 8.0, and
stored frozen. Alternatively, the truncated proteins were purified
directly from urea extracts by affinity chromatography on
calmodulin-agarose, as described previously (20). For the K860C mutant
10 mM
-mercaptoethanol was added into all extraction and
purification solutions to avoid cysteine oxidation. SDS-PAGE analysis
and determination of protein concentration were performed according to
standard protocols (29).
High Resolution Two-dimensional Gel Electrophoresis--
The
whole cell extracts of the respective clones expressing the truncated
proteins were prepared from exponentially growing cultures induced by
IPTG (1 mM) for 3 h. Total protein samples (20 µg)
were analyzed by large format two-dimensional (IEF/SDS-PAGE) gel
electrophoresis (30) using the Investigator system (Oxford Glycosystems). In the first dimension, proteins were separated according to their isoelectric point under denaturing conditions, for
16 h at 18,000 V-h, on a high-resolution 18-cm-long capillary tube
gels, containing 2.3% ampholites (Sigma) admixed in pH ranges ratio
(3-10):(4-6):(5-7) = 1:2:2. In the second dimension, the proteins
were separated by SDS-PAGE on 7.5% acrylamide slab gels (22 × 22 cm). The gels were stained with Coomassie Blue.
Upon initial experiments with purified proteins, the use of whole cell
extracts for two-dimensional electrophoresis was preferred, in order to
avoid prolonged incubation of ACT in 8 M urea solutions during purification. This was because products of urea decomposition may cause secondary covalent modifications, such as carbamylation, leading to uncontrolled changes of the proteins isoelectric points. Moreover, the whole cell extract samples gave better reproducibility of
resolution and protein positioning in two-dimensional electrophoresis of the differently acylated ACT
protein forms. ACT
constituted generally about 5-10% total cell proteins in the sample and could be
unambiguously identified on the gels by their characteristic size and
by comparison with samples devoid of the ACT
-derived material.
Moreover, the spots corresponding to the invariant cell proteins could
be conveniently used as internal positioning standards for definition
of charge/migration differences and relative positioning of the
acylated ACT
proteins.
Mass Spectrometric Analysis of Protein Acylation--
The
location and identity of the acyl modifications were analyzed by liquid
chromatography/MS/MS and MALDI-TOF MS techniques essentially as
described previously (26). Tryptic and Asp-N fragments of the analyzed
proteins were generated according to standard protocols and separated
on 50-µm inner diameter × 12-cm capillary column packed with
Magic 5 µm 200 Å C18 material (Michrom BioResources, Auburn, CA), at
a flow rate of 150 nl·min
1, with a 0-100%
acetonitrile gradient (1% acetic acid) over 40 min. Peptide analysis
by microcapillary HPLC coupled to a Finnigan TSQ 7000 electrospray
tandem quadrupole mass spectrometer was performed as described in
detail elsewhere (31, 32).
Positive ion MALDI mass spectra were measured on a PerSeptive Voyager
DE linear time-of-flight mass spectrometer equipped with a gridless
delayed extraction ion source. Ion acceleration voltage was 20 kV, and
guide wire voltage was set to 0.02%. For delayed extraction, an 8% kV
potential difference between the probe and the extraction lens was
applied with a time delay of 20 ns after each laser pulse. Samples
in
-cyano-4-hydroxycinnamic acid (premix sample preparation
technique) (33) were irradiated by 337-nm photons. Typically 20-50
shots were summed into a single mass spectrum. Spectra were calibrated
externally using the average [M + H]+ ion of a peptide
standard (insulin, Aldrich).
Identities of the C16:0 and C16:1 cis
D9 fatty-acyl groups were
confirmed by capillary gas chromatography/MS with electron impact
ionization after a one-step extraction and derivatization procedure
(34).
Assay of AC, Cell Binding, and Cytotoxic and Hemolytic
Activities--
Adenylate cyclase activities were measured as
described previously in the presence of 1 µM calmodulin
(35). One unit of AC activity corresponds to 1 µmol of cAMP formed
per min at 30 °C, pH 8.0. Cell-invasive AC was determined as the
amount of AC that becomes protected against externally added trypsin
upon internalization into erythrocytes in 30 min of incubation (11),
and the hemolytic activity was measured as the hemoglobin released upon
incubation of washed sheep erythrocytes (5 × 108/ml)
with the toxins for 270 min, respectively (11). Erythrocyte binding of
the toxins was determined as described in detail previously (36).
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RESULTS AND DISCUSSION |
Independent Acylation of the Lys860 and
Lys983 Residues of ACT in Vivo--
In contrast to the
essential acylation of lysine 983, the additional CyaC-mediated
acylation of lysine 860, occurring in E. coli K12 host
background, is not required for ACT activity and interferes with the
hemolytic (channel-forming) activity of r-Ec-ACT (20, 26).
It was important to determine whether this aberrant acylation can be
prevented by substitutions of lysine 860 and whether the activities of
the mutant mono-acylated toxins produced by E. coli would
resemble those of the mono-acylated wild-type ACT from
Bordetella (Bp-ACT). Therefore, the lysine 860 was replaced with arginine, leucine, and/or cysteine residues by PCR
mutagenesis, and the mutant proteins were produced in an E. coli K12 strain (XL1) in the presence, or in the absence, of CyaC.
The acylation status of the obtained mutants was analyzed by
two-dimensional electrophoresis (two-dimensional IEF/SDS-PAGE) by the
method of O'Farrell (30). This approach was already successfully used
for the analysis of HlyC-dependent acylation of HlyA (24). It allows to detect fatty-acylation of the respective lysines as loss
of one or two positive charges, leading to shifts in the isoelectric
point of the toxin. Indeed, nonacylated ACT, bi-acylated ACT, and
mono-acylated K860R mutant ACT could be resolved by this technique,
although less well than reported previously for HlyA (data not shown).
Despite use of 18-cm-long high-resolution IEF tube gels, with a pH
gradient set from pH 4 to 6, quantitative and reproducible resolution
of proACT from the mono- and bi-acylated ACT forms was difficult to
achieve. This could be expected, because the size of the ACT protein is
almost twice the size of HlyaA (177 versus 110 kDa), and the
theoretical isoelectric points of the three ACT forms under denaturing
conditions differ only by 0.01 pH unit (4.27, 4.26, and 4.25 for the
non-, mono-, and bi-acylated ACT, respectively). To circumvent this
problem, the 70-kDa-long, acidic repeats were deleted from the
r-Ec-ACT and its mutant variants. The resulting ACT
proteins, with a carboxyl-terminal aspartate residue at position 1008, had theoretical isoelectric points of 5.41, 5.35, and 5.29 in the non-,
mono-, and bi-acylated forms, respectively. These proteins could be
well resolved by the two-dimensional electrophoresis, as demonstrated
in Fig. 1.

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Fig. 1.
Analysis of ACT acylation by
two-dimensional electrophoresis. Fifty microgram of total protein
extracts of IPTG-induced cells producing the ACT proteins were
prepared and analyzed by two-dimensional (IEF/SDS-PAGE)
electrophoresis, as described under "Experimental Procedures." In
general, binary or tertiary mixtures of extracts were prepared by
directly mixing equal volumes of extracts containing similar amounts of
total cell proteins. For extracts with important differences in the
relative content of the ACT -derived proteins (i.e.
proACT -K860R versus ACT -K860R), the volumes of
extracts in the mixture were adjusted to yield comparable levels of the
ACT -derived proteins in the mixture. The gels were stained with
Coomassie Blue and gel sections comprising only the spots of the
various forms of ACT -derived proteins are shown. The sections were
aligned in columns so that the position of the spots relative to the
invariant cell proteins (positioning standards) in the original
individual gels was respected. Therefore, the relative position of the
spots in the columns reflects their relative position in respect to the
anode (+) and cathode ( ) in the original pH gradients after
isoelectric focusing.
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The proACT
protein, produced in the absence of CyaC, migrated as a
single spot at a position of the nonacylated form, while the ACT
,
produced in the presence of CyaC, resolved in two spots corresponding
to the major, bi-acylated form (about 80% total) and a mono-acylated
form (about 20% total) of ACT
, respectively (Fig. 1). When the
proACT
and ACT
proteins were mixed in a roughly equimolar ratio,
two major spots of the bi-acylated and nonacylated forms were resolved
with the minor spot of the mono-acylated ACT
protein positioned
between them. This observation was in good agreement with the
semiquantitative estimations from mass spectrometric analysis of
CyaC-activated intact r-Ec-ACT (26), where about 60%
r-Ec-ACT was estimated to be bi-acylated and the rest was mono-acylated. These results demonstrate that removal of the repeat portion downstream to residue 1008 did not have any effect on the
efficiency of the CyaC-mediated acylation of the Lys983 and
Lys860 residues of ACT. Therefore, the two-dimensional
electrophoresis of the COOH-terminally truncated proteins could be used
as a suitable assay for acylation of the Lys860 mutants of
ACT. It is noteworthy, however, that in some gels trailing of a small
portion (<10%) of the ACT
-derived material was observed and
yielded two very minor satellite spots, corresponding to one more
and/or one less positive charge than the major protein species in the
sample, respectively. It was not determined whether occurrence of these
minor spots was due to handling of samples or whether it was due to
intrinsic microheterogeneity of the ACT
proteins in the preparations
and could be observed due to high resolving power of the applied
separation technique.
When the K860R mutant forms of ACT
were produced in the absence
(proACT
-K860R) and in the presence (ACT
-K860R) of CyaC, each
migrated as a single major spot in two-dimensional electrophoresis (Fig. 1). When the two proteins were mixed, they resolved
quantitatively in two distinct spots separated by a distance
corresponding to a single charge difference. Because the K860R
substitution does not alter the net charge of the nonacylated protein,
this result is consistent with the conclusion that the substitution of
lysine 860 by an arginine prevented the acylation at residue 860 and that quantitative acylation of Lys983 of the mutant protein
still occurred in presence of CyaC. Indeed, as expected due to equal
charges, the proACT
-K860R and proACT
proteins produced in the
absence of CyaC co-migrated as a single spot, while ACT
-K860R
produced in the presence of CyaC resolved quantitatively also from the
nonacylated proACT
(Fig. 1). Furthermore, ACT
-K860R, if acylated
on Lys983, is expected to bear one positive charge more
than bi-acylated ACT
. Indeed, when ACT
and ACT
-K860R were
mixed, they resolved in two spots, with the ACT
-K860R mutant
migrating at the position of the mono-acylated form of ACT
. Finally,
when ACT
-K860R protein was mixed with proACT
and ACT
, it
migrated at the position of mono-acylated form of ACT
, in between
the nonacylated proACT
and the bi-acylated form of ACT
(Fig. 1).
This demonstrated that the K860R mutant was still quantitatively
mono-acylated by CyaC, most likely on Lys983.
The same acylation pattern was found also with the K860L mutant (Fig.
1). The ACT
-K860L protein expressed in the presence of CyaC migrated
at the same position as the bi-acylated ACT
. This is consistent with
its mono-acylation on lysine 983, because the replacement of lysine 860 by leucine removes one positive charge, as does the acylation of lysine
860. Indeed, the nonacylated proACT
-K860L and proACT
resolved as
two spots differing by one charge, as did proACT
-K860L and
ACT
-K860L. In contrast, the ACT
-K860L produced in the presence of
CyaC and the nonacylated proACT
resolved as two spots at a distance
corresponding to a difference of two charges. Consistently, the
ACT
-K860R resolved as a third spot between ACT
-K860L and
proACT
. These results clearly indicate that also the ACT
-K860L
mutant was quantitatively acylated on lysine 983.
Both Palmitic and Palmitoleic Acids Are Used for Acylation of
Lys983 of ACT--
It was important to prove that the
acylation of the ACT-K860R mutant was indeed located on lysine 983 and
that identical fatty-acyl residues were used for modification of the
wild-type and mutant proteins. For this, the same approach was used as
that originally applied for determination of the chemical nature and
location of the fatty-acyl modification of ACT from
Bordetella (23). Tryptic and Asp-N proteolytic fragments of
the full-length r-Ec-ACT and of the truncated ACT
forms
of both the wild-type and the K860R proteins, respectively, were
analyzed for acylation by MALDI-TOF MS and/or microcapillary HPLC
coupled to electrospray ionization tandem mass spectrometry, as
described previously (26). Representative mass spectra are shown in
Fig. 2, and the results of the MS
analysis of the various proteins are summarized in Table
I. These results fully confirm the
acylation patterns observed by two-dimensional gel electrophoresis. As
expected, no acylation was detected on the proACT and proACT
proteins produced in the absence of CyaC, while acylation was found on
both the Lys983 and Lys860 residues of the
r-Ec-ACT and ACT
proteins produced in presence of CyaC.
More importantly, no acylation of the arginine residue Arg860 of the ACT-K860R and ACT
-K860R mutants could be
detected, and the acyl residues in this mutant proteins were entirely
located on the Lys983 residue. Finally, both wild-type and
K860R mutant proteins were acylated by palmitoyl (C16:0) and
palmitoleil (cis
9 C16:1) fatty-acyl groups. No evidence of
myristoylation was observed at either lysine in any of the proteins,
quite unlike our previously reported results (26).

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Fig. 2.
Analysis of ACT acylation by mass
spectrometric analysis of proteolytic fragments. A,
reconstructed ion chromatogram from the microcapillary ESI LC-MS Auto
CID analysis (43) of a whole ACT protein. Note the base-line HPLC
resolution of both peptides acylated at lysine 983 (inset).
B, CID mass spectrum of the [M + 2H]2+ ion at
m/z 810. The yi and bi series ions
(see Ref. 7 for the nomenclature) confirm the location of epsilon
amino-linked palmitoleic species in the tryptic peptide. C,
CID mass spectrum of the [M + 2H]2+ ion (m/z
811) of the palmitoylated peptide.
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The sum of this data demonstrates that the CyaC-mediated acylation of
truncated ACT
protein in E. coli is identical to that of
the full-length ACT protein. The results show further that two-dimensional electrophoresis of whole cell samples containing the
ACT
protein can be used as a straightforward and reliable assay for
the acylating activity of CyaC in vivo. Collectively, these
results demonstrate that the substitution of the lysine 860 by arginine
did not have any effect on CyaC-mediated acylation of
Lys983 and that acylation of Lys860 and
Lys983 residues of ACT occurs independently in
vivo. This is similar to the previously observed independent
acylation at the two sites of HlyA, where only about 20-30 flanking
residues around each acylated lysine were required for productive
modification to take place both in vitro and in
vivo (24, 25).
Functional Characterization of the Mutants--
For comparison of
the biological activities, the mutant proteins and intact
r-Ec-ACT were purified close to homogeneity, as shown in
Fig. 3. Interestingly, all substitutions,
even the most conservative K860R replacement, caused a strong decrease
in toxin activities, reducing to about 10% the cell-invasive AC
activity of the K860R mutant and to about 5% those of the K860L and
K860C mutants, as shown in Table II. The
difference between the activities of the K860R mutant and those of
K860L and K860C mutants was small, but reproducible. This suggests that
presence of a positively charged amino acid at position 860 is
important for toxin function, albeit even an arginine residue could not
functionally replace the lysine 860.

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Fig. 3.
SDS-PAGE analysis of purified ACT and mutant
proteins. The proteins were expressed in the presence of the
activating protein CyaC in recombinant E. coli K12 strains
and purified from urea extracts of cell debris by DEAE- and
phenyl-Sepharose chromatography. Two micrograms of proteins were
separated on 7.5% acrylamide gel and visualized by Coomassie staining.
wt, wild-type.
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The mutant proteins were defective in both the target cell association
and in membrane translocation of the bound toxin, as shown further in
Table II. Tight cell association was reduced to 23% for the K860R
mutant and to 15% for the other two mutants, as compared with intact
r-Ec-ACT. Moreover, while under given conditions about 60%
cell-bound r-Ec-ACT had the AC domain translocated into
cells and had the AC domain protected against externally added trypsin,
only 30% bound K860R and 17% K860L and K860C ACT, respectively, was
translocated under identical conditions. Hence, the translocation
efficiency of K860R ACT was half, and that of both K860L and K860C was
about 28%, of the translocation efficiency of the intact
r-Ec-ACT, respectively. These data suggested that lysine 860 plays an important role in both the membrane insertion of ACT and
translocation of its AC domain into cells.
The acylation of Lys860 affects selectively the hemolytic
(channel-forming) activity of the toxin, which exhibits different
calcium requirements as the invasive AC activity (37-40). It was,
therefore, important to compare the calcium and concentration
dependencies of the K860R mutant with those of intact
r-Ec-ACT. No substantial difference of the two proteins in
function of free calcium concentration could, however, be observed when
the amount of the K860R protein in the comparative assays was raised
seven times in respect to intact ACT, in order to measure both toxin
activities in a similar readout range (Fig.
4).

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Fig. 4.
Effect of calcium on biological activity of
the ACT-K860R mutant toxins. Sheep erythrocytes (5 × 108/ml) were incubated at 37 °C with purified ACT ( ,
0.5 unit/ml) or ACT-K860R ( , 3.3 units/ml) in 10 mM
Tris-HCl, pH 8.0, 150 mM NaCl, and various free calcium
concentrations. After 30 min aliquots were taken for determination of
tightly cell-associated AC activity (A) and of the AC
activity internalized into erythrocytes and protected against
externally added trypsin (B). The hemolysis (C)
was measured photometrically at 541 nm, as hemoglobin released after
270 min of incubation. Each point is the mean of duplicate
determinations, and this graph is representative of three separate
experiments. In order to compensate for the lower biological activity
of the mutant toxin and to obtain for both toxins a readout in the same
range, the input concentration of the ACT-K860R mutant was 7-fold
higher as that of intact ACT (3.3 units/ml versus 0.5 unit/ml, respectively).
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It appears, therefore, that the K860R mutant toxin still exhibits
similar calcium requirements for action as the intact ACT. This
indicates that the K860R substitution did not affect the toxin
structure in a gross way, causing generalized misfolding of the
protein. Such effect of a conservative substitution appears unlikely.
Moreover, in contrast to what would be expected for a misfolded
protein, no difficulties with mutant toxin production and/or reduced
stability during purification were observed (Fig. 3), indicating that
the sensitivity of the mutant ACT to the endogenous proteases of
E. coli was not increased and the yields of intact and
mutant toxins were also very similar (data not shown). The residual
activity of the K860R mutant reaching up to 10% that of intact ACT
also suggests that the mutation caused a specific defect of membrane
interaction efficiency of ACT.
Conclusions--
The results reported here suggest that the lysine
860 (Lys860) residue is by itself an important functional
residue involved in membrane insertion and translocation of ACT,
regardless of its acylation status. It is intriguing that even a
conservative substitution of Lys860 by an arginine residue
caused a strong decrease in all toxin activities, while modification of
Lys860 in E. coli by a bulky and highly
hydrophobic palmitoyl residue causes only a selective reduction of the
channel-forming activity of r-Ec-ACT and does not affect the
membrane insertion and AC delivery capacities of the toxin (5, 10, 20,
26).
Acylation of Lys860 is not required for ACT function (23,
26). In the E. coli
-hemolysin (HlyA), however, the
corresponding Lys563 residue (Lys564 in some
alleles) at the first acylation site of HlyA was shown to be
preferentially acylated by HlyC in vitro (25), and it is
quantitatively acylated also in vivo (24). Interestingly, like with the ACT-K860R mutant described here, some of the
Lys563 substitutions in HlyA, such as Lys563
Cys, still allowed rather high hemolytic (20%) and especially leukotoxic activities of HlyA (60%) (41). Moreover, some reported substitutions at the second acylation site of HlyA on
Lys689 (Lys690 in some HlyA variants), such as
Lys689
Arg, also allowed substantial hemolytic (11%)
and leukotoxic (41%) activities of HlyA (41). The requirement for
quantitative acylation of HlyA on both the Lys563 and
Lys690 was also recently questioned by the analysis of HlyA
acylated by mutant HlyC (42). Guzmán-Verri et al. (42)
demonstrated that HlyA preparations, which contain only trace amounts
of doubly acylated HlyA and consist predominantly of a mixture of
mono-acylated HlyA with the nonacylated proHlyA, still exhibited
significant hemolytic activities, ranging from 27 to 60% of the
activity of preparations containing exclusively the doubly acylated
HlyA. It was, however, not investigated whether only one of the two potential acylation sites was preferentially modified in the
mono-acylated HlyA or whether the produced HlyA was a mixture of toxins
acylated at either the Lys564 or the Lys690
residues. These observations indicate, nevertheless, that as in the
case of Lys860 substitution in ACT, the loss of activity
upon substitution of Lys563 (Lys564) in HlyA
might also be due to structural effect of replacement of an essential
residue, rather than a mere consequence of the loss of the modification
at the first acylation site of HlyA (Lys563).
It remains unclear why double acylation of HlyA at Lys563
and Lys689 is optimal for hemolytic and cytotoxic activity
of HlyA, while single acylation at the corresponding Lys983
is optimal for full hemolytic activity of B. pertussis ACT.
The different acylation patterns of the recombinant r-Ec-ACT
and the native Bp-ACT show that maximal caution and detailed
characterization of fatty-acylated recombinant RTX toxins are required
prior to their use in functional studies. Nevertheless, understanding
of reasons why the second CyaC-mediated acylation of ACT on
Lys860 occurs only in E. coli K12 would
undoubtedly contribute important new insights into the process of
fatty-acyl activation of RTX toxins. It will be important do decipher
the mechanism by which the acylation of Lys860 interferes
selectively with only the hemolytic (channel-forming) activity of the
toxin and not with its membrane insertion and translocation capacities.
Detailed analysis of this intriguing artifact is likely to advance the
understanding of mechanism of action of ACT within target membranes. It
is intriguing that despite identical culture protocols, expression
plasmids, and E. coli strain used for production of ACT,
myristoylation of Lys983 was not present in the new batches
of toxins analyzed here, while myristoyl was found to constitute about
13% fatty acid residues bound to Lys983 in previous
batches of recombinant ACT (26). Similarly, presence of palmitoleil
(cis
9 C16:1) fatty-acyl groups was not found in previously analyzed
batches of ACT (26). It can only be speculated that subtle differences
in the physiological state of the producing culture, due to limited
reproducibility of different batches of complex culture media and/or
culture conditions, may affect the exact chemical nature of acyl groups
used for modification of ACT by CyaC. This hypothesis would deserve a
systematic testing. Our results suggest a remarkable flexibility of the
modifying enzyme (CyaC) in using fatty-acyl substrates and indicate
that various acyl residues may also be attached to naturally produced RTX toxins. It will be important to evaluate the significance of such
mixed acylation for biological activity and function of these toxins in
bacterial pathogenesis.