(Received for publication, June 15, 1995)
From the
Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.
Adenylate cyclase (AC) ()toxin is a bifunctional
177-kDa bacterial protein that contains a calmodulin-activated
adenylate cyclase catalytic domain and a pore-forming domain. It
exhibits a cell-invasive activity by delivering its catalytic domain to
the interior of target cells to elicit supraphysiologic cAMP
accumulation(1, 2, 3, 4) . The
pore-forming capacity of the molecule is responsible for hemolytic
activity against sheep erythrocytes and is entirely independent of the
presence and activities of the catalytic
domain(5, 6, 7, 8, 9) .
The AC toxin gene (cyaA) has been cloned and sequenced by
Glaser et al.(10, 11) and the AC toxin locus
was noted to have homologies with the Escherichia coli hemolysin operon and that of other RTX toxins. These toxins are
characterized by a set of glycine- and aspartate-rich nonapeptide
repeats(12, 13) . The formation of biologically active
AC toxin requires an accessory protein expressed from an upstream gene, cyaC(14) . That protein, CyaC, appears to be involved
in posttranslational activation of AC toxin. Recently, tandem mass
spectrometry was used to determine that the modification on native AC
toxin from Bordetella pertussis (Bp-CyaA) consists of
palmitoylation on the -amino group of
Lys
(15) . In vitro random chemical
acylation has also been shown to confer limited cell-invasive and
hemolytic activities on CyaA(16) .
It has been observed previously that recombinant AC toxin expressed in E. coli exhibits cell-invasive activity identical to the native toxin, but hemolytic activity which is severalfold reduced(17) . In order to determine whether the nature of the acylation could be the basis of this functional difference, wild type AC toxin from B. pertussis and recombinant AC toxin from E. coli were analyzed by mass spectrometry. We report here that despite the presence of the same requisite CyaC, the pattern and chemical nature of the acylation are different when AC toxin (CyaA) is expressed in these two organisms.
The fermentor cultures of E. coli strains were supplied by the Service of Fermentations of Institut Pasteur (Paris). The r-CyaA and r-proCyaA were extracted from E. coli cell debris after French press disruption, as described previously(17) . Bp-CyaA was extracted from washed B. pertussis cells with 4 M urea as described previously(5) . The different CyaAs were purified close to homogeneity by a combination of ion-exchange chromatography (19) and affinity chromatography(17) . In the final step CyaAs were eluted from calmodulin-agarose columns with 8 M urea, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and frozen at -70 °C.
CyaC-activated recombinant AC toxin (r-CyaA) was produced in E. coli(17, 18) and compared with Bp-CyaA
extracted from the natural producer B. pertussis. It was found
that the partially purified r-CyaA had about 4-fold lower specific
hemolytic activity on sheep erythrocytes and 4-10-fold lower
specific pore-forming activity in artificial planar lipid bilayers than
Bp-CyaA (9) , whereas the cell-invasive activity of both
proteins was equal. In the present studies, r-CyaA and Bp-CyaA were
mixed at different molar ratios, so as to yield a fixed total CyaA
concentration of 1 unit/ml, and the cell-invasive and hemolytic
activities of the mixtures were determined. The cell-invasive AC
activity was constant regardless of the molar ratio of the proteins in
the mixture (Fig. 1). In contrast, the hemolytic activity of the
mixture increased with the increasing proportion of the Bp-CyaA present
in the mixture, yielding an upwardly concave curve. Because both
cell-invasive and hemolytic activities depend on the posttranslational
fatty-acylation of Lys of CyaA(14, 15) ,
we asked whether or not the difference in the hemolytic activities of
r-CyaA and Bp-CyaA could be accounted for by differences in the
chemical nature and/or location of their posttranslational
modifications.
Figure 1:
Comparison of cell-invasive and
hemolytic activities of r-CyaA and Bp-CyaA. Toxin dilutions and/or
mixtures were prepared in 50 mM Tris-HCl, pH 8.0, 8 M urea, and 2 mM EDTA. R-CyaA and Bp-CyaA were mixed at
various molar ratios to obtain the final solutions at 100 units/ml (200
µg/ml) of total CyaA, and the toxin mixtures were directly diluted
to 1 unit/ml (100-fold) into prewarmed suspensions of washed sheep
erythrocytes (5 10
/ml) in TNC (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM
CaCl
). The suspensions were incubated at 37 °C. After
30 min, aliquots of the suspensions were chilled on ice and washed with
cold TNE buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl,
2 mM EDTA). Then L-1-tosylamido-2-phenylethyl
chloromethyl ketone-trypsin was added to 40 µg/ml, and the
suspensions were incubated at 37 °C for 10 min in order to destroy
the AC toxin remaining outside the erythrocytes. Upon addition of
soybean trypsin inhibitor (2-fold excess) the erythrocytes were washed
again, lysed in 50 mM Tris, pH 8.0, 0.2 mM
CaCl
, 0.1% Nonidet P-40 and the adenylate cyclase enzymatic
activity, which had penetrated into the erythrocytes and was protected
against externally added trypsin, was measured. The extent of
erythrocyte lysis was determined after 130 min of incubation by
spectrophotometrically measuring the released hemoglobin in cell-free
incubation supernatants at 541 nm. Detergent lysed erythrocyte
suspension was used to determine the 100% lysis
value.
When characterized by mass spectrometry, both native
Bp-CyaA and r-CyaA matched the amino acid sequence deduced from the
published nucleotide sequence of the cyaA gene (10, 17) and exhibited the following posttranslational
modifications. Bp-CyaA has previously been shown to bear a single
modification consisting of a palmitoylation at the -amino group of
Lys
(15) . As expected, this acylation was
observed only when the toxin was produced in the presence of a
functional CyaC protein and was not observed in
Bp-proCyaA(15) . The pattern of acylation in the r-CyaA,
however, differed from that observed in the B.
pertussis-derived protein. Both palmitoylation (87%) and
myristoylation (13%) were observed at Lys
(n = 3, RSD = 23%). About 67% (n = 3,
RSD = 32%) of the r-CyaA molecules were found to bear a second
modification at Lys
, consisting entirely of
palmitoylation, with the remaining Lys
residues
unmodified (Fig. 2). Acylation at Lys
was also
strictly CyaC-dependent.
Figure 2:
Most
of Lys residues of r-CyaA are palmitoylated. A,
CAD spectrum of the tryptic fragment of r-CyaA spanning residues
Thr
to Lys
and containing palmitoylation at
Lys
. The parent ion was [M +
2H]
, m/z 941. For definitions of b and
y ions, see (30) . B, MALDI-TOF MS spectrum of the
late eluting microbore HPLC fraction containing palmitoylated
Lys
r-CyaA, observed at m/z 5359 and
encompassing residues Asp
to Gln
(DIASRKGERPALTFITPLAAPGEEQRRRTKTGK
SEFTTFVEIVGKQ),
with a predicted average mass (protonated) of 5358. C,
MALDI-TOF MS spectrum of the fraction containing unmodified Lys
at m/z 5120. The predicted average mass was 5120. The
peaks observed in the MALDI spectra at m/z 4284 and 8566 are
from bovine ubiquitin, which was added to the samples as an internal
standard for mass calibration.
Both Lys and Lys
of CyaA lie in regions highly conserved among all RTX toxins.
Although acylation of the two corresponding lysine residues of the
homologous E. coli
-hemolysin (HlyA) has been observed in vitro(26) , the pattern of in vivo acylation of the naturally occurring HlyA remains unknown.
Nevertheless, these data led to the suggestion that both Lys
and Lys
of CyaA might be acylated(26) .
This, however, appears to be the case only for r-CyaA produced in E. coli and not for the naturally occurring Bp-CyaA produced
by B. pertussis. An abundant tryptic fragment, corresponding
to residues 861-872 of Bp-CyaA, was identified by MALDI-TOF MS
and sequenced by tandem MS (Fig. 3). This fragment would not be
present in tryptic digests if Lys
was acylated, thereby
eliminating the tryptic cleavage site. The tryptic peptide containing
unmodified Lys
and spanning residues Thr
to
Lys
was not recovered by our HPLC
procedures(15, 21) . A weak signal (signal/noise
5) at m/z 1882 was observed by MALDI-TOF, in an HPLC fraction
corresponding to the expected elution time of the tryptic palmitoylated
Lys
-containing peptide. The calculated average mass for
this peptide was 1882.3. The predicted HPLC behavior was based on the
retention times of a synthetic acyl peptide standard (same structure as
that shown in Fig. 2A) and the tryptic acyl peptide
isolated from the Lys
site in r-CyaA (Fig. 2A). The amount recovered from Bp-CyaA was
insufficient to generate a CAD spectrum. Only one abundant Asp-N
fragment was isolated from this site, observed [M +
H]
at m/z 5120, consistent with
unmodified Lys
(see the sequence given in the legend for Fig. 2). Based on the results discussed above, we estimate that
Bp-CyaA palmitoylated at Lys
represents less than 5% of
the total protein.
Figure 3:
Lys of Bp-CyaA is
essentially unmodified. CAD spectrum of the unmodified fragment
isolated from a tryptic digest of Bp-CyaA, which could only result from
cleavage at an unmodified Lys
. This fragment spans
residues Ser
to Lys
. The parent ion was
[M + 2H]
, m/z 679.
Because of its conservation in other RTX
toxins(15, 26) , the Lys site of r-CyaA
was also investigated with respect to potential modifications. As was
reported for Bp-CyaA(15) , only unmodified Lys
was observed.
It remains unclear why in the presence of the same CyaC protein, the CyaA is differently acylated in E. coli and in B. pertussis. One hypothesis is that acylation of AC toxin is not catalyzed by CyaC itself, but rather that it is catalyzed by an unidentified transacylase which uses acyl-ACP as substrate and CyaC as a co-factor. This hypothetical transacylase might have a slightly different specificity in E. coli and in B. pertussis. Indeed, the in vitro acylation experiments involving the HlyA and the CyaC homolog, HlyC, indicated that HlyC is not a conventional enzyme(27, 28) . In fact, stoichiometric amounts of HlyC were required for activation of proHlyA. Moreover, although acyl-ACP and HlyA could be labeled in vitro by a radioactive acyl chain, a radioactive acyl-HlyC intermediate was not observed. It is conceivable that the transacylating enzyme might have been present, as a minor component, in the partially purified preparations of HlyC, proHlyA, and/or acyl-ACP of E. coli, used for the in vitro acylation system. An alternative hypothesis is that CyaC may itself be catalyzing the acylation of CyaA, and when expressed in E. coli, its specificity may be affected by some alteration in the state of the overproduced r-proCyaA substrate.
The differential effect of altered
acylation on toxin function, namely a reduction in hemolytic activity,
but no effect on invasive activity, provides evidence against the
hypothesis that the pore involved in hemolytic activity has a role in
delivery of the catalytic domain(9, 29) . Since it
appears from their invasive activities that Bp-CyaA and r-CyaA are
comparable in their propensity to insert into the membrane, the
differences in their acylation state must affect a subsequent step in
toxin action. The most likely candidate for this functional defect is
the oligomerization of CyaA molecules to form the hemolytic pore.
Intoxication of target cells occurs as a linear function of AC toxin
concentration(18) , indicating a monomolecular mechanism for
delivery of the catalytic domain. ()In contrast,
channel-forming and hemolytic activities are a non-linear function of
toxin concentration exhibiting a cooperativity coefficient suggestive
of a toxin tetramer(8, 18) . On the basis of these
data, we propose that CyaA molecules in different states of
oligomerization, but especially monomers, can deliver their catalytic
domains into cells, whereas oligomerization of AC toxin is a
prerequisite for formation of hemolytic channels.
The data
suggesting that overacylated r-CyaA is selectively impaired in the
formation of the CyaA channels (Fig. 1) indicate that besides
being essential for the interaction of CyaA with the membrane, the
acylation of CyaA may also be involved in oligomerization of the CyaA
molecules and formation of CyaA channels. It remains to be determined
how the excess acylation on Lys of r-CyaA can impair its
channel-forming activity. Defining this mechanism will contribute to
our general understanding of the role of fatty-acylation in
protein-membrane and protein-protein interactions.