Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 452-458
doi:10.1111/j.1745-7270.2008.00415.x
C-terminus of TRAP in Staphylococcus
can enhance the activity of lysozyme and lysostaphin
Guang Yang1,
Yaping Gao1, Jiannan Feng1,
Yong Huang2, Shaohua Li1,
Yu Liu1, Chuan Liu1,
Ming Fan1, Beifen Shen1,
and Ningsheng Shao1*
1 Beijing Institute of Basic Medical Sciences, Beijing
100850, China
2 Department of Chemistry, Peking University,
Beijing 100086, China
Received: February
19, 2008
Accepted: March 17,
2008
This work was
supported by a grant from the National Natural Sciences Foundation of China
(No. 30700007)
*Corresponding
author: Tel/Fax, 86-10-68163140; E-mail, [email protected]
In Staphylococcus
aureus, the target of RNAIII activating protein (TRAP) is a
membrane-associated protein whose C-terminus can be used as a vaccine to
provide protection against staphylococcal infection. Here, we show for the
first time by surface plasmon resonance and enzyme-linked immunosorbent assay
that TRAP can specifically bind lysozyme and lysostaphin through its C-terminus
(amino acids 155–167)
and enhance lysozomal activities in vitro. It was also found that the traP
mutant strain is more resistant to lysostaphin than wild-type. Our previous
data showed that the C-terminus of TRAP might be extracellular. So our results
suggested that the C-terminus of TRAP could act as the specific targeting
protein of the lysozyme/lysostaphin on the S. aureus cell wall
and the biological significance of the interaction might be to facilitate
lysozyme/lysostaphin-mediated cell lysis.
Keywords Staphylococcus aureus; TRAP; lysozyme; lysostaphin
Staphylococcus aureus is a major
pathogen. Pathogenic effects are largely due to the production of bacterial
toxins [1]. The target of RNAIII activating protein (TRAP) plays an important
role in the regulation of S. aureus exoprotein secretion in a yet
unknown manner and has a high degree of sequence similarity among strains [2–5]. The
phosphorylation of TRAP activates the production of RNAIII, an RNA molecule
that regulates the production of toxins [6]. TRAP is located on the bacterial
cell wall, but has no predicted transmembrane domain and the mode by which it
is bound to the membrane is not yet known [6]. From our previous data, we
identified the C-terminal sequence [amino acids (aa) 155–167] of TRAP as
an antigen epitope where antibodies against the epitope could protect mice from
infections caused by S. aureus. So it was suggested that the
C-terminus of TRAP should be extracellular. By sequence analysis, it was found
that the sequence of the C-terminus is conserved among strains [7]. Lysostaphin secreted by Staphylococcus simulans is a
bacteriolytic enzyme that cleaves the pentaglycine cross-bridges of
staphylococcal peptidoglycans, specifically that of the target organism S.
aureus [8]. The mature form of lysostaphin encompasses two domains, the
glycyl-glycine endopeptidase domain that cleaves oligoglycine peptides [9], and
a C-terminal cell wall-targeting domain (CWT) [10].Lysostaphin binds S. aureus cells and cleaves
pentaglycine cross-bridges within peptidoglycan, thereby removing the cell wall
envelope and precipitating osmotic rupture of staphylococci [8,11,12]. Hen egg-white lysozyme (lysozyme) was the first enzyme to have its
3-D structure determined by X-ray diffraction techniques [13]. The natural
substrate of lysozyme is the peptidoglycan cell wall of bacteria. The
peptidoglycan cell wall is composed of cross-linked oligosaccharides consisting
of alternating 2-acetamido-2-deoxy-glucopyranoside (NAG) and
2-acetamido-2-deoxy-3-O-lactyl-glucopyranoside (NAM). It was reported that a
catalytically competent covalent intermediate forms during the catalytic cycle
of lysozyme [13].Here we show that TRAP can specifically bind lysostaphin, and that
the traP disrupted strain is not as sensitive to lysostaphin as the
wild-type strain. We also found that TRAP could bind lysozyme and enhance its
activity. The C-terminus of TRAP was identified as the binding site of
lysozyme, consistent with our previous data that the C-terminus of TRAP is
expected to be extracellular. The binding of TRAP to lysostaphin/lysozyme might
be helpful in specifically targeting the enzyme to S. aureus.
Materials and Methods
Bacterial strains
Staphylococcus aureus wild-type 8325-4,
mutant 8325-4 DtraP minus strain, RN6390B, and RN6911 (agr minus) were kindly
provided by Dr. Naomi Balaban (Tufts University, North Crafton, USA). Escherichia
coli BL21 was the host strain for pET-28a (Invitrogen, Grand Island, USA). S.
aureus was grown in tryptone soya broth.
Expression of TRAP (167 aa)
and truncated TRAP mutant (TRAPm, 154 aa)
The traP gene was obtained by PCR using S. aureus
8325-4 chromosomal DNA as the template. The primers for wild-type traP
were p1 (upstream 5-GGAATTCCATATGAAGAAACTATATACA-3) and p2 (downstream
5-CCCAAGCTTCTATTCTTTTATTGGGTATAG-3). The primers for traP
mutant were p1 (upstream) and p3 (downstream 5-CCCAAGCTTCTATGAATGTTGTCCGCTTGAACC-3).
The cleavage site of restrictive enzymes NdeI and HindIII
is underlined. The TRAP mutant does not have the sequence coding for the
C-terminus (aa 155–167) of TRAP. The two PCR amplicons were cloned into pET-28a. The
plasmids were transformed into E. coli BL21 and protein expression was
induced by the addition of isopropyl-b–D-thiogalactoside. The recombinant
proteins were purified by Ni affinity chromatography according to the
manufacturer’s instructions (GE Healthcare, New York, USA). The proteins were
eluted with 0.5 M imidazole.
Cell lysis
induced by lysostaphin
Both the bacteria 8325-4 and 8325-4 DtraP were cultured in
tryptone soya broth overnight at 37 ?C. The same amounts of bacteria (1?108 cells/ml, 100 ml) were
incubated with lysostaphin (2 mg/ml, 100 ml) at 37 ?C for 10 min. The surviving bacteria were counted on the
agar plates and expressed as c.f.u.. The survival ratio was calculated by the
survival amounts compared to the total input.
Immobilization
of protein on sensor chip
Immobilization
of protein on sensor chip
To immobilize proteins on sensor chips, the carboxymethylated dextran
surface was first activated by injecting 50 ml of the 0.1 M N-ethyl-N-(3-diethylaminopropyl
) carbodiimide/0.1 M N-hydroxysuccinimide (1:1) mixture. The protein (10
mg/ml,
50 ml)
in HBS-EP buffer [0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20
(pH 7.4)] was then injected over the activated surface, followed by the
injection of 50 ml of 1 M ethanolamine to deactivate the remaining active carboxyl
groups. The sensor chip was then washed with 10 ml of 10 mM Gly/HCl (pH 2.2)
to remove remaining non-covalently bound TRAP. The sensor chip was washed
overnight before the experiment with HBS-EP buffer to ensure a stable baseline.
The immobilization procedures were carried out at 25 ?C and at a constant flow
rate of 5 ml/min HBS-EP buffer.
Real-time association and
dissociation measurements
All binding experiments were carried out at 25 ?C with a constant
flow rate of 30 ml/min HBS-EP buffer. Thirty microliters of different concentrations
of the analyte was injected over the sensor surface with an immobilized
protein, followed by a 240 s wash with HBS-EP buffer. The sensor surface was
then regenerated by washing with 30 ml of 10 mM Gly/HCl (pH 2.2). To correct for
non-specific binding and bulk refractive index change, a blank channel without
protein on the sensor chip surface was used and run simultaneously for each
experiment. The sensorgrams for all binding interactions were recorded in real
time and were analyzed after subtracting the sensorgram from the blank channel.
The sensorgram data were analyzed using BIAevaluation Software version 4.0
(GE Healthcare). The 1:1 Langmuir binding model was chosen to globally fit the
data by choosing fit kinetics simultaneous ka/kd. The degree of
randomness of the residual plot and the reduced c2-value were used to
assess the appropriateness of a model to the sensor data. Therefore, integrated
rate equations for this model were used for fitting the sensorgram data to
derive binding kinetic constants ka (association rate constant) and kd (dissociation rate constant), and thermodynamic constants KA (association equilibrium constant) and KD (dissociation equilibrium constant).
Enzyme-linked immunosorbent
assay (ELISA)
The specific binding of TRAP and TRAPm to lysozyme (Sigma, St. Louis, USA) was tested by ELISA. Microtiter
96-well plates (Apogent Technologies, Portsmouth, USA) were coated with TRAP or
TRAPm [2 mg/well in 0.1 M NaHCO3 (pH 9.6)] at 4 ?C overnight. Unbound proteins were removed, and the
wells were blocked with 3% bovine serum albumin in phosphate-buffered saline at
37 ?C for 1 h. Lysozyme (5 mg/well) was added into each well and incubated for 2 h at 37 ?C.
Plates were washed with the washing buffer (phosphate-buffered saline
containing 0.05% Tween-20) five times. The anti-lysozyme antibodies (1:1000)
(prepared in our laboratory) were then added and incubated for 1 h at 37 ?C.
After washing, the horseradish peroxidase-labeled anti-rabbit antibodies were
added, and the binding was detected using 3,3‘,5,5‘-tetramethylbenzidine
substrate (Sigma). Results were expressed as the reading at 450 nm. ELISA was
also carried out by coating the same plates with lysozyme followed by
incubation with TRAP and TRAPm. The binding was
determined by adding anti-TRAP antibodies using the method described above.
Binding of peptide to lysozyme
Briefly, the peptide in accordance to the C-terminus of TRAP (aa 155–167) was
synthesized and coated in 96-well plates (10 mg/well). Lysozyme (5 mg/well) was
added. The binding of peptide to lysozyme was detected by the methods described
above.
Lysozyme activity test in
vitro
The activities of lysozyme were tested by the lysozyme detection kit
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the
bacterial suspension (0.25 mg/ml) was prepared by diluting the powder of Micrococcus
cell wall in bacterial powder dissolvent (provided in the kit) and placed on
ice as the substrate of lysozyme. Either TRAP (0.5 mg/ml, 100 ml) or the
peptide (0.1 mg/ml, 100 ml) was added to the standard enzyme (2500 U/ml, 100 ml) and incubated
at room temperature for 10 min. Mixtures of TRAP/lysozyme and peptide/lysozyme
were placed on ice for 5 min. Then 0.2 ml mixture was incubated with 2 ml
bacterial suspension in tubes at 37 ?C for 15 min. The tubes
were then placed on ice to stop the reaction. The transmittance of each tube
was measured at 530 nm.
Statistical analysis
Statistical analysis was carried out using Student’s t-test
by Excel version 2003 (Microsoft, Washington, USA). Significance was accepted
when the P<0.05.
Results
TRAP specifically binds
lysostaphin
The recombinant TRAP was expressed and purified as described in our
previous work [7]. The binding of TRAP to lysostaphin (Sigma) was determined by
ELISA and by biosensor measurements. Results showed that TRAP could
specifically bind to lysostaphin in vitro [Fig. 1(A,B), Table
1]. At the same time, we observed that the TRAP cells were not easily
disrupted by lysostaphin compared to the parent TRAP+ strain. Indeed, when the cells were incubated with lysostaphin for
3 h, more TRAP–
bacteria survived the treatment [Fig. 1(C)], once again suggesting that
TRAP is also necessary for the binding and activity of lysostaphin in vitro.
Both TRAP and its C-terminus
peptide can specifically bind with lysozyme
The interaction kinetics between TRAP protein and lysozyme was
measured with a surface plasmon resonance biosensor. TRAP was immobilized on the
sensor chip surface. The binding and dissociation of various concentrations of
lysozyme and different immobilized TRAP were measured and recorded as
sensorgrams by the BIAcore3000 instrument (GE Healthcare) [Fig. 2(A)].
It was found that TRAP could bind lysozyme with high affinity. Our previous results showed that the C-terminus of TRAP (aa 156–167) is an
epitope that could induce the production of protective antiserum against S.
aureus infection [7], suggesting that the C-terminus might be extracellular.
Therefore, the binding of the peptide to immobilized lysozyme was carried out [Fig.
2(B)]. Table 2 shows the kinetic parameters obtained. As shown in Table
2, the KD value for peptide and
lysozyme (5.44?10–6) is two orders of magnitude higher than the KD for TRAP and lysozyme (9.86?10–8). The difference in binding properties between TRAP and the peptide
is mainly presented in the association processing. The association rate
constant ka for peptide and lysozyme
(2.73?103) was much lower than the ka for TRAP and lysozyme (4.99?105). The magnitude of the
dissociation rate constant kd is in the same order. So TRAP and the peptide have almost the same
dissociation properties for lysozyme.It is clear that both TRAP and the C-terminus peptide can
specifically bind to lysozyme. The peptide can therefore be considered as one
possible binding site in TRAP interacting with lysozyme.
TRAPm
with C-terminus deletion could not interact with lysozyme
The C-terminus deletion of TRAPm was expressed and purified (data not shown). The binding of
TRAP/TRAPm to lysozyme was measured
with ELISA. TRAP and TRAPm were used to coat
96-well plates, following the addition of lysozyme. Alternatively, lysozyme was
used to coat a 96-well plate, followed by incubation with TRAP/TRAPm. The results showed that TRAP bound to lysozyme, but TRAPm did not [Fig. 3(A,B)]. Then we tested the activity of
lysozyme after interaction with TRAP/TRAPm. The same amounts of TRAP or TRAPm were incubated with lysozyme. The activity of lysozyme was measured
according to the protocol of the kit. The results showed that the TRAP protein
increased the activities of lysozyme in a dose-dependent manner, whereas TRAPm had no such effect [Fig. 3(C,D)], suggesting the C-terminus
peptide is important for this activity.
TRAP C-terminus peptide (aa
155–167) can specifically bind to
lysozyme and increase lysozyme activity
The C-terminus of TRAP was predicted as the binding domain of
lysozyme, so the binding of the peptide [corresponding to the C-terminus of
TRAP (aa 155–167)] and lysozyme was determined by ELISA using antibodies against
lysozyme. As shown in Fig. 4(A), the peptide could specifically bind
lysozyme. Different concentrations of the peptide were then incubated with
lysozyme and the transmittance of bacterial suspension was measured. Results
showed that the peptide had the same activity-enhancement effect on lysozyme as
the whole TRAP [Fig. 4(B)].
Discussion
TRAP is a 167 aa conserved staphylococcal membrane-associated protein
[4,6]. The exact function of the protein is not yet known [19–21] but TRAP has
been shown to be important for virulence as the highly conserved C-terminus of
TRAP (aa 155–167) can stimulate mice to produce protective antiserum against S.
aureus infections [7]. This result also suggested that the C-terminus of
TRAP is available to extracellular interactions [7]. Interestingly, TRAP
expression was induced after S. aureus was treated by cell wall
active agents [14], suggesting that TRAP is also involved in stress response. It was found that there is a mutation in agrA in the traP
mutant strain [20]. So we checked if the different sensitivity to lysostaphin
in the traP mutant strain is caused by agrA mutation. The results showed
that there is no significant difference between RN6911 (agr minus) and RN6390B
(data not shown). It is therefore suggested that the agr system could not
influence the sensitivity to lysostaphin in S. aureus.Our results showed that both the intact protein and the peptide
derived from the TRAP C-terminus could specifically bind lysozyme and enhance
its activity. But it was reported that S. aureus was completely
resistant to lysozyme. Modifications in peptidoglycan by O acetylation, wall
teichoic acid, and a high degree of cross-linking contribute to this resistance
[15,16]. There is a question left as to the biological function of the
interaction between lysozyme and TRAP on the cell wall of S. aureus.
One explanation could be that TRAP on S. aureus could enhance the
activity of lysozyme secreted by the host to kill other microbes that compete
for limited resources, just like bacteria secretes bacteriocins [17]. Lysostaphin could specifically hydrolyze the cell wall envelope. The
information for target cells is encoded within the C-terminal residues of
lysostaphin [10]. In the present study, it was found that the CWT domain of
lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also
serves as substrate for its glycyl-glycine endopeptidase domain [18]. The mechanism
of how the substrate recognized by CWT could be hydrolyzed by the endopeptidase
domain is still not clear. Our results suggested that TRAP could be another
target of lysostaphin. But the binding domain on lysostaphin remains unclear.There is no sequence homology between lysozyme and lysostaphin. Why
should both of them interact with TRAP? In this experiment, it was found that
the antibodies against lysozyme could specifically bind lysostaphin. It is
suggested that lysostaphin might have the same epitope as, or an epitope
similar to that of, lysozyme. The details of this epitope will be the focus of
further studies.
Acknowledgements
We thank Dr. Naomi Balaban (Tufts University, North Crafton, USA),
who kindly provided the bacterial strains and reviewed the manuscript
carefully. We thank colleagues of our laboratory for their help.
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