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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 452-458 |
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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-GGAATTC�CATATG�AAGAAACTATATACA-3) and p2 (downstream 5-C�C�C��AAGCTTCTATTCTTT�TATTGGG�TATAG-3). The primers for traP mutant were p1 (upstream) and p3 (downstream 5-CCCAAGCTTCTATGAATGTTGT�CCG�CTTGAACC-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
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-diethyla�minopropyl ) 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'-tetramethyl�benzidine 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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