Staphylococcus lysin and use thereof

Abstract

The present invention discloses a lysin that is capable of killing Staphylococcus and the use thereof, belonging to the field of biological agents. The present invention discloses the amino acid sequence and the encoding gene sequence of the lysin. This lysin keeps active in a wide range of pH. It has lytic activity against Staphylococcus in pH 4-11. The recombinant protease constructed by the encoding gene can be solubly expressed in E. coli strain BL21 (DE3). The lysin can be used to effectively kill multiple species Staphylococcus in vitro, including methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) isolated in clinics. This lysin can be used as an antibiotic for the treatment of staphylococcal infections in vivo. This lysin is also able to rapidly lyse staphylococcal cell wall; as a result, intracellular substances such as ATP and DNA are released. Those released substances can be used to detect the type of Staphylococcus.

Claims

1. A recombinant Staphylococcus lysin protein, wherein the amino acid sequence of said lysin is shown in SEQ.ID.NO.2.

2. A gene encoding the recombinant Staphylococcus lysin protein according to claim 1, wherein the DNA sequence of said gene is shown in SEQ.ID.NO.1.

3. A method of soluble expression and purification of the recombinant Staphylococcus lysin protein according to claim 1, wherein the gene according to claim 2 is cloned and then ligated with the expression vector pBAD24 to form an expression plasmid, the expression plasmid is transferred into E. coli strain BL21(DE3); expression of the expression plasmid is induced in the E. coli strain BL21(DE3) by using 0.2% L-arabinose, the E. coli strain BL21(DE3) are collected and sonicated, forming a supernatant the supernatant is precipitated using 33% ammonium sulfate to form a precipitate, the precipitate is dissolved in PBS and then dialyzed against PBS overnight to form a crude extract solution, the crude extract solution after dialysis or the supernatant after sonication is passed through a HiTrap Q Sepharose FF column to collect an effluent, the effluent is then passed through a HiTrap SP Sepharose FF column to obtain a sample, the sample is gradient-eluted by 1M NaCl and elution peaks are collected in fractions, and the fractions with activity are combined and dialyzed against PBS overnight to obtain a sample of the purified lysin protein.

4. A method for killing Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus equorum, Staphylococcus capitis, Staphylococcus albus, Staphylococcus xylosus, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus chromogens, or Staphylococcus aureus in vitro comprising the use of the mixing an effective amount of recombinant Staphylococcus lysin protein according to claim 1 with a Staphylococcus bacteria suspension.

5. The method according to claim 4, wherein the Staphylococcus aureus include methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

6. A pharmaceutical product comprising the recombinant Staphylococcus lysin protein of claim 1 as an active ingredient.

7. A method for detection and characterization of Staphylococcus via ATP or DNA released from the Staphylococcus wherein the method comprises: mixing a Staphylococcus sample with the recombinant Staphylococcus lysin protein according to claim 1 to form a Staphylococcus mixture; adding luciferase and its substrate to the Staphylococcus mixture, incubating the mixture at 37 C., and detecting a change of fluorescence intensity of the Staphylococcus mixture using a microplate reader, wherein a Staphylococcus sample without the recombinant Staphylococcus lysin protein according to claim 1 is a negative control.

8. A method for detection and characterization of Staphylococcus, wherein the method comprises: mixing a Staphylococcus sample with the recombinant Staphylococcus lysin protein according to claim 1 to form a Staphylococcus mixture; incubating the mixture at 37 C. for 10-15 min, centrifuging the mixture at 10,000 g for 1 min to form a pellet and supernatant, collecting the supernatant as a template, contacting the supernatant with two pairs of primers to determine if the Staphylococcus sample is a Methicillin-resistant Staphylococcus aureus strain, wherein the first pair of primers is FemB-F (SEQ.ID.NO.5) and FemB-R (SEQ.ID.NO.6) to amplify femB gene; the second pair of primers is MecA-F (SEQ.ID.NO.7) and MecA-R (SEQ.ID.NO.8) to amplify mecA gene; amplifying by PCR the femB gene and the mecA gene, wherein the amplifying comprises incubating the supernatant and two pairs of primers 1) at 94 C. for 5 minutes; 2) performing 30 cycles of 94 C. for 30 seconds, 50 C. for 45 seconds, 72 C. for 60 seconds, and 3) 72 C. for 10 minutes to form a PCR product, and analyzing the PCR product by electrophoresis to identify whether a strain is Methicillin-resistant Staphylococcus aureus or not.

9. The method according to claim 8, for confirmed Methicillin resistant Staphylococcus aureus strain, further comprising: contacting the supernatant with 4 pairs of primers, wherein the pairs of primers is (SEQ.ID.NO.9) and 3 (SEQ.ID.NO.10) to amplify the ccrA2-B gene; ccrCF (SEQ.ID.NO.11) and ccrCR (SEQ.ID.NO.12) to amplify the ccrC gene; 1272F1 (SEQ.ID.NO.13) and 1272R1 (SEQ.ID.NO.14) to amplify the IS1272 gene; and 5RmecA (SEQ.ID.NO.15) and 5R413 (SEQ.ID.NO.16) to amplify the mecA-IS431 gene; amplifying by PCR the ccrA2-B gene, the ccrC gene, the IS1272 gene and the mecA-IS431 gene to form a PCR product, and analyzing the PCR product by electrophoresis for Staphylococcal Cassette Chromosome Mec typing of the Methicillin resistant Staphylococcus aureus strain.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is the results of PCR amplification of ClyO gene. In the figure, the lane M represents standard DNA marker, the size of the bands is shown on the left. The lane O is the band of amplified ClyO.

(2) FIG. 2 is the results of ClyO killing Staphylococcus aureus standard strain CCTCC AB91118. The dotted line shows the change trend of OD.sub.600 absorbance over time after Staphylococcus aureus being mixed with ClyO. The solid line shows the change trend of OD.sub.600 absorbance over time after Staphylococcus aureus being mixed with buffer. The hollow circle shows the change trend of Log CFU over time after Staphylococcus aureus being mixed with buffer.

(3) FIG. 3 is the results of specificity of ClyO killing various Staphylococcus strains and against different variety of bacterial strains in vitro. The vertical coordinates show the decrease rate of OD.sub.600 absorbance after incubating the mixtures of various bacterial strain and ClyO at 37 C. for 30 min.

(4) FIG. 4 is the results of ClyO killing MRSA in milk. The vertical coordinates show the decrease of the log value after incubating the mixtures of various bacterial strains and ClyO at 37 C. for 60 min.

(5) FIG. 5 is the results of ClyO eliminating MRSA on the infected skin of scalded mice. For each group (20 mice), high concentration of MRSA was inoculated on the scalded skin, then the mice were treated with PBS and 100 mg ClyO after 24 h. After treatment for 24 h, skins were cut off and the MRSA numbers were calculated in each tissue sample by the method of serial dilution followed by colony-counting on the plate. No treatment was done in the control group after inoculation of the bacteria.

(6) FIG. 6 is the results of ClyO killing Staphylococcus aureus in vivo. For each group (20 mice), lethal dose of Staphylococcus aureus was injected respectively. For the experimental group 1 mg ClyO was injected intraperitoneally after 3 h. For the control group, same volume of PBS was injected intraperitoneally after 3 h. The survival rate for each group was observed every day.

(7) FIG. 7 is the tested cytotoxicity result of ClyO. No significant cytotoxicity was observed for ClyO at a concentration of 0.1 mg/ml, 0.5 mg/ml, and 1 mg/ml on Caco-2 cells respectively. Ionomycin and mitomycin C were used as positive controls which were known to be toxic to cells.

(8) FIG. 8 is the results of rapid detection of Staphylococcus aureus type based on ATP release using ClyO. The black solid line represents the blank control. The rising curve represents different Staphylococcus aureus strains.

(9) FIG. 9 is the PCR detection results of identifying MRSA using ClyO. The position represented by triangle is femB gene, the position represented by the star is mecA gene. MRSA strains contains both femB and mecA genes, while MSSA strains contain only femB. Other Staphylococcus aureus strains only contain mecA.

(10) FIG. 10 is the PCR detection results of MRSA strain typing using ClyO. The size of the band obtained by PCR SCCmec type I strain is 415 bp. the size of the band obtained by SCCmec type II strain PCR is 937 bp. The size of the band obtained by SCCmec type III strain PCR is 518 bp. The size of the two bands obtained by SCCmec type IV strains PCR are 415 bp and 937 bp respectively. The size of the two bands obtained by SCCmec type V strains PCR are 518 bp and 359 bp respectively.

(11) FIG. 11 is the results of reduction of the number of Staphylococcus aureus after mimic skin disinfection using ClyO. The Staphylococcus aureus can be completely eliminated by ClyO when the inoculated amount of bacteria is less than 100 CFU.

DETAILED DESCRIPTION OF THE INVENTION

(12) The present invention is further explained by the following examples, but the present invention is not to be limited by this.

(13) The present inventors designed and artificially synthesised the gene sequence that is able to express a kind of Staphylococcus lysin ClyO, and obtained this enzyme by in vitro recombination and expression in E. coli, through analysis of the amino acid sequences of the catalytic domain and the cell structure domain of Staphylococcus phage lysin.

(14) The following methods used in examples were standard experimental methods without special description. All primers used in experiments were provided by Invitrogen (Shanghai). DNA sequencing was performed by Invitrogen (Shanghai). The standard S. aureus CCTCC AB91118 strain was purchased from Culture Collection Center of University of Wuhan, S. epidermidis ATCC 12228 strain and reference strain were purchased from Guangdong Culture Collection Center. Other strains were isolated in clinics from several hospitals in Wuhan, and were identified by Biolog Automatic Analyzer for Microbes. The drug resistance of clinical bacterial strains was verified by methicillin paper disk dilution method. The clinical isolates of S. aureus were streaked on the Baird-Parker agar (purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.); then single colony was picked, and then cultured overnight in TSB medium for testing use.

EXAMPLE 1

Construction of the Lysin being Able to Kill Staphylococcus

(15) 1) DNA sequences of ClyO gene expressing lysin ClyO was totally synthesised by Sangon Biotech (Shanghai) Co., Ltd. The synthesised sequences was inserted into plasmid pUC57. The ClyO gene was used as a template, and restriction enzyme sites NcoI and XhoI were introduced at two terminals of the target gene. Primers were designed as below:

(16) TABLE-US-00001 Forwardprimer: (SEQ.ID.NO.3) 5-TTAACCATGGGCATGGCACTGCCTAAAACG-3 NcoI Reverseprimer: (SEQ.ID.NO.4) 5-ATATCTCGAGTTTAAATGTACCCCAAGC-3 XhoI

(17) 2 l of gene was used as a template, then 1 g of each primer was added for PCR amplification, and the programme of PCR amplification was described as below: 1) 94 C., 5 min; 2) 94 C., 30 sec, 62 C., 45 sec, 72 C., 45 sec, 30 cycles; 3) 72 C., 10 min.

(18) PCR products were electrophoresed and recovered, electrophoretic pattern was shown in FIG. 1. The size of ClyO gene was 777 bp, which was consistent with the size of designed lysin gene.

(19) 2) The ClyO gene was ligated with expression plasmid pBAD24 to obtain recombinant plasmid pB-ClyO, and then this recombinant plasmid was transformed into E. coli strain BL21(DE3).

(20) 3) Expression and Purification of ClyO

(21) Expression strain BL21(DE3)/pB-ClyO was induced by using 0.2% L-arabinose under low temperature. Bacteria were collected, and then broke by sonication. The supernatant was precipitated out by 33% ammonium sulfate, and the precipitates were dissolved in PBS, then dialysed against PBS overnight. The dialysate has obvious anti-bacterial activity.

(22) The crude extract solution after dialysis or the supernatant after sonication was passed through a HiTrap Q Sepharose FF column (GE Healthcare), and the effluent was collected. The effluent was then passed through a HiTrap SP Sepharose FF column, then samples were gradient-eluted by 1M NaCl, and elution peaks were collected in sections. The fractions with activity were combined, and then dialysed against PBS overnight; the resulting sample was the purified enzyme.

EXAMPLE 2

Verification of the Effect of ClyO on Killing Standard S. aureus Strain CCTCC AB91118

(23) S. aureus was cultured overnight and collected by centrifugation, and then washed with PBS once, re-suspended in PBS. A quantity of ClyO was mixed with the above bacteria, at the same time, the change of absorbance at 600 nm was monitored by a microplate reader. The mixtures of buffer and S. aureus bacteria were used as a negative control. The final lysis curve was obtained as shown in FIG. 2. At the same time, the number of viable bacteria treated at different time was counted on plates after serial dilution. This result showed that CCTCC AB91118 strain was rapidly lysed by ClyO, as a result, turbidity of bacteria suspension dropped, which was shown as the absorbance at 600 nm was rapidly declined.

EXAMPLE 3

Verification of the Specificity of ClyO Against Various Staphylococci In Vitro and Other Different Type of Bacteria

(24) To verify the lytic spectrum of ClyO, several non-Staphylococcus strains stored in the lab of the inventors and clinical isolates including S. aureus, S. epidermidis, S. saprophyticus, S. equorum, S. sciuri, S. haemolyticus, S. chromogenes and some other strains were selected and tested. Firstly, every testing strain was cultured overnight, and then collected by centrifugation, and washed with PBS once, then re-suspended in PBS. A quantity of ClyO was mixed with the above bacteria; at the same time, the change of absorbance at 600 nm was monitored for 30 min by a microplate reader. The declining rate of OD.sub.600 was used to indicate the lytic effect of ClyO against different strains. The mixtures of buffer and bacteria suspensions were used as a negative control. The killing effect obtained from the experiment was shown in FIG. 3. This result showed that ClyO was able to rapidly kill various clinically isolated Staphylococci strains, but showed no effect on other types of bacteria.

EXAMPLE 4

The Effect of ClyO on Eliminating of Various Staphylococcal Strains in Milk

(25) Various types of MRSA isolated clinically were cultured overnight, then collected by centrifugation, followed by PBS wash once, and then re-suspended in sterile pasteurized milk. A quantity of ClyO was mixed with the above bacterial suspension and mixed well respectively, the reaction suspension was placed at 37 C. for 60 minutes, and then the number of viable bacteria was calculated by number counting on plates by serial dilution. The killing effect obtained from the experiment was shown in FIG. 4. This result showed that ClyO was able to rapidly kill various Staphylococci in milk.

EXAMPLE 5

The Effect of ClyO on Eliminating Infection Caused by MRSA on Scalded Mice Skin Tissue

(26) Female BALB/c mice (6 weeks old, weighing 20-22 g) were used in the experiment. Backs of experimental mice (60 mice) were scalded by exposure of the naked skin in water at 80 C. for 10 s. Then 110.sup.7 CFU MRSA was inoculated on scalded wounds. After 24 h, mice were divided into three groups with 20 mice in each group. The experimental groups were treated with 100 mg ClyO or sterile PBS respectively, while the control group was left without treatment. The viable bacteria load per unit of the scald skin tissue was calculated after 24 h. The results were shown in FIG. 5. This result showed that ClyO was able to effectively reduce the viable MRSA load on scalded skin tissues.

EXAMPLE 6

Verification of the Effect of ClyO on Killing Staphylococcus In Vivo

(27) Female BALB/c mice (6 weeks old, weighing 20-22 g) were used in the experiment. 40 experimental mice were injected intraperitoneally with 610.sup.7 CFU S. aureus stain WHS11081 isolated clinically. After three hours, mice were divided into 2 groups, and each group had 20 mice. Mice in the experimental group were injected intraperitoneally with 1000 g of ClyO, mice in the control group were injected with PBS. The survival rates were observed every day. The results were shown in FIG. 6. The results showed that ClyO was able to kill MRSA in vivo effectively in mice, thus the survival rates of mice were increased.

EXAMPLE 7

Test of the Cytotoxicity of ClyO

(28) Caco-2 cells were inoculated into 96 well plates at a concentration of 510.sup.3 cells per well. After being cultured for 24 hours, A quantity of concentration of ClyO (0.1-1 mg/ml), Ionomycin (15 mg/ml) and mitomycin (15 mg/ml) were added into wells. Cells were further cultured for 24 hours. A staining regent WST-8 was added into wells after the incubation was finished, and then the absorbance at 450 nm was measured after standing still. The results were shown in FIG. 7. This result showed that there was no cytotoxicity caused by ClyO even at high concentration, but Ionomycin and mitomycin as positive controls showed higher cytotoxicity.

EXAMPLE 8

Use of ClyO for Rapid Detection of Staphylococcus Based on Release of ATP

(29) S. aureus and E. coli were cultured overnight and collected by centrifugation, and then washed with PBS once, and then re-suspended in PBS. A quantity of ClyO was mixed with the above bacterial suspension, at the same time 0.25 M luciferase and 5 M fluorescein substrate containing 12.5 M coenzyme A were added. The reaction mixture was placed at 37 C. after being mixed well, and then the fluorescence intensity of the mixture was detected by a microplate reader. The results were shown in FIG. 8. This result showed that tested staphylococcal cultures were fluoresced at different degree. Obviously, it was caused by the lysis of the cell wall of Staphylococcus using ClyO which leaded to the release of ATP from cells. Luciferase could use the released ATP to catalyze the luminescence of the substrate. However, in E. coli solution being used as the blank control, no luminescence light was detected, because no ATP was released from E. coli that was not able to be lysed by ClyO.

EXAMPLE 9

PCR Identification of the Effect of ClyO on MRSA Strain

(30) Various S. aureus isolated in clinics were cultured overnight and collected by centrifugation, and then washed with PBS once, and then re-suspended in PBS. A quantity of ClyO was mixed with the above bacterial suspension, then lysed at 37 C. for 10-15 min. Lytic solution was then centrifuged at 10,000 g for 1 min. 1 L of supernatant was used as template, and 1 g primers was added respectively for PCR amplification. Primers used to amplify femB gene were: FemB-F (5-TTACAGAGTTAACTGTTACC-3) (SEQ.ID.NO.5) and FemB-R (5-ATACAAATCCAGCACGCTCT-3) (SEQ.ID.NO.6); primers used to amplify mecA gene were MecA-F (5-GTAGAAATGACTGAACGTCCGATAA-3) (SEQ.ID.NO.7) and MecA-R (5-CCAATTCCACATTGTTTCGGTCTAA-3) (SEQ.ID.NO.8). The PCR amplification program was as follows: 1) 94 , 5 min; 2) 94 C., 30 sec, 50 C., 45 sec, 72 C., 60 sec, 30 cycles; 3) 72 C., 10 min. PCR products were analysed by electrophoresis, and the electrophoretic pattern was shown in FIG. 9. The size of femB gene is 651 bp, and the size of mecA gene is 310 bp. MRSA strains contain both genes, and MSSA strains contain only femB. Other types of S. aureus strains only contain mecA. This result showed that it was convenient to identify MRSA strain by PCR by using the lytic supernatants of S. aureus after ClyO treatment.

EXAMPLE 10

Verification of PCR Identification of MRSA Strain Typing by ClyO

(31) MRSA isolated in clinics were cultured overnight and collected by centrifugation, and then washed with PBS once, and then re-suspended in PBS. A certain amount of ClyO was mixed with the above bacterial suspension, and lysed at 37 C. for 10-15 min, and then centrifuged at 10,000 g for 1 min. 1 L of supernatant was used as a template, and 1 g of primers required for typing were added for PCR amplification. The PCR amplification program was follows: 1) 94 C., 5 min; 2) 94 C., 30 sec, 50 C., 30 sec, 72 C., 30 sec, 30 Cycles; 3) 72 C., 10 min. PCR products were analyzed by electrophoresis, and the electrophoretic pattern was shown in FIG. 10. The results showed that the supernatant after ClyO lysis could be conveniently used as the template for PCR analysis of MRSA typing. Different types of MRSA could be well characterized. The 4 pairs of primers used for MRSA typing and their characterizations were as follows:

(32) TABLE-US-00002 Size Target SCCmectype Primer Sequence(5-3) (bp) gene I II III IV V ATTGCCTTGATAATA 937 ccrA2-B X X GCCYTCT (SEQIDNO:9) 3 TAAAGGCATCAATG CACAAACACT (SEQIDNO:10) ccrCF CGTCTATTACAAGAT 518 ccrC X X GTTAAGGATAAT (SEQIDNO:11) ccrCR CCTTTATAGACTGGA TTATTCAAAATAT (SEQIDNO:12) 1272F1 GCCACTCATAACAT 415 IS1272 X X ATGGAA (SEQIDNO:13) 1272R1 CATCCGAGTGAAAC CCAAA (SEQIDNO:14) 5RmecA TATACCAAACCCGA 359 mecA- X CAACTAC IS431 (SEQIDNO:15) 5R413 CGGCTACAGTGATA ACATCC (SEQIDNO:16)

EXAMPLE 11

The Effect of ClyO on Reducing the Number of Staphylococcus after Simulated Skin Disinfection

(33) Different concentrations of S. epidermidis were spread on naked skin of mice, and was dried in the air naturally for test. During test, the experimental group was firstly wiped with PBS solution containing 10 g/ml ClyO, after 10 s-2 min, the skin was wiped with 75% ethanol again. The tampon was put in the liquid medium for Staphylococcus culture after repeating several times, after being cultured overnight, the bacterial culture was diluted for plate counting. For the negative control group, the skin was only wiped with 75% ethanol. The tampon was put in the liquid medium for Staphylococcus culture after repeating several times. After being cultured overnight, the bacterial culture was diluted for plate counting. The resulting statistical graph was shown in FIG. 11. The result showed that ClyO was able to effectively eliminate S. epidermidis on surface of skin.