Proteins for use in human and animal Staphyococcus infections
The present invention relates to a polypeptide termed ply_pitti26 comprising the sequence as depicted in SEQ ID NO:1 as well as variants of this polypeptide. Furthermore, the present invention relates to nucleic acids and vectors encoding for said polypeptide and variants thereof as well as host cells comprising these nucleic acids and/or vectors. Finally, the present invention relates to the uses of said polypeptide, variants thereof, nucleic acid sequences, vectors and host cells, in particular for the treatment or prophylaxis of a subject infected by or exposed to Staphylococci.
1. A method for diagnosing staphylococcal contamination of medicine, food, feedstock or an environment, said method comprising:
i) a contacting a medicine, food, feedstock or environmental sample with a polypeptide comprising the sequence as depicted in SEQ ID NO:1 or variant thereof, said variant being:
a) a polypeptide comprising a sequence in which the CBD of SEQ ID NO:1 has been replaced by a CBD domain of another Staphylococci specific endolysin; or
b) a polypeptide comprising the sequence as depicted in SEQ ID NO:1 but lacking 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 N-terminal amino acid residues of SEQ ID NO:1; or
c) a polypeptide comprising the sequence of SEQ ID NO:1 except for at least the first N-terminal amino acid and at most the first 28 N-terminal amino acids of SEQ ID NO:1 and wherein the sequence as depicted in SEQ ID NO:1 exhibits single or multiple substitutions with regrard to SEQ ID NO:1, wherein
i) the substituted residues are selected from the following amino acid residues of SEQ ID NO:1: F19, W22, W36, F42, Y44, L55, L56, F67, L74, Y78, W107, Y115, I116, Y119, W123, W128, W137, W139, W154, E163, R167, E179, E189, Y200, Y275, Y276, C282, F300 and C303, or
ii) one or more substitutions are selected from the group comprising: W22R, F42A, Y44A, F67T, Y1155, W123M, W137A, W139A, W154H, E163A, E179Q, E179A, E187Q, Y200A, Y200H, Y275A, Y275M, Y276A, C282A, F300A, C3035, W310A and W310M, or
iii) one or more substitutions are selected from the group comprising: F67T+Y1155, F67T+W137A, F67T+W139A, F67T+W154H, Y1155+W137A, Y1155+W139A, E163Q+R169A, E163A+R169A, E163Q+R167A+E189Q, E163A+R167A+E189Q, E163Q+R167A+E179Q+E189Q, E163Q+R167A+E179A+E189Q, E163A+R167A+E179Q+E189Q, E163A+R167A+E179A+E189Q, Y200A+Y275A, Y200A+Y276A, Y200A+C282A, Y200A+F300A, Y275A+Y276A, L55H+L56T+E163A+R167A+Y200H, E163A+R167A+E179A+E189Q+Y200H, L55H+L56T+E163A+R167A+E179A+E189Q+Y200H, S237L+R354Q+A367V, and L55H+L56T+E163A+R167A+Y200H+S237L+R354Q+A367V; or
d) a polypeptide comprising one or more amino acid substitutions in the sequence of SEQ ID NO:1, wherein
i) the substituted residues are selected from the following amino acid residues of SEQ ID NO:1: F19, W22, W36, F42, Y44, L55, L56, F67, L74, Y78, W107, Y115, I116, Y119, W123, W128, W137, W139, W154, E163, R167, E179, E189, Y200, Y275, Y276, C282, F300 and C303, or
ii) one or more substitutions are selected from the group comprising: W22R, F42A, Y44A, F67T, Y115S, W123M, W137A, W139A, W154H, E163A, E179Q, E179A, E187Q, Y200A, Y200H, Y275A, Y275M, Y276A, C282A, F300A, C303S, W310A and W310M, or
iii) one or more substitutions are selected from the group comprising:
F67T+Y115S, F67T+W137A, F67T+W139A, F67T+W154H, Y115S+W137A, Y115S+W139A, E163Q+R169A, E163A+R169A, E163Q+R167A+E189Q, E163A+R167A+E189Q, E163Q+R167A+E179Q+E189Q, E163Q+R167A+E179A+E189Q, E163A+R167A+E179Q+E189Q, E163A+R167A+E179A+E189Q, Y200A+Y275A, Y200A+Y276A, Y200A+C282A, Y200A+F300A, Y275A+Y276A, L55H+L56T+E163A+R167A+Y200H, E163A+R167A+E179A+E189Q+Y200H, L55H+L56T+E163A+R167A+E179A+E189Q+Y200H, S237L+R354Q+A367V, and L55H+L56T+E163A+R167A+Y200H+S237L+R354Q+A367V; or
e) a polypeptide comprising in addition to the sequence of SEQ ID NO: 1 sequences representing marker moieties, tags or other functional polypeptide sequences; or
f) a polypeptide comprising one or more additional amino acid residues inserted into the amino acid sequence according to SEQ ID NO:1; or
g) a polypeptide comprising a polypeptide sequence representing any combination of variants a), b), c), d), e) and f),
wherein said polypeptide or variant thereof lyses Staphylococcus if present in said medicine, food, feedstock or an environment, and
ii) specifically detecting Staphylococcus bacteria using nucleic acid based methods.
2. A diagnostic kit comprising a polypeptide comprising the sequence as depicted in SEQ ID NO:1 or variant thereof, said variant being:
a) a polypeptide comprising a sequence in which the CBD of SEQ ID NO:1 has been replaced by a CBD domain of another Staphylococci specific endolysin; or
b) a polypeptide comprising the sequence as depicted in SEQ ID NO:1 but lacking 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 N-terminal amino acid residues of SEQ ID NO:1; or
c) a polypeptide comprising the sequence of SEQ ID NO:1 except for at least the first N-terminal amino acid and at most the first 28 N-terminal amino acids of SEQ ID NO:1 and wherein the sequence as depicted in SEQ ID NO:1 exhibits single or multiple substitutions with regrard to SEQ ID NO:1, wherein
i) the substituted residues are selected from the following amino acid residues of SEQ ID NO:1: F19, W22, W36, F42, Y44, L55, L56, F67, L74, Y78, W107, Y115, I116, Y119, W123, W128, W137, W139, W154, E163, R167, E179, E189, Y200, Y275, Y276, C282, F300 and C303, or
ii) one or more substitutions are selected from the group comprising: W22R, F42A, Y44A, F67T, Y115S, W123M, W137A, W139A, W154H, E163A, E179Q, E179A, E187Q, Y200A, Y20014, Y275A, Y275M, Y276A, C282A, F300A, C303S, W310A and W310M, or
iii) one or more substitutions are selected from the group comprising: F67T+Y115S, F67T+W137A, F67T+W139A, F67T+W154H, Y115S W137A, Y115S+W139A, E163Q+R169A, E163A+R169A, E163Q+R167A+E189Q, E163A+R167A E189Q, E163Q+R167A+E179Q+E189Q, E163Q+R167A+E179A+E189Q, E163A+R167A+E179Q E189Q, E163A+R167A+E179A+E189Q, Y200A+Y275A, Y200A+Y276A, Y200A+C282A, Y200A+E300A, Y275A+Y276A, L551-1+L56T+E163A+R167A+Y200H, E163A+R167A+E179A+E189Q+Y200H, L55H+L56T+E163A+R167A+E179A+E189Q Y200H, S237L+R354Q+A367V, and L55H+L56T+E163A+R167A+Y200H+S237L+R354Q+A367V; or
d) a polypeptide comprising one or more amino acid substitutions in the sequence of SEQ ID NO:1, wherein
i) the substituted residues are selected from the following amino acid residues of SEQ ID NO:1: F19, W22, W36, F42, Y44, L55, L56, F67, L74, Y78, W107, Y115, I116, Y119, W123, W128, W137, W139, W154, E163, R167, E179, E189, Y200, Y275, Y276, C282, F300 and C303, or
ii) wherein one or more substitutions are selected from the group comprising: W22R, F42A, Y44A, F67T, Y115S, W123M, W137A, W139A, W154, E163A, E179Q, E179A, E187Q, Y200A, Y200H, Y275A, Y275M, Y276A, C282A, F300A, C303S, W310A and W310M, or
iii) one or more substitutions are selected from the group comprising: F67T+Y115S, F67T+W137A, E67T+W139A, F67T+W154H, Y115S+W137A, Y115S+W139A, E163Q+R169A, E163A+R169A, E163Q+R167A+E189Q, E163A+R167A+E189Q, E163Q+R167A+E179Q+E189Q, E163Q R167A+E179A+E189Q, E163A+R167A+E179Q+E189Q, E163A+R167A+E179A+E189Q, Y200A+Y275A, Y200A+Y276A, Y200A+C282A, Y200A+F300A, Y275A+Y276A, 155H+L56T+E163A+R167A+Y200H, E163A+R167A+E179A+E189Q+Y20011, L5511+L56T+E163A+R167A+E179A+E189Q+Y200H, S237L R354Q+A367V, and L55H+L56T+E163A+R167A+Y200H+S237L+R354Q+A367V; or
e) a polypeptide comprising in addition to the sequence of SEQ ID NO: 1 sequences representing marker moieties, tags or other functional polypeptide sequences; or
f) a polypeptide comprising one or more additional amino acid residues inserted into the amino acid sequence according to SEQ ID NO: 1; or
g) a polypeptide comprising a polypeptide sequence representing any combination of variants a), b), c), d), e) and f).
3. The kit according to claim 2 , wherein the polypeptide lyses S. aureus, S. aureus (MRSA), S. epidermidis, S. haemolyticus, S. simulans, S. saprophyticus, S. chromogenes, S. hyicus, S. warneri and/or S. xylosus.
4. The kit according to claim 2 , wherein the variant of the polypeptide comprises the sequence as depicted in SEQ ID NO:1 comprises an endolysin cell binding domain of the SH3 type.
5. The kit according to claim 2 , wherein the variant of the polypeptide comprises the sequence as depicted in SEQ ID NO: 1 comprises a CBD domain selected from the endolysin CBD domains of ply_USA or ply_pitti20.
6. The kit according to claim 5 , wherein the variant of the polypeptide comprises the sequence as depicted in SEQ ID NO:1 comprises a CBD domain as denoted in SEQ ID NOs: 3 or 5.
7. The kit according to claim 6 , wherein the polypeptide comprises the sequence as denoted in SEQ ID NOs: 7 or 11.
8. The kit according to claim 2 c)i), wherein replacements of amino acids residues F, W, Y, I, L are exchanged for amino acids residues R, D, F, N, K, Q, H, S, T, M, G or A.
9. The kit according to claim 2 , wherein the polypeptide comprises biotin or Streptavidin as additional marker moiety.
10. The kit according to claim 2 , wherein the polypeptide further comprises a HA-tag, His-tag, Strep-tag, Myc-tag or GST-tag.
11. The kit according to claim 10 , wherein the polypeptide comprises the sequence as denoted in in SEQ ID NO: 9 or SEQ ID NO: 13.
12. The kit according to claim 2 , in part d)i), wherein replacements of amino acids residues F, W, Y, I, L are exchanged for amino acids residues R, D, E, N, Q, H, S, T, M, G or A.