Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-02
2003-05-20
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S029000
Reexamination Certificate
active
06566062
ABSTRACT:
The present invention relates to a method for identifying a nucleic acid which codes for a polypeptide factor affecting the covalent bonding of polypeptides to the surface of Gram-positive bacteria, and to the nucleic acids obtainable by said method.
In view of the increased occurrence of antibiotic-resistant strains, infections in humans caused by Gram-positive bacteria are an increasing therapeutic challenge. The pathogenesis of these organisms is associated with a wide variety of bacterial surface proteins. Thus, pathogenicity factors anchored to the cell wall are known which promote bacterial adhesion by the binding to extracellular matrix components of the host tissues, such as collagen. Other factors bind serum components, such as IgG, and thus conceal the authentic bacterial surface from the host's immune system. Therefore, selective inhibition of the binding reaction of these proteins to the bacterial cell wall is of great medical interest.
Schneewind et al. (Cell, Vol. 70, p. 267-281, 1992) have studied the anchoring mechanism of protein A in the cell wall of Staphylococci. Protein A belongs to a growing class of surface proteins of Gram-positive bacteria which are characterized by a succession of the characteristic sequence motif LPXTG, followed by a group of 15-22 hydrophobic amino acids, and a C-terminal group of 5-12 charged amino acids. The conservation of these elements is considered an indication of a common export mechanism of these proteins in different Gram-positive species. In order to establish the localization of protein A (discrimination between protein A anchored in the cell wall and secreted protein A) in
S. aureus,
the authors employ radioactive labeling methods. The importance of the above mentioned sequence elements to cell wall anchoring is supported by using hybrid proteins and through mutagenesis of the LPXTG motif and of the C terminus. However, Schneewind et al. are concerned neither with enzymes which might catalyze the anchoring of the surface proteins, nor with their inhibition.
The cell wall anchoring elements in surface proteins of Gram-positive bacteria are also the subject of another article by Schneewind et al. (EMBO J., Vol. 12, p. 4803-4811, 1993). It is shown that enterotoxin B, a protein normally secreted into the medium, can be anchored in the Staphylococcus cell wall through C-terminal fusion to the protein A anchoring signal. The results support the hypothesis that the cell wall sorting is accompanied by a proteolytic cleavage of the polypeptide chain at the C terminus. Presumably, the LPXTG motif is the site of such cleavage and covalent binding to the cell wall while the charged sequence segment serves as a retention signal during the cell wall sorting. The relevance of the geometrical length of the hydrophobic domain, which is dependent on the folding properties, is confirmed by experiments.
An article by Samuelson et al. (J. Bacteriol., Vol. 177, No. 6, p. 1470-1476, 1995) is concerned with the cellular surface display of recombinant proteins on
Staphylococcus carnosus.
The surface display of the malaria peptide M3 is effected using the promoter, secretion signal and propeptide region of the lipase gene of
S. hyicus
and the cell wall anchoring regions of protein A of
S. aureus.
The hybrid protein structure further includes a serum albumin binding protein which serves for the detection of the recombinant surface-anchored proteins in a colorimetric sandwich assay. Further detection methods comprise immunogold electron microscopy, immunofluorescence assays and fluorescence-activated cell sorting (FACS). Samuelson et al. are not concerned with the exact molecular mechanisms of cell wall anchoring either.
The structure of the cell wall anchor of the surface proteins in
Staphylococcus aureus
is the subject of a report by Schneewind et al. (Science, Vol. 268, p. 103-106, 1995). The authors use a combination of molecular-biological and mass-spectrometric techniques and are able to show that after cleavage of the surface protein between threonine and glycine of the conserved LPXTG motif, the carboxy group of threonine is covalently bonded via transpeptidization to the murein sacculus with the free amino group of the cell wall pentaglycine. However, Schneewind et al. also fail to identify or characterize the protein believed to be responsible for proteolysis and transpeptidization, the so-called sortase.
Strau&bgr;and Götz (Molecular Microbiology, Vol. 21, p. 491-500, 1996) are concerned with the in vivo immobilization of enzymatically active polypeptides on the cellular surface of
Staphylococcus carnosus.
They have constructed a hybrid protein which consists of
Staphylococcus hyicus
lipase and the C-terminal region of
Staphylococcus aureus
fibronectin binding protein B (FnBPB). To study the cell wall association of the prolipase, or the proLipFnBPB hybrid, the authors use a prolipase-specific antiserum in an immunofluorescence assay and immunoblotting. Further examinations have demonstrated that a distance of about 90 amino acids between the C terminus of the enzyme and the cell wall sorting signal is evidently indispensable to an efficient folding of the lipase into its active conformation. The influence of greater distances has been examined on fusions of proLip and the C-terminal region of
S. aureus
protein A (proLipSPA, spacer with 165 amino acids) and
S. aureus
fibronectin binding protein A (proLipFnBPA, spacer with 223 amino acids). Additional experiments were performed with
E. coli
&bgr;-lactamase as the reporter molecule.
International Patent Application PCT/US 96/14154 (International Publication No. WO 97/08553) describes a method for the stable non-covalent display of proteins, peptides and other substances on the surface of Gram-positive bacteria. Comparative studies between the non-covalent display process and the covalent display process, which has been described in more detail above, were performed. When the C-terminal sorting signal of protein A, which results in covalent display, was replaced by the cell wall targeting signal of lysostaphin (SPA
CWT
), an essentially unchanged binding intensity of FITC-labeled IgG to the Staphylococcus surface could be observed.
U.S. Pat. No. 5,616,686 discloses a polypeptide consisting of about 6 to 20 amino acids which contains as an integral part a peptide construct which is responsible for the anchoring of virulence-determining proteins on the surface of Gram-positive bacteria. In particular, this construct is characterized by containing the amino acids L, P, T and G at positions 1, 2, 4 and 5, respectively, of the amino acid sequence. Due to the homology of these peptides with the sequences of the virulence determinants in the wild type surface proteins, the former presumably react with enzymes involved in anchoring. The result is that the virulence determinants of the bacteria cannot be anchored or can be anchored only to a lesser extent, and thus the progress of the infection is prevented. However, the enzyme or enzymes involved in surface anchoring are not characterized in this patent specification either.
International Patent Application PCT/US 93/02355 (International Publication No. WO 93/18163) is concerned with the provision of fusion proteins which contain at least the anchoring region of Gram-positive surface proteins as well as varying proteins, polypeptides or peptides, especially those having a therapeutic effect in humans and animals. The anchoring region comprises an LPSTGE segment, a spacer segment of the sequence TAN, a hydrophobic segment consisting of 20 amino acids, and a charged segment with the sequence KRKEEN.
Therefore, it is desirable to provide a method which allows the identification of nucleic acids in Gram-positive bacteria which code for those polypeptide factors which directly or indirectly affect the covalent bonding of polypeptides to the surface of the Gram-positive bacteria.
Surprisingly, this object is achieved by the method of the present invention.
“Polypeptides” within the meaning of the invention means polymers usu
Götz Friedrich
Pohlner Johannes
Strauss Andreas
Thumm Gunther
Evotec BioSystems AG
Katcheves Konstantina
Yucel Remy
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