Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors

Abstract

A protein obtainable from a non pathogenic microorganism, said protein having mucosa binding promoting activity and a molecular weight of 20-40 kD is disclosed. Application of such a protein or a peptide derived therefrom in a method of screening non pathogenic microorganisms for a microorganism capable of specifically binding mucosa, said method comprising detection in a manner known per se. of the presence of a particular protein on or in a microorganism or in a culture of microorganisms, said particular protein being the already defined protein. Kits suitable for such a screening method are also disclosed. Use of a component selected from the group of components comprising a protein or peptide as defined an expression vector comprising nucleic acid encoding such protein or peptide a recombinant. microorganism or a part of said microorganism expressing such protein or peptide, said part expressing mucosa binding promoting activity a non pathogenic microorganism capable of expressing such protein or peptide or a part of said microorganism, said part expressing mucosa binding promoting activity as pharmaceutically active component in a pharmaceutical composition for prophylaxis and/or treatment of disease or illness associated with a mucosa colonising pathogenic microorganism. Use of such components as food additive and compositions comprising such components are described.

1 . An isolated and purified protein or polypeptide having mucosa binding activity and a molecular weight of 20-40 kD as measured by non-gradient denaturing SDS-PAGE, using a calibration curve obtained with standard proteins, and gel filtration chromatography, relative to the standard curve, or a functionally equivalent polypeptide thereof having mucosa binding activity, wherein said protein or polypeptide comprises one or more of the following amino acid sequences:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                     I) 
                   AASAVNSELVHK 
                   (SEQ ID No.21) 
                 
                     
                     
                 
                     
                    II) 
                   ANFVPTK 
                   (SEQ ID No.22) 
                 
                     
                     
                 
                     
                   III) 
                   DTAIQSSYNK 
                   (SEQ ID No.23) 
                 
                     
                     
                 
                     
                    IV) 
                   ISALFNK 
                   (SEQ ID No.24) 
                 
                     
                     
                 
                     
                     V) 
                   IAGTGTNNA, 
                   (SEQ ID No.25) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and wherein said mucosa binding activity comprises binding to a receptor-which is present on the mucosa and is recognized by a pathogenic microorganism:  
     
     
         2 . A protein or polypeptide according to  claim 1 , wherein said mucosa binding activity is equal to or exceeds that of  L. fermenltur  104R 29 kD adherence factor with the amino acid sequence of SEQ ID No. 2.  
     
     
         3 . A protein or polypeptide according to  claim 1 , wherein said protein or polypeptide comprises SEQ ID No. 6.  
     
     
         4 . A protein or polypeptide according to  claim 1 , wherein said protein or polypeptide comprises SEQ ID No. 13.  
     
     
         5 . A protein or polypeptide according to  claim 1 , wherein said protein or polypeptide comprises SEQ ID No. 6 and SEQ ID No. 13.  
     
     
         6 . A protein or polypeptide according to  claim 1 , wherein the protein or polypeptide comprises one or more of the following amino acid sequences:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                    II) 
                   ANFVPTK 
                   (SEQ ID No.22) 
                 
                     
                     
                 
                     
                   III) 
                   DTAIQSSYNK 
                   (SEQ ID No.23) 
                 
                     
                     
                 
                     
                    IV) 
                   ISALFNK 
                   (SEQ ID No.24) 
                 
                     
                     
                 
                     
                     V) 
                   IAGTGTNNA. 
                   (SEQ ID No.25) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . A protein or polypeptide according to  claim 1 , wherein said protein or polypeptide comprises two structurally identifiable domains.  
     
     
         8 . A protein or polypeptide according to  claim 1 , wherein said protein or polypeptide comprises the amino acid sequence of L. fermentum 104R adherence factor of 29 kD of SEQ ID No. 2.  
     
     
         9 . A protein or polypeptide according to  claim 1 , obtainable by binding to an antibody either polyclonal or monoclonal raised against the protein or polypeptide with the amino acid sequence of  L. fermentum  104R adherence factor of 29 kD of SEQ ID No. 2.  
     
     
         10 . A polypeptide fragment of a protein or polypeptide according to  claim 1 , said fragment having mucosa binding activity, having a length of less than 60 amino acids and comprising one or more of the following amino acid sequences:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                     I) 
                   AASAVNSELVHK 
                   (SEQ ID No.21) 
                 
                     
                     
                 
                     
                    II) 
                   ANFVPTK 
                   (SEQ ID No.22) 
                 
                     
                     
                 
                     
                   III) 
                   DTAIQSSYNK 
                   (SEQ ID No.23) 
                 
                     
                     
                 
                     
                    IV) 
                   ISALENK 
                   (SEQ ID No.24) 
                 
                     
                     
                 
                     
                     V) 
                   IAGTGTNNA. 
                   (SEQ ID No.25) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . A polypeptide fragment according to  claim 1 , saids fragment having a length of less than 40 amino acids.  
     
     
         12 . A recombinant protein or polypeptide comprising the amino acid sequence of a protein or polypeptide according to  claim 1 .  
     
     
         13 . A recombinant polypeptide fragment comprising the amino acid sequence of a polypeptide fragment according to  claim 10 .  
     
     
         14 . A fusion protein or polypeptide comprising a protein or polypeptide having mucosa binding activity according to  claim 1  attached to a drug, an immunomodulator or antigen.  
     
     
         15 . A fusion polypeptide fragment comprising a polypeptide fragment having mucosa binding activity according to  claim 10  attached to a drug, an immunomodulator or antigen.  
     
     
         16 . A composition comprising a protein or polypeptide having mucosa binding activity according to  claim 1  as a pharmaceutically active component and a pharmaceutically acceptable carrier in a pharmaceutically acceptable dosage form.  
     
     
         17 . A composition comprising a polypeptide fragment having mucosa binding activity according to  claim 10  as a pharmaceutically active component and a pharmaceutically acceptable carrier in a pharmaceutically acceptable dosage form.  
     
     
         18 . Food product comprising a protein or polypeptide having mucosa binding activity according to  claim 1  as an additive.  
     
     
         19 . Food product comprising a polypeptide fraqment having mucosa binding activity according to  claim 10  as an additive.  
     
     
         20 . A method of screening non-pathogenic microorganisms for a microorganism capable of specifically binding mucosa, said method comprising detection of the presence of a particular protein or polypeptide on or in a microorganism or in a culture of said microorganism, said particular protein or polypeptide being a protein or polypeptide according to  claim 1.