Recombinant microorganisms expressing an oligosaccharide receptor mimic

Abstract

Chimeric carbohydrates produced by recombinant microorganism carrying exogenous glycosyl transferases act with or without exogenous enzymes required for synthesis or nucleotide synthesis precursors. These recombinant microorganism can be used as a means for competitively inhibiting the binding of toxins or adhesins to receptors of mucosal surfaces, especially gastrointestinal surface. In particular chimeric sugar moieties have been made for lipopolysaccharides, in recombinant microorganism that present multiple copies of the oligosaccharides. The oligosacchide moieties so presented act as receptor mimic for toxins and adhesins. A number have been synthesise and have been shown to confer protection against attack by pathogenic organisms or their products in vitro and an in vivo.

1 - 116 . (canceled)  
     
     
         117 . A method of preventing or treating a pathogenic microorganism infection of the gastrointestinal tract, the method including the step of administering to the gastrointestinal tract an effective amount of a recombinant bacterium that displays on its surface a binding moiety that act as a receptor mimic, or a fraction of said recombinant bacterium comprising said binding moiety, the binding moiety being a receptor mimic of a receptor for a toxin of the pathogenic microorganism or an adhesin of the pathogenic microorganism, wherein the binding moiety consists of an oligosaccharide which comprises a sugar residue that is attached to an acceptor moiety by a glycosyltransferease that is encoded by an exogenous nucleic acid which is present in the bacterium said oligosaccharide forming part of a lipopolysaccharide molecule, the recombinant bacterium selected from the group of species consisting of  Escherichia coli  and  Salmonella enterica    
     
     
         118 . The method of  claim 117 , wherein the receptor mimic is a mimic of the receptor of a toxin.  
     
     
         119 . The method of  claim 118 , wherein the toxin is selected from the group consisting of shiga toxins, clostridial toxins, cholera toxins,  E. coli  enterotoxins, and staphylococcal enterotoxins.  
     
     
         120 . The method of  claim 119 , wherein the toxin is selected from the group consisting of cholera toxin,  E. coli  heat labile enterotoxin types I and II, and ST toxins.  
     
     
         121 . The method of  claim 119 , wherein the toxin is a shiga toxin.  
     
     
         122 . The method of  claim 121 , wherein the shiga toxin is selected from the group consisting of, Stx, Stx1, Stx2, Stx2c, Stx2d, and Stx2e.  
     
     
         123 . The method of  claim 119 , wherein the toxin is a clostridial toxin.  
     
     
         124 . The method of  claim 117 , wherein the receptor mimic is partially or wholly formed within a sugar moiety of selected from the group consisting of: 
 Galα[1→4]Galβ[1→4]Glc,    Galα[1→4]Galβ,    GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc,    Galβ[1→4]GlcNAc,    Galα[1→3]Galβ[1→4]Glc,    Galα[1→3]Galβ[143 4]GlcNAc,    Galβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc,    Glcα[1→6]Glc,    Glcα[1→6]Glcα[1→6]Glc,    NeuNAc,    Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc, 
 I  
 NeuNAcα[2→3] 
   Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc,    GalNAcβ[1→4]Gal,    GalNAc,    Gal,    NeuGc→GM 3 , and    NeuNAc→-GM 3 .    
     
     
         125 . The method of  claim 117 , wherein the receptor mimic of the purified carbohydrate is partially or wholly formed within a sugar moiety selected from the group consisting of 
 Galα[1→4]Galβ[1→4]Glc,    GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc, and    Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc.    
     
     
         126 . The method of  claim 117 , wherein the binding moiety comprises Galα[1→4]Galα[1→4]Glc.  
     
     
         127 . The method of  claim 117 , wherein the binding moiety comprises GalNAcβ[→3]Galα[1→4]Galβ[1→4]Glc.  
     
     
         128 . The method of  claim 117 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of: Galβ[1→4]GlcNAc, Galβ[1→4]GalNAcα[1→3]Galβ[1→4]Glc, Galα[1→3]Galβ[1→4]GalNAc and Galα[1→3]Galβ[1→4]Glc.  
     
     
         129 . The method of  claim 117 , wherein the binding moiety comprises NeuNAc.  
     
     
         130 . The method of  claim 117 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of Glcα[1→6]Glc and Glcα[1→6]Glcα[1→6]Glc.  
     
     
         131 . The method of  claim 117 , wherein the binding moiety comprises the oligosaccharide: 
 Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc. 
 I  
 NeuNAcα[2→3] 
   
     
     
         132 . The method of  claim 117 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of NeuGc→GM 3  and NeuNAc→GM 3 .  
     
     
         133 . The method of  claim 117 , wherein the binding moiety is a mimic of natural receptor for adhesins or toxins produced by a microorganism selected from a group of genera consisting of  Escherichia, Salmonella, Shigella, Citrobacter, Helicobacter, Yersinia, Vibrio, Aeromonas, Campylobacter, Pseudomonas, Pasteurella, Neisseria, Haemophilus, Klebsiella, Staphylococcus, Streptococcus, Clostridium, rotavirus,  and  Entamoeba.    
     
     
         134 . The method of  claim 117 , wherein the microorganism further comprises one or more exogenous enzymes involved in synthesis of a nucleotide sugar which serves as a donor for the glycosyltransferase.  
     
     
         135 . The method of  claim 134 , wherein the nucleotide sugar is selected from the group consisting of GDP-Man, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, CMP-sialic acid, GDP-Fuc, and UDP-xylose.  
     
     
         136 . The method of  claim 134 , wherein the enzyme is a nucleotide sugar synthetase.  
     
     
         137 . The method of  claim 134 , wherein the enzyme is involved in synthesis of a nucleotide that comprises the nucleotide sugar.  
     
     
         138 . The method of  claim 134 , wherein the enzyme is involved in synthesis of a sugar that comprises the nucleotide sugar.  
     
     
         139 . The method of  claim 134 , wherein the one or more sugars transferred to the acceptor molecule by the exogenous glycosyltransferases make up the entirety of the receptor mimic.  
     
     
         140 . The method of in  claim 117 , wherein a combination of sugars of the acceptor molecule and the one or more sugars transferred to the acceptor molecule by the exogenous transferases make up the entirety of the receptor mimic.  
     
     
         141 . The method of  claim 140 , wherein the acceptor molecule is all or a portion of the core of the lipopolysaccharide.  
     
     
         142 . The method of  claim 117 , wherein one or more exogenous nucleotide sugar precursor synthesizing enzymes are also expressed by said organism, said sugar precursor enzymes forming precursors to make up said chimeric carbohydrate.  
     
     
         143 . The method of  claim 117 , wherein the delivery microorganism is non harmful and live.  
     
     
         144 . The method of  claim 117 , wherein the administration is orally.  
     
     
         145 . The method of  claim 144 , wherein the delivery microorganism is protected by a protective capsule or held within a protective matrix.  
     
     
         146 . The method of  claim 117 , wherein the delivery microorganism is killed.  
     
     
         147 . The method of  claim 146 , wherein the delivery microorganism is killed by treatment with a chemical agent selected from the group consisting of formalin, or thiomersal, or a bactericidal antibiotic, or by exposure to heat or to UV irradiation.  
     
     
         148 . A method of testing for the presence of a toxin or a pathogenic microorganism in a sample, the method comprising: 
 contacting a sample with a recombinant bacterium that displays on its surface a binding moiety that act as a receptor mimic, or a fraction of said recombinant bacterium comprising said binding moiety,    either the recombinant microorganism or fraction thereof, or the sample being immobilized;    washing off unbound purified carbohydrate or toxin or pathogenic microorganism; and adding detection means to detect bound purified carbohydrate and the toxin or pathogenic microorganism,    the binding moiety of the recombinant microorganism being a receptor mimic of a receptor for the toxin or an adhesin or toxin of the pathogenic microorganism, wherein the binding moiety consists of an oligosaccharide which comprises a sugar residue that is attached to an acceptor moiety by a glycosyltransferease that is encoded by an exogenous nucleic acid which is present in the bacterium said oligosaccharide forming part of a lipopolysaccharide molecule, the recombinant bacterium selected from the group of species consisting of  Escherichia coli  and  Salmonella enterica.      
     
     
         149 . The method of testing as in  claim 148 , wherein the recombinant bacterium or fraction thereof is immobilised on the support.  
     
     
         150 . The method of testing as in  claim 148 , wherein the receptor mimic is a mimic of the receptor of a toxin.  
     
     
         151 . The method of testing as in  claim 150 , wherein the toxin is selected from the group consisting of shiga toxins, clostridial toxins, cholera toxins,  E. coli  enterotoxins, and staphylococcal enterotoxins.  
     
     
         152 . The method of testing as in  claim 150 , wherein the toxin is a shiga toxin.  
     
     
         153 . The method of testing as in  claim 150 , wherein the toxin is a clostridial toxin.  
     
     
         154 . The method of testing as in  claim 148 , wherein the receptor mimic is partially or wholly formed within a sugar moiety selected from the group consisting of 
 Galα[1→4]Galβ[1→4]Glc,    Galα[1→4]Galβ,    GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc,    Galβ[1→4]GlcNAc,    Galα[1→3]Galβ[1→4]Glc,    Galα[1→3]Galβ[1→4]GlcNAc,    Galβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc,    Glcα[1→6]Glc,    Glcα[1→6]Glcα[1→6]Glc,    NeuNAc,    Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc, 
 I  
 NeuNAcα[2→3] 
   Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc,    GalNAcβ[1→4]Gal,    GalNAc,    Gal,    NeuGc→GM 3 , and    NeuNAc→GM 3 .    
     
     
         155 . The method of testing as in  claim 148 , wherein the receptor mimic of the purified carbohydrate is partially or wholly formed within a sugar moiety selected from the group consisting of 
 Galα[1→4]Galβ[1→4]Glc,    GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc; and    Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc.    
     
     
         156 . The method of testing as in  claim 148 , wherein the binding moiety comprises Galα[1→4]Galβ[1→4]Glc.  
     
     
         157 . The method of testing as in  claim 148 , wherein the binding moiety comprises GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc.  
     
     
         158 . The method of testing as in  claim 148 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of: Galβ[1→4]GlcNAc, Galβ[1→4]GalNAcβ[1→3]Galβ[1→4]Glc, Galα[1→3]Galβ[1→4]GalNAc and Galα[1→3]Galβ[1→4]Glc.  
     
     
         159 . The method of testing as in  claim 148 , wherein the binding moiety comprises NeuNAc.  
     
     
         160 . The method of testing as in  claim 148 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of Glcα[1→6]Glc and Glcα[1→6]Glcα[1→6]Glc.  
     
     
         161 . The method of testing as in  claim 148 , wherein the binding moiety comprises the oligosaccharide: 
 Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc. 
 I  
 NeuNAcα[2→3] 
   
     
     
         162 . The method of testing as in  claim 148 , wherein the binding moiety comprises an oligosaccharide selected from the group consisting of NeuGc→GM 3  and NeuNAc→GM 3 .