Attaching substances to microorganisms

Abstract

The invention relates to surface display of proteins on microorganisms via the targeting and anchoring of heterologous proteins to the outer surface of cells such as yeast, fungi, mammalian, plant cells, and bacteria. The invention provides a proteinaceous substance comprising a reactive group and at least one attaching peptide including a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence listed in FIG. 10 and further includes a method for attaching a proteinaceous substance to the cell wall of a microorganism comprising the use of the attaching peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a divisional of U.S. Ser. No. 09 / 554,354, filed May 12, 2000, which is a national entry of PCT International Patent Application No. PCT / NL98 / 00655, filed on Nov. 12, 1998, (corresponding to PCT International Publication No. WO 99 / 25836, the contents of which is incorporated by this reference as is the sequence listing in U.S. Ser. No. 09 / 554,354).TECHNICAL FIELD [0002] The invention relates to surface display of proteins on microorganisms via the targeting and anchoring of heterologous proteins to the outer surface of cells such as yeast, fungi, mammalian, plant cells, and bacteria. BACKGROUND [0003] Heterologous surface display of proteins (Stahl and Uhlen, TIETECH May 1997, 15, 185-192) on recombinant microorganisms via the targeting and anchoring of heterologous proteins to the outer surface of host-cells such as yeast, fungi, mammalian and plant cells, and bacteria has been possible for several years. Display of h...

AI Technical Summary

Benefits of technology

[0019] Anchoring of recombinant proteins to non-recombinant microorganisms such as lactococci or other, bacteria or fungi, is especially attractive if the use of recombinant bacteria is not desired, e.g., in food processes or as pharmaceuticals for medical use such as in vaccines or in antibacterial therapy. The invention provides, for example, vaccine delivery or immunization via microorganisms which are labeled with distinct antigenic determinants and which may be directed against a wide variety of diseases. A protective immune response can, for example, be elicited by oral delivery of a bacterial vaccine provided by the invention wherein tetanus toxin fragment C is attached via a protein provided by the invention to a non-recombinant Lactococcus lactis. Such immunogens expressed by microorganisms provided by the invention are presented in particulate form to the antigen-presenting cells (for example M-cells) of the immune system and are, therefore, less likely to induce tolerance than soluble antigens. In addition, the existence of a common mucosal immune system permits immunization on one specific mucosal surface to induce secretion of antigen-specific IgA and other specific immune responses at distant mucosal sites. The invention solves the drawback of earlier bacterial vaccines whereby the potential to flourish on mucosal surfaces of the attenuated or recombinant bacterial strain used can cause problems such as inflammation and disease in itself, potentially leading to fever and bacteraemia or to the induction of immune tolerance. Also, the invention avoids the potential risks that are involved when using recombinant DNA-containing bacterial vectors for vaccination. In yet another possible vaccine and vaccine use provided by the invention, certain (killed) micro-organisms with adjuvant properties (such as the mycobacteria used in BCG) are labeled or loaded with a protein or substance composed of an antigenic determinant and an attaching peptide. These microorganisms then function as adjuvant, thereby greatly enhancing the immune response directed against the specific antigenic determinant. Yet another use provided by the invention comprises anchoring proteins from the outside to a microorganism which provides a means to present proteins or peptides which normally cannot be overexpressed and / or secreted by the microorganism. For example, the subunit B of cholera toxin (CTB) can be overproduced in E. coli but expression in L. lactis has been unsuccessful until now. The adjuvant activity of CTB in experimental recombinant vaccines is well documented and the ability of CTB or part thereof to bind to GM1 ganglioside on eucaryotic cell surfaces is of interest with respect to the use of L. lactis or other gram-positives in vaccines which specifically require targeting to mucosal surfaces. Yet another medical use provided by the invention is the addition of purified antigen::cA fusion proteins in vivo by parenteral administration into the bloodstream of humans or animals to combat bacterial infections. In this case, the antigen::cA fusion protein is used as a “flag” for the immune system. The antigenic determinant of a protein provided by the invention being a subunit of a vaccine regularly used for the immunization of humans (preferably children) or animals, e.g. a subunit of the Rubella, Pertusis, Poliomyelitis, tetanus or measles vaccine. After delivery into the bloodstream, the “flag” will bind through the AcmA repeats to the pathogenic bacterium present in the blood. A “flag” protein provided by the invention will then activate a memory response, i.e., the response to the antigenic determinant present in the protein. The antibodies thus produced recognise the “flag”-labeled bacteria, which will then be neutralised by the immune system. In this way, the protein is used to stimulate a pre-existing memory immune response, non-related to the bacterial infection, to clear bacterial infections from the system. Yet another use (which alternatively may be considered medical use or food use) provided by the invention is the use wherein a protein provided by the invention has the ability to bind to cells, such as mucosal cells, e.g. of the gut. The reactive group of such a protein is in such a case, for example, partly or wholly derived from a fimbriae protein or another gut attachment protein as is present, for example, in various E. coli strains. Microorganisms to which such a protein is attached will specifically home or bind to certain areas of the gut, a property which, for example, is beneficial for certain bacterial strains (i.e., lactococcal or lactobacillar strains) used as a probiotic. In another food or use of food provided by the invention, the protein or substance is composed of a food additive (such as an enzyme or flavor compound) which affects quality, flavor, shelf-life, food value or texture, joined with an attaching peptide, and subsequently attached or anchored to a microorganism which is then mixed with the foodstuff. The anchoring of such proteins to a bacterial carrier offers the additional advantage that the additive can be targeted to a solid bacteria-containing matrix (e.g. curd) in a process for the preparation of food, e.g. cheese or tofu. Yet another use of a proteinaceous substance or microorganism provided by the invention is the use of bacterial surface display in generating whole-cell bioadsorbents or biofilters for environmental purposes, microbiocatalysts, and diagnostic tools.

Problems solved by technology

A drawback to this approach is the potential of the bacterial strain to cause inflammation and disease in itself, potentially leading to fever and bacteraemia.

However, a potential problem with such recombinant organisms is that they may colonize the mucosal surfaces, thereby generating a long-term exposure to the target antigens expressed and released by these recombinant microorganisms.

In addition, the mere fact alone that such organisms are genetically modified and contain recombinant nucleic acid is meeting considerable opposition from the lay public as a whole, stemming from a low level of general acceptance for products containing recombinant DNA or RNA.

However, as explained above, present techniques of heterologous surface display of proteins in general entail the use of anchoring or targeting proteins that are specific and selective for a limited set of microorganisms which in general are of recombinant or pathogenic nature, thereby greatly restricting their potential applications.

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