PubMed · 11190672
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A P May, E Y Jones. 2001. Sialoadhesin structure.. https://doi.org/10.1007/978-3-540-46410-5_8
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In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.
An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.
We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion with N-chlorosuccinimide but not in isotype distribution or overall reactivity with S. mutans or intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with either S. mutans alone or S. mutans coated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlapping spaP subclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface of S. mutans alters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.