Dental plaque is a complex biofilm characterized by a pioneer community that is dominated by streptococci. As plaque reaches its final climax community it acquires and increasingly more filamentous composition. During maturation the invading filamentous bacteria must adhere to the early plaque colonizer. It is widely believed that the adhesins responsible for this adherence may be fimbriae or located on the fimbriae of the streptococci. One model employed to address this question is comprised of streptococci species associated with these corncob structures have been identifies as Streptococcus crista. Mixing S. crista and the gram-negative anaerobic microorganism Fusobacterium nucleatum in vitro, duplicates the formation of corncobs. The plaque climax community contains large numbers of F. Nucleatum. Microscopic studies have shown that the binding of S. crista to the fusobacteria occurs through hair-like processes, called fimbriae, are the adhesins that mediate corncob formation. We propose that the simplicity of the corncob unit, its prevalence in dental plaque, its distinctive morphology, our ability to produce the structure in vitro, and the observation that attachment occurs only through the fimbriae suggests that it is an ideal model for the study of the role of fimbriae in the binding process. Our discovery of the natural genetic transformability of S. crista CC5A will be applied to the creation of binding-deficient mutants by trasposon insertional inactivation and random insertion-duplication. Mutants will be characterized by binding assays and immunoelectron microscopy. Fimbriae and fimbrial-associated adhesin loci in these mutants will be identified and rapidly cloned by a long inverse polymerase chain reaction (LIPCR) method and the genes responsible for adhesion will be mapped using a mini-lambda delta shuttle mutagenesis strategy. These genes will be expressed in E. coli to obtain purified protein for binding inhibition and cell surface localization studies. In an alternative approach, the corncob receptor for F. crista adhesins(s). These innovative approaches will be important in understanding the molecular components of the fimbriae involved in attachment and thus provide a rational basis for chemotheraputic approaches to the control of the plaque biofilm.
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