The long-range, health-related goal of this proposal is to develop species-specific, cell wall-hydrolyzing enzymes encoded in the genomes of phages specific for Streptococcus mutans and Actinomyces naeslundii as new therapeutic treatments for dental caries. S. mutans is the primary etiological agent of human enamel caries, whereas A. naeslundii (A. viscosus), an early colonizer of dental plaque, has long been believed to be involved in gingivitis and root surface (cementum) caries. The major objective of the research proposed herein is to isolate, purify and characterize the enzymes which enable phages specific for these species to lyse their host cells. The lysis genes of two previously studied phages which are specific for S. mutans and A. naeslundii will be isolated, cloned and sequenced. To accomplish this, the complete genomes of these two phages will be sequenced, which will allow direct PCR subcloning of their holin and endolysin genes and characterization of their respective products. The DNA sequences of these holin/lysin gene pairs will provide information on their regulatory mechanisms and further our knowledge of the evolutionary relatedness of these viral proteins. Comparative analyses of their deduced primary amino acid sequences may also reveal conserved protein domains that are important in determining their structural and functional properties. Additional cloning experiments will be employed to isolate holin genes, and nearby endolysin genes, from these two phages and three additional oral phages, in order to develop a generally applicable method for directly isolating oral phage lysis genes. Phage DNA libraries will be constructed in a phage vector having a defective holin gene, which will allow recombinants expressing oral phage holins to be selected by complementation (plaque formation) of the defect in the phage vector. Inserts will then be sequenced to identify the phage holin genes and primers complementary to the ends of these genes will then be used to sequence, directly from the phage genomes, the adjacent, downstream endolysin genes. Selected endolysin genes will be subcloned, by PCR, from phage genomic DNAs, or from recombinant phage vectors, into expression vectors and introduced into E. coli in order to isolate and purify their gene products. The enzymatic activities of these proteins will then be extensively characterized, including determining the specific bonds which they cleave in the cell walls of their respective hosts. Purified preparations of these lytic enzymes might ultimately be used to kill, in a species-specific manner, S. mutans and A. naeslundii in dental plaque.
| Delisle, Allan L; Guo, Ming; Chalmers, Natalia I et al. (2012) Biology and genome sequence of Streptococcus mutans phage M102AD. Appl Environ Microbiol 78:2264-71 |
| Delisle, Allan L; Barcak, Gerard J; Guo, Ming (2006) Isolation and expression of the lysis genes of Actinomyces naeslundii phage Av-1. Appl Environ Microbiol 72:1110-7 |
