The goal of this R21 project is to contribute to an understanding of the efficacies of hypohalite host defense factors toward pathogenic oral bacteria. Within the oral cavity, hypochlorite (OCI-) and hypothiocyanite (OSCN-) are produced by enzymes. Nevertheless, we have discovered a facile non-enzymic pathway that produces hypothiocyanite by oxidation of thiocyanate (SCN-) with hypochlorous acid (HOCI), a reaction that preserves the oxidizing equivalents of hypochlorite by transferring them to hypothiocyanite, a more discriminate biocide that is not lethal to mammalian cells. Remarkably little is known about the biocidal mechanisms of the hypohalites, due in part to their extraordinary reactivities and the fact that they produce cascades of derivative reactive intermediates with largely unknown physiologic properties. In addition to the complexities of the dynamic chemistry, the issue of biocidal mechanisms is further complicated by the heterogeneity of the oral cavity, where complex biofilms develop in chemically distinct environments. Recent studies have suggested that the genetic response of E. coli is different for the hypochlorite as compared to the hypothiocyanite system. Given that hypochlorite and hypothiocyanite apparently control spatially distinct regions of the oral cavity, but interracial regions near the gingival margin likely exhibit a concentration gradient of these hypohalites, we posit that these two biocides act in concert. The Specific Aims of this project are: 1) to develop protocols for assessing the biocidal efficacies of the hypochlorite and hypothiocyanite chemical systems and selected secondary derivatives toward a library of oral bacteria (and some controls); and 2) employing this protocol and the same library of oral bacteria, investigate synergistic effects of the primary and secondary biocides. These goals will be achieved through the application of whole-cell chemical-quench-flow and the introduction of chemical insults (some generated in real time) vis-a-vis a bioreactor system. We note that all of the experiments we describe in this R21 application involve planktonic cultures, which we believe is a logical first step before tackling the complexities of heterogeneous systems. Nevertheless, the methods that will be developed are expected to be adaptable to future studies that will include mixed-cultures and heterogeneous systems (including biofilms). Long-range Benefit: An understanding of the capacity of human defense mechanisms to control pathogenic oral microbes is essential for the improvement of therapies for oral infectious diseases. The ultimate objective of our research is the development of specific approaches to reduce pathogenic flora without altering the normal commensal (non-injurious) flora of the oral cavity.

R21DE016889

2007-02-01

2010-01-31