Targeted Delivery of a Photosensitizer to Aggregatibacter actinomycetemcomitans Biofilm

The ability to selectively target specific biofilm species with antimicrobials would enable control over biofilm consortium composition, with medical applications in treatment of infections on mucosal surfaces that are colonized by a mixture of beneficial and pathogenic microorganisms. We functionalized a genetically engineered multimeric protein with both a targeting moiety (biotin) and either a fluorophore or a photosensitizer (SnCe6). Biofilm microcolonies of Aggregatibacter actinomycetemcomitans, a periodontal pathogen, were targeted with the multifunctional dodecamer. Streptavidin was used to couple biotinylated dodecamer to a biotinylated anti-A. actinomycetemcomitans antibody. This modular targeting approach enabled us to increase the loading of photosensitizer onto the cells by a cycle of amplification. Scanning laser confocal microscopy was used to characterize transport of fluorescently tagged dodecamer into the microcolonies and targeting of the cells with biotin-labeled, fluorescently tagged dodecamer. Light-induced activity of the targeted photosensitizer reduced the viability of A. actinomycetemcomitans biofilm, as indicated by membrane permeability to propidium iodide. The functionalized multimeric protein promises to be a useful tool for controlling periodontal biofilm consortia and offers a modular design whereby moieties that target different species can be readily combined with the functionalized protein construct.Therapeutic tools that enabled targeted delivery of antimicrobials to specific species in biofilms would expand our capability to treat chronic infections associated with mucosal surfaces where complete sterility is not the natural healthy condition. The oral cavity and the gut are prime examples of mucosal surfaces at which the immune system maintains a healthy microbial presence (3, 50). Mucosal surfaces in the oral cavity are nonsterile environments colonized by biofilm consortia (38). In the gut, some commensals are true symbionts (30, 36), and similarly, it is hypothesized that oral commensals may aid in maintaining a healthy immune homeostasis (35). The implication is that treatment of oral diseases such as periodontal disease can be made more effective by selective elimination of pathogens. By extension, we may also find that tools that enable specific targeting of biofilm pathogens provide effective treatments for chronic infections of the gut (31).A class of multimeric proteins that self-assemble into highly symmetric quaternary structures (7, 52) shows promise for development into delivery vehicles for therapeutic (15, 16, 44) and imaging agents (41, 51). We selected one of these proteins, a relatively small, spherical, 9-nm-diameter dodecamer, originally isolated from Listeria inocula (LiDps) (21), for targeted delivery of a photosensitizer to Aggregatibacter actinomycetemcomitans biofilm. We genetically added a tetrapeptide to the C terminus of each of the 12 monomeric 18-kDa subunits of LiDps. Cysteines incorporated into this tetrapeptide were labeled with biotin. Since the C terminus is presented on the exterior surface of the assembled dodecamer, these biotins were readily available for binding to streptavidin, a universal coupling protein. An SnCe6 photosensitizer was covalently linked to intrinsic lysines.Photosensitizers are compounds that produce reactive oxygen species (ROS) upon excitation by light (25). Antimicrobial photodynamic therapy (PDT) has been shown to be effective against oral pathogens (6, 29, 54). SnCe6 has been used previously for targeted antimicrobial PDT (10-12). The primary modes of action of photosensitizers are membrane disruption and, if the photosensitizer is internalized, DNA damage (19). The antimicrobial activity of ROS produced by light-activated photosensitizers is relatively localized. This is especially the case for singlet oxygen (diffusion length of <50 nm), which is thought to be the primary ROS causing cell death (32). The localized action of photosensitizers makes them attractive agents for targeted antimicrobial therapy.The LiDps genetic construct, dual functionalized with SnCe6 and biotin, is a modular unit that can be combined with any targeting moiety that can be biotinylated. This would accommodate a variety of approaches that have been used to target microbial pathogens, including lectin/carbohydrate interactions (53), antimicrobial peptides (8, 22), and the lysins isolated from bacteriophage (14). We chose to use an antibody as the targeting moiety for these studies since monoclonal antibodies are available for a number of the prevalent periodontal pathogens (5, 9, 17). In addition, antibodies offer a wide range of potential cell surface targets combined with the possibility to engineer high binding affinities (4).A. actinomycetemcomitans is a Gram-negative periodontal pathogen implicated as the primary etiological agent in localized aggressive periodontitis (13, 37). Rough colony variants isolated from sites of infection form discrete microcolonies when cultured in vitro which exhibit distinctive cohesive as well as adhesive properties (27). We characterized transport of the LiDps dodecamer to the base of A. actinomycetemcomitans microcolonies, demonstrated targeting of the LiDps to the cells in the microcolonies, and showed that SnCe6 targeted to 24-h biofilms produced light-activated membrane disruption. The modular design enabled us to use cycles of alternating exposure to the streptavidin and the LiDps dodecamer to increase the loading of targeted photosensitizer onto the cells.