Converting staphylococcal pathogenicity islands from malevolence to benevolence

Project summary/AbstractStaphylococcus aureus, causes a wide variety of life-threatening infections, many of which cannot be treatedeffectively, owing to antibiotic resistance. Consequently, there is an urgent need for new ways to treat theseinfections, which annually cause some 18,000 deaths in the US. We have developed a novel non-antibioticmethod, for treating staphylococcal infections based on the highly mobile staphylococcal pathogenicity islands(SaPIs). The SaPIs are ~15 kb genetic elements that are stably inserted in the staph chromosome but can beinduced by ?helper? phages to excise and replicate. The replicated SaPI DNA is packaged in infectious phage-like particles which are released upon phage-induced lysis. The SaPIs carry and disseminate genes encodingsuperantigen toxins and other virulence factors. Instead of working on the prevention of SaPI spread, we hitupon the idea of exploiting SaPI spread by converting these agents of disease into agents of therapy ?antibacterial drones (ABDs). To create the ABDs, we have re-engineered the SaPIs, deleting their naturalcargo (toxin genes), increasing their packaging capacity from 15 to >40 kb, and inserting antibacterial modules.We have also modified the helper phage so that ABD particles are produced in the absence of functionalphage. The ABD particles are administered to an infected animal (or plant), where they attach to the infectingbacteria, insert their DNA, express their antibacterial cargo genes and thus abrogate the infection. As proof ofprinciple, we have begun by incorporating into ABDs either CRISPR/cas9 or CRISPR/dcas9 modules withspacers targeting a chromosomal gene or the promoter region of a global virulence regulator, respectively.Preliminary studies have shown that the CRISPR/cas9-containing ABD kills S. aureus in vitro by DNAcleavage, blocks the development of a subcutaneous S. aureus abscess, and rescues mice given a lethal doseof S. aureus intraperitoneally. The CRISPR/dcas9 containing ABD blocks the expression of staphylococcalvirulence in vitro and blocks the formation of a subcutaneous abscess in vivo. This proposal outlines ourprogram to develop the ABD system and validate our underlying hypothesis that SaPIs can be converted toversatile and fully effective anti-staphylococcal therapeutic agents. There are 3 specific aims. In Aim I we willconstruct and test ABDs designed to treat the wide array of infections caused by S. aureus. In Aim II, we willfocus on expanding the host range of the ABDs to target diverse S. aureus strains. In Aim III, we willencapsidate the ABD particles for increased efficacy, test for ABD resistance and immunogenicity, and refineour procedures for producing and preserving high titer ABD preparations.