INFECTIOUS DISEASES CAUSE BILLION-DOLLAR LOSSES ANNUALLY TO THE AQUACULTURE INDUSTRY AND NEGATIVELY IMPACT GROWTH AND SUSTAINABILITY BOTH IN THE US AND GLOBALLY. WHILE ANTIBIOTICS ARE EFFECTIVE AGAINST MANY BACTERIAL DISEASES OF FISH, THERE ARE FEARS THAT THEIR USE IN AQUACULTURE MAY CAUSE SERIOUS ENVIRONMENTAL AND HUMAN HEALTH PROBLEMS RELATED TO THE POTENTIAL FOR FARM-TO-CLINIC ANTIBIOTIC-RESISTANCE TRANSFER. STREPTOCOCCUS INIAE (A GRAM-POSITIVE BACTERIUM) IS RECOGNIZED AS AN EMERGING PATHOGEN OF WILD AND CULTURED FISH WITH ~30 SPECIES OF SUSCEPTIBLE FISH, INCLUDING FARM-RAISED TILAPIA AND HYBRID STRIPED BASS. S. INIAE CAN RESIDE BOTH WITHIN (INTRACELLULAR) AND OUTSIDE (EXTRACELLULAR) INFECTED FISH CELLS. THERE IS A NEED FOR NOVEL (NON-ANTIBIOTIC) ANTIMICROBIALS THAT ARE REFRACTORY TO RESISTANCE DEVELOPMENT THAT CAN KILL BOTH INTRACELLULAR AND EXTRACELLULAR BACTERIA. PHAGE (BACTERIAL VIRUS) ENDOLYSINS ARE CELL WALL DEGRADING ENZYMES (PEPTIDOGLYCAN HYDROLASES, PGHS) THAT DIGEST THE MAJOR STRUCTURAL COMPONENT OF THE BACTERIAL CELL WALL (PEPTIDOGLYCAN), ALLOWING NEWLY FORMED PHAGE TO ESCAPE THE HOST CELL AND INFECT NEW HOST CELLS. IN GRAM-POSITIVE PATHOGENS (INCLUDING S. INIAE), THE PEPTIDOGLYCAN IS SURFACE EXPOSED. WHEN THE PURIFIED ENDOLYSIN IS EXPOSED EXTERNALLY, IT CAN DEGRADE THE CELL WALL AND CAUSE DEATH TO THE PATHOGEN. THIS ABILITY ALLOWS US TO USE THEM AS 'ALTERNATIVE ANTIBIOTICS'. WE HYPOTHESIZE THAT PHAGE ENDOLYSINS CAN BE IDENTIFIED FROM PUBLIC DATA SETS AND PREVENT AND/OR ERADICATE S. INIAE INFECTIONS IN FISH. COEVOLUTION OF PHAGE AND HOST ENSURES THAT THESE ENZYMES ARE HIGHLY REFRACTORY TO RESISTANCE DEVELOPMENT IN THE HOST CELL. ONCE IDENTIFIED, THE PGH PROTEINS WILL BE FUSED TO KNOWN 'PROTEIN TRANSDUCTION DOMAINS' (SHORT PROTEIN DOMAINS THAT HELP TRANSPORT PROTEINS INTO ANIMAL CELLS), ALLOWING THE ENZYME TO LYSE S. INIAE THAT RESIDES BOTH INSIDE AND OUTSIDE OF FISH CELLS. WE WILL IDENTIFY OPTIMAL (HIGH ACTIVITY) FUSION CONSTRUCTS VIA ANTI-S. INIAE ASSAYS WITH CULTURED FISH CELLS AND TEST FOR TARGET AND NON-TARGET LYTIC ACTIVITY ON MULTIPLE S. INIAE AND BACTERIAL COMMENSAL STRAINS ISOLATED FROM FARM RAISED TILAPIA. THIS PROPOSAL WILL GENERATE PGH-ANTIMICROBIALS HIGHLY SPECIFIC FOR S. INIAE FOR USE IN STRATEGIES TO PROTECT FARM-RAISED FISH FROM DISEASE.