The halophilic bacteria Vibrio vulnificus and Vibrio damsela are found in marine and estuarine environments and are increasingly recognized etiological agents of severe wound infections and life-threatening septicemia in humans. The long-term objective of the research described in this application is to identify and characterize the potential extracellular virulence factors of the bacteria in order to aid the diagnosis, prevention, and treatment of the diseases they cause. Previous studies by the PI have focused on the purification and characterization of lethal, tissue-damaging cytolytic toxins (membrane-damaging toxins) produced by V. vulnificus and V. damsela, and a lethal, tissue-damaging elastolytic protease produced by V. vulnificus. The specific aim of the research described in the application is to evaluate the importance of the previously isolated cytolysins and the elastase in the pathogenesis of experimental diseases caused by the bacteria. Studies to determine whether in vivo production and action of the three tissue-damaging substances are important virulence factors of the bacteria will include: (i) determining whether active and passive immunization against the isolated cytolysins and/or elastase protects mice against tissue damage or death after challenge with the bacteria, (ii) determining whether isogenic mutants specifically lacking the ability to express the putative virulence factors are less virulent for mice than are the parental, wild-type strains, (iii) determining whether parenteral administration of the isolated products enhances the virulence of the isogenic mutants for mice, (iv) using light and electron microscopy to determine whether the tissue damage elicited by the isolated products mimics that produced in mice by the replicating bacteria, and (v) determining whether the products are synthesized in vivo during the development of the infectious diseases in mice.