Construction of Highly Pathogenic Photobacterium Damselae Subsp. Damselae Mutant Library and Identification of Genes Involved in Hemolysis Regulatory

Abstract

Photobacterium damselae subsp. damselae (PDD) is a highly virulent pathogen within marine aquaculture settings, capable of infecting a diverse array of marine fish species and leading to hemorrhagic septicemia and mortality. Hemolysin is known as an important pathogenic factor of PDD. To investigate genes involved in hemolysin synthesis and secretion by PDD, we created a library of Mini-Tn10 transposon random insertion mutants using transposon insertion and biparental membrane grafting method. Mutant strains with significant differences in hemolytic phenotypes were screened from the library using the blood plate culture method. Whole-Genome Sequencing was used to identify the insertion site of Mini-Tn10 on the genome of PDD in the mutant strains and to clarify the potential gene classes that influence the synthesis and secretion of hemolysin. To verify the function of the insertion gene, the insertion gene deletion strains were constructed in this study using the homologous recombination technique and compared with the hemolytic phenotype of the wild strain. The results showed that Mini-Tn10 was able to mutagenize PDD successfully to construct a library including 3648 PDD mutant strains, from which screened six genes associated with hemolytic phenotypes, hlyA, epsD, epsF, dorA, Int and hypothetical gene, which encoded hemolysins, type II secretion system channel proteins, and molybdopterin-dependent oxidoreductase, tyrosine-type recombinase and hypothetical protein in the gene annotations, respectively. There was no hemolysis in PDD after the knockdown of the hlyA, epsD, and epsF genes, and the dorA, Int, and hypothetical gene deletion strain still had hemolytic activity, which was weaker than the wild strain. In conclusion, interference or disruption of these genes by Mini-Tn10 may inhibit the expression and secretion of hemolysin in PDD, leading to a decrease in hemolytic capacity. The results of this study will lay a theoretical foundation for the in-depth analysis of the pathogenesis of PDD.

Keywords: Photobacterium damselae subsp. damselae, Mini-Tn10, transposon mutant library, hemolytic phenotype, gene function