A Study Of The Antimicrobial Components In The Neutrophil Extracellular Traps Of Cynoglossus Semilaevis

Neutrophil extracellular traps(NETs)are a form of extracellular bacterial killing structure of neutrophils observed in higher and lower vertebrates,the latter including the teleost fish tongue sole Cynoglossus semilaevis.Histone and elastase are main components of NETs presence in high local concentrations which enables NETs to kill microorganisms in higher vertebrates.However,the bactericidal mechanism of fish NETs is unknown.In the present study,we examined the potential contribution of histones and elastases to the antibacterial effect of tongue sole NETs.For this purpose,two histones(Cs H2 B and Cs H4)and two elastases(Cs Ela1 and Cs Ela2)were cloned from tongue sole and investigated.The histones and elastases possess the conserved domain structures characteristic of that of histones H2B/H4 and trypsin-like serine protease,respectively.Recombinant Cs H2 B,Cs H4,Cs Ela1,and Cs Ela2 bound a wide range of Gram-negative and Gram-positive bacteria,and some of the bound bacteria were inhibited in growth by the bound proteins.Cs H2 B,Cs H4,Cs Ela1,and Cs Ela2 were all localized in NETs induced by various stimuli including bacterial pathogen.Treatment of NETs with antibodies targeting Cs H2 B,Cs H4,Cs Ela1,and Cs Ela2 significantly reduced the antimicrobial effect of NETs.These results indicate that histones and chymotrypsin-like elastases are components of teleost NETs that contribute to the antimicrobial activity of NETs.