In mammals neutrophil elastase is
In mammals, neutrophil elastase is a trypsin-like serine protease stored in the azurophilic granules of naïve neutrophils , . The catalytic activity of neutrophil elastase relies on a His57–Asp102–Ser195 triad (chymotrypsin numbering system) that functions by transfer of electrons from the carboxyl group of Asp to the oxygen of Ser, which becomes a nucleophile and attacks the preferred peptide bond . Neutrophil elastase is a powerful protease capable of degrading a variety of soluble and insoluble substrates . The main intracellular physiological function of neutrophil elastase is the degradation of foreign organic 58 5 phagocytosed by neutrophils, including virulence factors of bacteria , , .
Neutrophil extracellular traps (NETs) are a novel extracellular bacterial killing mechanism of neutrophils . NETs are composed of a backbone of DNA associated with various neutrophil-derived proteins, including histone, elastase, cathepsin, and myeloperoxidase , , . It has been proposed that the presence of high local concentrations of antimicrobial proteins enables NETs to kill microorganisms .
Recently, NETs-like structures have been reported in several teleost species including turbot, tongue sole, fathead minnows, carp, and zebrafish , , , , . In turbot and tongue sole, antimicrobial activities of NETs were observed , , however, the composition and mechanism of the involving antimicrobials remain unknown. In the present study, with an aim to identify antimicrobial factors that contribute to the bacterial inhibition/killing effect of fish NETs, we examined two histones and two elastases from tongue sole for their antibacterial potentials and involvement in NETs.
Materials and methods
Discussion H2B and H4 are known to participate in antimicrobial defense toward both Gram-positive and Gram-negative bacteria . H2B has been reported to exhibit antimicrobial activity in channel catfish , human placenta , and murine macrophages , and H4-derived fragments were shown to be present in an active fraction against B. megaterium . A mixture of H2B and H4 obtained from the hemocytes of Pacific shrimp displayed an activity against Gram-positive bacteria . In addition, all subtypes of mammalian histones, i.e. H1, H2A, H2B, H3, and H4, can bind bacterial LPS and LTA . In line with these observations, in our study, both ELISA and microscopy revealed binding of rCsH2B and rCsH4 to Gram-negative/Gram-positive bacteria. Interestingly, rCsH2B exhibited higher binding indexes to Gram-positive bacteria, while rCsH4 exhibited higher binding indexes to Gram-negative bacteria. These results are consistent with the previous reports that Lys-rich histone H2B and Arg-rich histones H4 had different binding affinities towards Gram-negative and Gram-positive bacteria , , . For both rCsH2B and rCsH4, we observed apparent inhibitory effects on the growth of some target bacteria, suggesting a capacity of these proteins to function as effectors of innate immunity. Like histones, neutrophil elastase is known to possess antibacterial activity. Reports have shown that elastase in neutrophils can proteolytically degrade the cell wall of Gram-negative bacteria , and that mice deficient in this enzyme displayed impaired killing of bacteria and fungi and reduced survival against Gram-negative infections . A recent study indicated that neutrophil elastase might have a profound effect in the gastrointestinal tract, where it could degrade the toxins of Shigella, Salmonella, and Yersinia, and prevented escape of Shigella from neutrophil phagosomes . In our study, rCsEla1 and rCsEla2 were found to exhibit comparable antibacterial properties and bind to a wide range of Gram-negative and Gram-positive bacteria. However, binding of these elastases to bacteria did not necessarily cause growth defect in the part of the bacteria, which was observed in only some of the bound bacteria. These results suggest the possibility that the target molecules of rCsEla1 and rCsEla2 in different bacteria may be different.