Characterisation Of Nucleases In Trichinella Spiralis And Its Role In The Evasion Of Macrophage Extracellular Traps-mediated Antimicrobial Activity

Extracellular traps (ETs) exist in vertebrate and invertebrate innate immune cellsincluding neutrophil, eosinophil, mast cells, and monocyte/macrophage, is a newinnate immune phagocytosis-independent extracellular defence mechanism which wasdiscovered in recent years. ETs are extracellular fibrous structure which is composedof DNA and granular proteins. The new mechanism was reported to ensnare variedkinds of invading microbes inducing bacteria, fungi, virus, and protozoa to preventlocal diffusion, and kill them with various ET-associated active effector molecules invitro, and also in vivo. But whether the mechanism existing in defence to bigpathogens like nematodes is still unknown. Trichinella spiralis (T. spiralis) is atypical pathogenic nematode. One of most important characteristics is inefficiency ofhost innate immune cells in the early innate immune response to T. spiralis infection.For instance, although T. spiralis invading to host leads to eosinophil assembling inintestine phase, lacks of following inflammatory and worm expelling. Furthermore, ithas been reported that extracellular nucleases were accounted as an importantvirulence factor in various bacteria species Group A Streptococcus, Staphylococcusaureus, and Streptococcus pneumoniae, allowing the evasion of the microbes fromETs via degrade DNA. It is noteworthy that more extensive expansion of the DNaseII-like protein family (estimated125genes) in the genome of the parasitic nematode T.spiralis comparison to the other nematodes, and almost half of those were classifiedas encoding excretion/secretion products (ESP). So, we deduced T. spiralis DNase IImay contribute to evasion of macrophage extracellular traps-mediated antimicrobialactivity.Firstly, murine macrophages cell line J774A.1was used to investigate whethermacrophages cast ETs in response to T. spiralis. T. spiralis ML and NBLco-incubated with macrophages did not stimulate macrophages develop extracellular fiber-like structures, but these structures were observed with the addition of ATA bylaser scanning confocal microscope and scanning electron microscopy, and adhered tothe surface of worm. The major ingredient of these structures was DNA anddesignated as macrophage extracellular traps (METs). Neither ROS production nornecrosis, apoptosis was observed in induced-T. spiralis METs formation process. Itshowed that approximately30-40%T. spiralis ML and NBL were killed by METs invitro.Secondly, the active nucleases under acidic (pH4, pH5, and pH6) and slightlyalkaline (pH8and pH9) in T. spiralis ESP was confirmed by agar diffusion, agarosegel electrophoresis, and acid dissolution method, and its optimum temperature was25°C,30°C, and37°C. The activity was inhibited by high concentrations of metalions including K+, Na+, Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+. The chelatorEDTA and G-actin had no effect on the activity, but a general nuclease inhibitor,ATA, inhibited the activity. DNA substrate cleavage and intermediate productterminal group analyse indicated that nuclease activity in ESP displayed DNase IIcharacteristic.1D nuclease zymography (SDS-PAGE) analysis showed one and twoactivity band existed in ML and Ad6/NBL ESP, respectively. Four protein spots withthe nucleases activity were identified from ML and Ad6/NBL ESP by2-dimensionalelectrophoresis (2-DE) nuclease zymography combined Nano LC-ESI/MS/MStechnique, respectively.Lastly, plancitoxin-1-like, only contains typical active site of DNase II (HKDmotif) in its C-terminus domain in125DNase II-like protein family, was cloned andexpressed in a prokaryotic expression system and its nuclease activity was measured.However, the sequences of plancitoxin-1cloned from T. spiralis were shorter210bpthan the predicted sequences in GenBank. Intriguingly, there were two HKD motifs inthe N-and C-termini in the cloned sequences. Western blot analysis showed that theantibody of the expression protein recognised crude antigens but not ES antigens, soplancitoxin-1-like was not identified from ESP by2-DE nuclease zymography andmass spectrometry. Plancitoxin-1-like was examined at all T. spiralis developmentalstages by Real-time quantitative PCR and immunolocalisation analysis. Asdemonstrated by DNase zymography, the expressed proteins as inclusion bodies displayed the cation-independent DNase II activity after renaturation in gel. theexpressed proteins displayed DNase activity, and had a narrow optimum pH range inslightly acidic conditions (pH4and pH5) and its optimum temperature was25°C,30°C, and37°C.The nuclease in T. spiralis ML and NBL ESP displayed DNase II characteristic.T. spiralis ML and NBL stimulated macrophage to release METs with the addition ofATA to the suppression of DNase activity. T. spiralis can be ensnared and killed byMETs, but the mechanism was not triggered after blocking with anti-T. spiralis serum.The active DNase II protein was identified from ESP by2D nuclease zymographycombined Nano LC-ESI/MS/MS technique, and plancitoxin-1-like contains typicalactive site of DNase II was classified as a somatic protein. Results suggest that thesecretion of DNase II in T. spiralis contributes to evasion of METs-mediatedantimicrobial activity.