Mechanism Study Of Aeromonas Hydrophlia Evasion From Fish Macrophage Extracellular Traps Killing Mediated By Nuclease

Aeromonas hydrophila is gram-negative bacteria,which is also an opportunistic pathogen to various species,from aquatic to terrestrial animals,including humans.It also infects most species of freshwater fishes.A.hydrophila causes septicemia by releaseing virulant factor such as adhesins,enterotoxins,hemolysin,aerolysin,and proteases during infection.This pathogen seriously affects the healthy development of aquaculture.Nuclease is the extracellular secretory product of A.hydrophila.In early studies,extracellular nucleases were thought to be involved in nutrient acquisition and the elimination of exogenous DNA.However the role of the nuclease in the pathogenesis of A.hydrophila is rarely studied.Phagocytes recruited to infection sites play important roles in the elimination of invading pathogens.In addition to phagocytosis,the release of extracellular traps（ETs）from immune cells is recently described as a new mechanism of the innate immune response to infection.After being stimulated by pathogenic microbes or chemical regents,neutrophil,eosinophil and mast cells release extracellular fibrous structure which is composed of DNA and granular proteins.This structure can entangle and kill various kinds of invading microbes.ETs also form physical barriers that make pathogenic microbes more easily to phagocytosis and to be removed by other immune cells.In order to escaping the capture and killing of ETs,p REvious studies showed that Streptococcus A,Staphylococcus aureus and Streptococcus pneumoniae could degrade the backbone DNA of ETs by secreting nuclease.In this study,A.hydrophila and fish head kidney macrophages are used as the research object.Firstly,in order to study the role of nuclease in the pathogenesis of A.hydrophila.A.hydrophila nuclease（ahn）mutant was constructed by homologous recombination.Blunt snout bream were used as infection model.Secondly,blunt snout bream and grass carp head-kidney macrophages were isolated,and were induced release ETs（extracellular traps）.The components and biological characteristics of the ETs of the blunt snout bream and grass carp head-kidney macrophages were further observed.Finally,we p REliminary elucidated the mechanism of A.hydrophila immune evasion from fish head-kidney macrophages extracellular traps mediate by nuclease.The results were as follows.1.The mutant strain（Δahn）of the A.hydrophila nuclease gene was successfully constructed by homologous recombination.The growth rate of mutantΔahn and wild-type strain Ah J-1 was determined.The results indicate that the deletion of ahn do not affect the growth of mutant strain.In addition,β-hemolytic phenotype experiment showed that the mutantΔahn and wild-type strain Ah J-1 have no significant difference ofβ-hemolytic capacity.The in vitro blunt snout breams head-kidney macrophages killing experiment certified that the anti-macrophages killing ability ofΔahn was significantly attenuated as compared with Ah J-1.Similarly,the Ah J-1 load in the ascites was significantly higher than that of mutantΔahn,indicating that the mutant could not effectively resist the killing of macrophages in the abdominal cavity.According to competitive growth assay in fish abdominal cavity,the competitive indices are less than 1,it was proved that the mutantΔahn showed a significant survival disadvantage compared with the WT strain Ah J-1,and could not resist the removal of the host innate immune system.Blunt snout bream were infected with equal dose of WT andΔahn separately.Bacteria loads recovered from the kidneys and livers from Ah J-1 infection group were significantly higher than that fromΔahn infection group.These results provide convincing evidence that ahn contributes to the systemic dissemination of A.hydrophila.Pathological examination of the kidneys revealed that renal tubular cell swelling and inflammatory cell infiltration were observed in the WT-infected blunt snout breams.The degree of infection was more severe than theΔahn infected group.In addition,the experiment of determining virulence of the WT strain andΔahn showed that the LD₅₀of theΔahn mutant was 6 times that of the WT strain Ah J-1,indicating that the virulence of the mutantΔahn is attenuated.These findings implicate ahn in A.hydrophila virulence,with important functions in pathogeneses.2.The fish head-kidney macrophages release extracellular fiber-like structure by stimulation of A.hydrophila,E.coli and PMA,which were observed by fluorescence microscopy and scanning electron microscope.The quantification of extracellular DNA also proved the above result.The composition of the blunt snout bream and grass carp head-kidney macrophages extracellular traps were studied.The result showed that the components of METs include the important antimicrobial protein of histone,in addition to the DNA.By PCR experiments,the result show that skeletal DNA of grass carp head-kidney METs derives from both the nuclear and the mitochondria.But for the blunt snout bream,the skeletal DNA of METs derives only from nuclear,indicating that the skeletal DNA has different resources in different fish species.In addition,the quantification of extracellular DNA was determined after addition of reactive oxygen free radical（ROS）inhibitor.The result showed that the amount of the grass carp and blunt snout breams macrophages extracellular DNA were increased,indicating that the production of METs do not rely on ROS.LDH assay showed that the LDH of the grass carp and blunt snout breams macrophages was increased after stimulation with A.hydrophila,and there was no significant change between PMA,E.coli group and control group,indicating that the release of ETs by fish head-kidney macrophage is not necessarily accompanied by cell necrosis.3 After ETs generating by the grass carp and blunt snout breams macrophages induced by the PMA,the culture supernatant of the WT strain Ah J-1 and mutantΔahn were added.The quantification of extracellular DNA showed that Ah J-1 supernatant reduced the production of extracellular DNA,the addition of the mutantΔahn supernatant has no effect on the production of extracellular DNA.In addition,comparing with the WT strain Ah J-1,the mutantΔahn is more easy to trap by the ETs.The above results demonstrate that ahn is critical for the ability of DNA degradation of extracellular products of A.hydrophila.Finally,the bactericidal assay by fish METs revealed that,the defense ability of mutant strainΔahn against fish METs significantly decreased as compared to the WT strain Ah J-1,.The current study verified that nuclease mediated the successful escape of A.hydrophila from the killing by ETs of fish macrophages.