Molecular Mechanism Of Phenazine Inhibits Innate Immunity Of C.elegans By Histone Deacetylase HDA-1

The innate immune system is first line of organism to defense against pathogens,and it has highly conserved regulatory mechanisms in nematodes,fruit flies,rodents and humans.MAPK signaling pathway,DAF-2 / DAF-16 signaling pathway and TGF-?signaling pathway have been found in C.elegans.Histone deacetylase hda-1 is involved in a series of cellular processes by regulating the level of histone acetylation.In this study,we used C.elegans as a model infection by P.aeruginosa to study the mechanism of histone deacetylase hda-1regulated host-pathogen interaction.Firstly,the survival experiment was conducted to screen 10 histone deacetylases in C.elegans.After knockdown of hda-1 by RNAi,we found that the survival rate of C.elegans increased during P.aeruginosa infection.After knockdown of hda-1 by RNAi in different tissues,it was found that hda-1 mainly played a role in the intestinal and epidermis.At the same time,we found that the mRNA level of hda-1 and expression of hda-1::gfp increased only in young worms during P.aeruginosa infection.The pathogenicity of P.aeruginosa is closely related to its virulence factor,and the activation of hda-1 may be related to virulence factor.Firstly,we separated the superscript of P.aeruginosa into components with molecular weights < 3KD,3-10 KD and >10KD by membrane separation and then tested them.We found that components with molecular weights < 3KD significantly promoted the expression of hda-1.Secondly,we used the LC-MS technique to detect the active components,and it was found that the active components mainly included phenazine,pyocyanin,6-methoxyquinoline,and pyroquilon.Finally,our result showed that phenazine could significantly activate hda-1.The lysine at the end of the histone can be modified by the histone deacetylase hda-1.We detected the effect of hda-1 on the acetylation levels of histone H4K5 ac,H4K8ac,H4K12 ac and H4K16 ac,and found that the acetylation levels of histone H4K5 ac and H4K8 ac significantly increased after inactivation of hda-1.Furthermore,we found that the acetylation levels of histone H4K5 ac and H4K8 ac decreased after infection by P.aeruginosa.In addition,the acetylation levels of histone H4K5 ac and H4K8 ac were significantly reduced after phenazine treatment.There was no change in the acetylation levels of histone H4K5 ac and H4K8 ac after Phz12-a fenazine-deficient strain infection.We used ChIP-seq and RNA-seq techniques to detect the downstream target genes of hda-1,and found that GO analysis enriched to the molecular functions of "structural constituent of cuticle" and "structural constituent of collagen and cuclamin-based cuticle".Those results indicated that phenazine secrected by P.aeruginosa activated hda-1,which reduced the acetylation levels of H4K5 ac and H4K8 ac and down-regulated expression of collage genes,and then inhibit the innate immunity of worms.This study explored host-microbial interactions from the perspective of epigenetics,it provided theoretical and experimental basis for us to research the role and mechanism of histone deacetylase in innate immunity,and also provided us with a new pathogen treatment strategy for infection.