Identification And Functional Study Of A Novel Bacterial Lysine Deacetylase,AhCobQ

Protein lysine acetylation modification is distributed in a variety of species and involved in many important biological processes.The molecular mechanism of this modification is a hot issue of current research of biological macromolecules.In terms of mechanism,on the one hand,proteins can acetylate lysine residues in proteins through multiple acetyltransferases or non-enzymatic chemical reactions;on the other hand,proteins require specific lysine deacetylase（KDACs）to reversibly regulate the function of acetylated proteins.Currently,KDACs in bacteria are all homologous to eukaryotes,including Zn²⁺-dependent Rp Lda（same as type II KDACs）,Zn²⁺-dependent Acu C（same as type I）,and NAD⁺-dependent sirtuins（same as type III）.Whether there are other types of KDACs is currently unknown.However,there are hundreds to thousands of protein lysine acetylation modification sites in different species,the number of known bacterial KDACs is obviously far from the number of modified substrates.Therefore,there are probably some unknown KDACs in bacteria that act on specific protein substrates to maintain the normal physiological functions of cells.In conclusion,finding new bacterial protein KDACs,discoverying their molecular regulation mechanism and functions is one of the important research contents in the field of protein acetylation modification regulatory mechanism.This thesis takes Aeromonas hydrophila as the research material,which is an important human-animal-fish comorbid pathogen that causes fish outbreaks in aquaculture.It was found that the Cob Q protein of A.hydrophila（Ah Cob Q,Uniport ID A0KI27,gene name AHA＿1389）is a kind of lysine deacetylase independent of Zn²⁺,NAD⁺and ATP.This protein has no homology with all KDACs currently known,and exists only in prokaryotes.It is a newly discovered lysine deacetylase.The main research contents of this thesis are as follows;（1）The ahcob Q deletion strains were constructed,and the lysine acetylation modification level of the whole bacterial protein of the ahcob Q mutant strain was detected by using the wild type and the classical lysine deacylase Ah Cob B gene deletion strain as the control.The results showed that bothΔahcob Q andΔahcob B could increase the acetylation level of whole bacterial protein.Therefore,we suspect that Cob Q is a lysine deacetylase;（2）Bioinformatics analysis showed that Ah Cob Q is composed of an NTPase domain（4-179aa）and an unknown functional domain（180-265aa）.Multiple sequence alignment analysis found that this protein has no homology with currently known KDACs,and there is no homologous protein in eukaryotes neither,suggesting that Ah Cob Q may be a new type of KDAC;（3）The detection of the physiological phenotype of the deletion bacteria showed that the ability of bacterial biofilm formation was enhanced after the deletion of ahcob B,whileΔahcob Q significantly improved the bacterial motility and iron ion utilization ability,and decreased the biofilm formation.The effects of Ah Cob Q and Ah Cob B on bacterial physiological phenotype are significantly different;（4）It was found that Ah Cob Q can deacetylate the acetylated BSA protein（Kac-BSA）independent of Zn²⁺,NAD⁺and ATP,and may rely on ferrous ion to perform the lysine deacetylase function by lysine deacetylase assay in vitro;（5）Synthesized lysine acetylated modified peptides were treated with KADCs and then identified by mass spectrometry to confirmed that Ah Cob Q could indeed deacetylate the synthesized acetylated modified peptides,thereby excluding the possibility of hydrolase;（6）Molecular biological techniques such as site-directed mutagenesis were used to determine that the deacetylase active region of Ah Cob Q was in the range of 195-245aa,which belonged to the unknown functional region,and L196was found to be one of the key enzyme active sites of Ah Cob Q.The above results indicate that Ah Cob Q can significantly remove the acetylation of Kac-BSA independent of Zn²⁺and NAD⁺.It has no obvious homology with the currently known KDACs,and is a new class of lysine deacetylases.（7）In order to better understanding the lysine deacetylase activity of Ah Cob Q protein and its biological functions,this thesis used quantitative modification proteomics technology to compare the acetylation modification protein substrates and modification sites among theΔahcob B,Δahacu C andΔahcob Q strains.The results showed that Ah Cob Q up-regulated 52 acetylation sites in 47 modified proteins.Among them,6 sites are co-regulated with Ah Cob B,9 are co-regulated with Ah Acu C,and 4modification sites are co-regulated by the three KDACs;（8）The following bioinformatics analysis showed that Ah Cob Q mainly affects the acetylation modification of energy metabolism and translation-related proteins;（9）It was found that Ah Cob Q can indeed deacetylate the above-mentioned acetylated substrate proteins in vitro by site-specific acetylation modification and Western blotting.It is worth noting that Ah Cob Q can not only remove the acetylation modification of a specific site alone,but also cooperate with other KDACs to jointly regulate the deacetylation modification of different modification sites of the same protein or the same site;（10）Through the enzyme activity assay,it was further found that Ah Cob Q can remove the acetylation modification of the K388 site in isocitrate dehydrogenase ICD,which is a key enzyme in the TCA cycle.Ah Cob Q can enhance the activity of this enzyme,thereby participating in the regulation of bacterial energy metabolism;（11）Finally,by intramuscular injection of gene deletion A.hydrophila strains to challenge zebrafish,it was found that Ah Cob Q is involved in the regulation of the virulence of A.hydrophila.Further using enzyme activity assay and competitive ELISA technique,it was found that Ah Cob Q did not affect the metabolic enzyme activity of enolase ENO,which is a bacterial virulence factor.However,Ah Cob Q can enhance the protein-protein interaction between ENO and human plasminogen（PLG）by removing the acetylation modification of ENO K195,suggesting that Ah Cob Q may positively regulate the interaction between ENO and PLG to promote the degradation of pathogenic bacteria and degrade the host’s extracellular matrix into the cell,thereby invading the host.In summary,this thesis found that Ah Cob Q is a novel lysine deacetylase,which can regulate the activities of various key enzymes and affect many biological processes such as bacterial energy metabolism,transcription and translation,and bacterial virulence.At the same time,the active region and active site of Ah Cob Q were determined,and a new molecular mechanism for regulating bacterial Kac proteins was proposed.The above results expand the understanding of the types and dependencies of bacterial lysine deacetylases,and deepen the understanding of how bacterial protein acetylation.It provides a new direction to better study how bacteria can adapt to change in the external environment and maintain the dynamic balance of bacterial niche by regulating the function of proteins.And provide new targets and ideas for the prevention and treatment of the pathogenic bacteria infection.