Quantitative Proteomics Analysis Reveal The Effect Of Acetylation Modification In Iron Homeostasis In Aeromonas Hydrophila

Aeromonas hydrophila is an opportunistic fish pathogen,commonly found in freshwater environments.It causes infection in fish,shellfish,grass carp,and shrimp,and can also cause a motile aeromonad septicemia and multifocal Aeromonas osteomyelitis.It has been associated with emerging infections such as prostatitis and hemolytic-uremic syndrome.Most of the pathogenic bacteria require iron for their growth and survival as well as for the maintenance of iron homeostasis.In iron deficient condition,bacteria acquire iron molecules from environments by the regulation of siderophore and other important proteins of iron import pathways.Modern omics technologies and tools facilitated the understanding of genes and protein's expression level.It has also expanded our approach to understanding the key metabolic pathways,small molecule exporting channels and protein trafficking pathways,in and outside of the cell.In proteomics study,recent advancement in science and technology extended our knowledge into a new scientific era,known as post-translational modifications(PTMs).PTMs play a fundamental role in the alteration of protein activity through chemical modification of specific amino acid residues reversible and involve in a variety of physiological activities.Nowadays,about 600 different kinds of PTMs are studied.Among them,acylation in lysine residues is a frequently occurred modification,such as methylation,propionylation,malonation,butyrylation,succinylation,and acetylation.There are many studies available about PTM modifications(phosphorylation,methylation and so on)in both prokaryotes and eukaryotes.Whereas,there is no published report about the role of protein acetylation modification on bacterial iron homeostasis mechanism and so far.There are several scientific questions need to be clarified:(1)Under iron limitation,how many proteins are altered in expressions and distribution in whole cell proteomics and acetylome analysis;(2)What is the function of lysine acetylation-PTM in the regulation of iron homeostasis?Therefore,the objectives of this study are quantitative proteomics,acetylome analysis,and their comparisons,and functional analysis of lysine modified protein under iron limitation conditions.To answer the above questions,the current study has been designed and studied in A.hydrophila ATCC7966.For the study of whole cell quantitative proteomics and acetylome analysis under iron-limited conditions.Three independent biological repeats of each sample with and without 200 ?M 2,2'-dipyridyl(DIP)iron limited treatment has been studied using label-free quantitative(LFQ)combined with high-resolution mass spectrometry methods.Furthermore,for the quantification and acetylome analysis,various statistical and bioinformatics tools were used.Raw data were analyzed by MaxQuant,proteomics software with the Andromeda.Then statistically and bioinformatically,proteomics data were compared and analyzed.The whole cell and lysine-acetylated proteins were studied to investigate the biological processes,cellular components,molecular functions,KEGG pathways and protein to protein interactions.Also,whole cell and lysine acetylome proteomics were compared and demonstrated from different angles of distribution using bioinformatics and statistical analysis.Moreover,the proteomics analysis was validated by co-immunoprecipitation and western blotting method.For the validity of proteomics results,protein specific antibodies were used and the expression of lysine acetylation modified proteins were analyzed using anti-lysine antibodies in iron limitation.Finally,post-translational modifications effect was studied by the site-direct mutagenesis mutants on lysine acetylated sites and the survival rates of these mutants were analyzed in iron limitations.The results were as below:1.The whole cell quantitative proteomics analysis was performed in iron-limited conditions by label-free quantitative(LFQ)combined with high-resolution mass spectrometry methods.We have identified 1376 proteins with a significant conservative threshold(confidence level?95%)and 172 altered proteins.Besides that,172 altered proteins were acknowledged with a fold change? 2.0 and p-value?0.05,which comprised 61 up-regulated and 111 down-regulated proteins.Bioinformatics analysis showed that differentially expressed proteins are involved in various biological,cellular and molecular functional processes,such as oxidoreductase process,cellular amino acid metabolic processes,oxidoreductase complex,macromolecule complex and iron-sulfur binding proteins as well as structural molecular activity.In addition,the KEGG and protein-protein interactions analysis in several metabolic pathways like biosynthesis of secondary metabolites,TCA cycle,carbon metabolism,metabolic pathways oxidative phosphorylation,biosynthesis of antibiotics purine metabolism,glycine,serine,and threonine metabolism,were analyzed and enriched.Furthermore,proteomics data has been validated by western blotting methods,results show that outer membrane protein OprM(AOKMB2),Acriflavin resistance protein AcrA(AOKMB4),hemolysin co-regulated protein(AOKJA9),and TonB-dependent siderophore receptor(AOKFG8)proteins up-regulated and flagellin protein(AOKIY3),pyruvate kinase protein pyk-2(AOkHY7),and catabolite gene activator protein Crp(AOKGX7)proteins were down-regulated,which were consistent with our proteomics results.2.For the study of the effect of acetylation modifications on iron homeostasis,lysine-acetylated proteins of A.hydrophila in iron-limited conditions were studied with a combination of high-affinity purification and highly sensitive mass spectrometry technologies.Overall,941 Kace proteins were identified via MaxQuant software with a highly conservative threshold(FDR<1%).Furthermore,1606(57.1%)Kace sites altered in 2810 total Kace sites and 720(76.5%)Kace proteins altered(fold change±2.0 and p-value<0.05)in total 941 Kace proteins with 50 up-regulated and 686 were down-regulated in iron-limited conditions.The altered acetylated proteins were classified into different groups of molecular functions,cellular processes and the biological processes by bioinformatics analysis.These acetylated proteins are involved in different metabolic pathways and protein-protein interaction networks.The various metabolic processes proteins are largely up-regulated including propanoate metabolism,butanoate metabolism,metabolic pathways,oxidative phosphorylation,riboflavin metabolism,and alanine metabolism.While some metabolic and transportation associated proteins were largely down-regulated such as metabolic pathways,ABC transporter,biosynthesis of secondary metabolites and microbial metabolism in a diverse environment.Furthermore,acetylated proteomics data has been confirmed by western blotting and immunoprecipitation(IP)methods under iron-limited condition.Six selected Kace proteins results analysis showed the significant down-regulation of LuxS(AOKG57),membrane protein insertase YidC(AOKQZ7),chaperone protein DnaK(AOKMI6),phosphate acetyltransferase(AOKGN7),cytochrome C(AOKLXO)and Fur(AOKIG7)in acetylation level but unchanged in whole cell protein level.In general,our results reveal that the lysine acetylation modification in protein plays an important role in the regulation of iron homeostasis in A.hydrophila.3.The lysine acetylation modifications effect was investigated in Crp under iron stress.The biological characteristics between wild-type,KO and lysine modified site mutants were compared under iron limitations.Our results suggest that KO strain has a slower growth rate than wild-type.Besides that,the lysine acetylated site mutants of ?Aha-crp at 103R/Q,133R/Q,155R/Q,and 191R/Q were generated.The growth survival rate of these two types of acetylated(R)and deacetylated(Q)mutants were detected and R type mutants significantly decreased their survival rates when compared with Q type mutants.These results indicate the lysine acetylation of Crp may play an important role in iron homeostasis as a positive regulator.Furthermore,the mRNA level expression of hfq and fur in serials of crp derivatives show that the acetylation modification on K103 and K191 sites may positively but site K133 negatively regulate the expression of general RNA-binding protein Hfq.And the acetylation modification on K103 and K191 sites may positively but site K133 and K155 negatively regulate the expression of ferric uptake regulation protein Fur under iron-limited condition.In conclusion,we systematically compared the differential expression of whole cell proteins and Kace modification proteins in the iron-limited condition in A.hydrophila using proteomics methods.The following survival rates of crp and its site direct mutagenesis derivatives reveal that the lysine acetylation modification on proteins may play important roles in bacterial iron homeostasis.