Study Of The Iron Homeostasis Regulation Of Megalobrama Amblycephala And Its Effect On The Virulence Of Aeromonas Hydrophila

The capability of organisms such as pathogens and their hosts to synergistically flourish in their native environment heavily relies on their ability to coexist in dynamic balance with one another.Alterations in any of these three factors may disrupt the delicate dynamic balance between them,evoking anadverse affects to the host’s health.Megalobrama amblycephala is one of the economic fishes in China,has a series of excellent properties,and is widely welcomed by the market.The process of breeding megalobrama amblycephala under poor management and overpopulated facilities can facilitate pathogen infection from bacteria such as Aeromonas hydrophila,causing huge losses in the yield of marketable fish.Aeromonas hydrophila is a common pathogenic bacterium for aquatic animals that is known to cause fulminant hemorrhagic disease.Its pathogenicity is closely related to virulence factors,and it is generally considered a result of synergistic and multiple factors.Iron is an essential element of organisms normally found in trace amounts,which is responsible for maintaining conventional enzyme activity and electron transfer.Due to iron’s finite quantities and scarcity in the environment,a high efficiency of iron intake is critical to the survival and virulence of invasive and pathogenic microorganisms.When iron nutrition is insufficient,it will have a great influence on the pathogenic bacteria and the fish.The iron homeostasis of A.hydrophila and M amblycephala have been well studied over the years,and the mechanism of storage,absorption and transport has been clearly defined,however,most of the studies are limited to virulent gene expression,alterations to membrane proteins,and iron metabolism.These studies are commonly performed under limited iron conditions,with few studies on virulence evaluation,whole gene and protein expression.As a result,the mechanisms of moolecules competing with iron in the host cells of M amblycephala are not yet clearly understood.In this study,the transferrin gene was cloned and analyzed.Different stress models were simulated,the changes of iron homeostasis and immunity indexes of M.amblycephala were observed.It expounded the molecular mechanism of iron competition between A.hydrophila and M amblycephala and provided a certain reference for the treatment of bacterial haemorrhage produced in practice.Based on the iron competition in A.hydrophila and M.amblycephala,the iron stress model was established,and the iron deficiency environment was simulated.With the analyses of transcriptomic and proteomic data,the effects of iron restriction on the growth and virulence of A.hydrophila were evaluated in a more comprehensive way.This study mainly includes five parts presented as follows:1 Molecular cloning and expression analysis of Megalobrama amblycephala transferrin geneIn this study,we identified and characterized the Transferrin(Tf)gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila.Furthermore,we studied the iron binding properties of recombinant Tf.Under the operation of biological technology such as RACE and RT-PCR,the full-length M.amblycephala Tf complementary DNA(cDNA)consisting of(GenBank accession no.:KX698308)of 2245 bp was cloned containing a 1953 bp open reading frame(ORF)encoding 650 amino acid residues and flanked by a 68 bp 5’and a 204 bp 3’ untranslated regions(UTR).Predicted conservative structure illustrated that M.amblycephala Tf consisted of two conservative Tf domains.Amino acid sequence alignment revealed that M amblycephala Tf had high similarity with that of cyprinids deposited in Genbank,and phylogenetic analysis showed that M.amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix.Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ,being significantly higher than other tissues(P<0.05).In the liver,Tf mRNA expression in fish artificially injected with the pathogenic bacteria A.hydrophila was significantly upregulated,reaching a peak at 12 h post injection(hpi)and then decreasing afterward.The expression in FeC13-injected fish showed a similar tendency but reached a peak at 8 hpi.Meanwhile,fish serum iron significantly decreased following A.hydrophila injection,but increased to peak at 4 hpi and then decreased in FeCl3-injected fish.The recombinant M.amblycephala Tf showed iron binding capacity using CAS analysis.It is found that Tf participates in innate immunity and responds to changes in external conditions,such as pathogenic bacterial infection and iron concentration.2 Effect of stress on expression of iron homeostasis related genes and immune indexes in Megalobrama amblycephalaThe purpose of this study was to investigate the effects of stress on iron homeostasis related genes and immune function in fish.Different stress models were established by intraperitoneal injection of cortisol and lipopolysaccharide(LPS).The blood and liver were sampled at different time points after injection,the iron content in serum and liver was determined by spectrophotometry,and the expression of hepcidin(Hep)and Tf were detected by quantitative real-time PCR.The results showed that compared with the control group,the plasma cortisol and blood glucose levels increased significantly(P<0.05),and the plasma iron content decreased first and then increased,while the iron content of the liver increased first and then declined after cortisol and LPS injection.The results of gene mRNA analysis showed that the expression of Hep gene and Tf gene in liver of M amblycephala were increased first and then decreased after treatment with cortisol and LPS.In order to study the molecular mechanism of immune response of peripheral blood leukocytes under stress condition,the appropriate concentration(the concentrations of cortisol,Deferoxamine(DFO)and LPS were respectively 4 μg/ml,20 μg/ml,62.5μg/ml)was selected by cell activity test.The Reactive oxygen species(ROS),respiratory burst and immune response related genes were measured at 0,6,12 and 24 h after stimulation of leukocytes,to investigate the effect of stress cortisol,DFO,LPS on the activity of immune cells in vitro.It was found that all three kinds of stresses led to the increase of ROS and respiratory burst,and the expression of four immune genes,Hep,il-l,il-6 and TNF-α were all up-regulated.In summary,moderate cortisol and LPS can induce stress response and change of iron homeostasis in M.amblycephala,which can be dealt with decreasing serum iron content,increasing liver iron storage,and promoting transcription of related iron metabolism genes.Moderate cortisol,DFO,LPS stress also increased the immune ability of peripheral blood leukocytes cells.3 The transcriptomic analyses of Aeromonas hydrophila in vitro in limited iron conditionsThis study aimed to evaluate the changes of the A.hydrophila transcriptome under iron restriction.Six concentrations(0,100,200,300,400,and 500 μmol/L)of 2,2’-bipyridine(Bip),a iron chelating agent,in normal tryptic soy broth medium were set to detect the optimal concentration according to the growth curve of A.hydrophila NJ-35.The results showed that Bip had a strong restriction on the growth of A.hydrophila and could delay the time of entering the logarithmic phase and reduce the maximum when compared with the control group.When the Bip concentration was 500 μM,the growth of A.hydrophila was totally inhibited for at least 24 hours.Due to the significant inhibition and higher cells concentration,200 μmol/L Bip was chosen as the proper iron-limitation concentration for transcriptome analyses.After filtering with FDR,1,204 genes were found to be differentially expressed between the control and iron-limitated groups.In comparison,the quantity of down-regulated differentially expressed genes detected(603)was greater than that of the up-regulated genes(601).GO and KEGG enrichment analyses were used to classify the enriched DEGs between the control and iron-limitated groups using bioinformatics methods,and these DEGs are mainly related to bacterial energy metabolism,electron transfer and iron transport related pathways.To verify the accuracy of the transcriptome data,20 virulence genes(13 up-regulated and seven down-regulated genes)together with reference genes(rpoB)were selected for investigation based on their expressions,which were measured by real-time quantitative PCR(RT-qPCR),and the reliability of the transcriptome result was proven.4 The proteomic analyses of Aeromonas hydrophila in vitro in limited iron conditionsThis study aimed to evaluate the changes of the A.hydrophila proteome under iron restriction.The experimental material was the same as the transcriptome research.A total of 328236 second-order mass spectrograms were obtained by iTraq analysis,and a total of 16417 peptide segments were identified.The total number of identified proteins was 2244,of which 2012 proteins were quantified.236 differentially expressed proteins were identified under iron restriction stress,90 of which were up-regulated and 146 were down-regulated.The number of proteins that are down-regulated was higher than that of up-regulated.The enrichment analysis of differentially expressed proteins showed that these proteins were mainly related to enterocectin biosynthesis,iron absorption,ion transport,protein transport,energy metabolism and so on.It was found that the differential proteins were mainly concentrated in the protein groups such as energy biosynthesis,ABC transporter,etc.,which further indicated that A.hydrophila synthesized a large number of iron-related proteins to compete for iron in the transport environment under iron-limited stress.It provides a solid foundation for further expounding the mechanism of iron tolerance in A.hydrophila through energy translocation into cells and maintenance of self-growth and reproduction.5 Comparative transcriptomic and proteomic analyses and enzyme activity changes of Aeromonas hydrophila in vitro in limited iron conditionsThe purpose of this study was to investigate the changes of integration analyses of the transcriptome and proteome and the virulence of A.hydrophila under iron restriction.Integration analyses of transcriptomic and proteomic data revealed that almost all of the mRNA and protein levels did not vary above 2-fold.Overall,680 transcriptomes showed DEGs with no difference in proteins,while 35 transcriptomes showed different proteins but no difference in genes.Conversely,the expression of six genes and proteins was opposite.In summary,most of the trends in DEP abundance were consistent with the DEG data.Through clustering of virulence genes and proteins in A.hydrophila in iron-limited medium,and the results indicated a comprehensive impact and clear changes in the regulation of virulence factors.Determination of iron concentration showed that A.hydrophila enhanced iron absorption and transport under iron deficiency stress,leading to significantly higher intracellular iron content than normal group(P<0.05).Through the detection of virulence factors of A.hydrophila,we found that the total protease activity was significantly increased to 0.36 under iron deficiency condition,compared with the normal group 0.105(P<0.05),the lipase activity and blood-plate hemolysis has been increased,but the difference was not significant.Interestingly,the swimming ability of the bacteria was strong under iron limiting conditions,which could reflect attempts to move to areas with more suitable conditions.Through the artificial infection experiment showed that compared with the normal group,A.hydrophila cultured under iron limitation has stronger lethality on healthy M.amblycephala.In summary,the virulence of A.hydrophila was enhanced under the iron restriction.