| Aeromonas hydrophila is a Gram-negative bacteria which is widely spread in aquatic environments.This bacterium can infect most freshwater fish and can cause hemorrhagic sepsis in fish,leading to large economic losses in the global aquaculture industry.TonB system is the energy supply system that most gram-negative bacteria use for nutrient absorption.TonB systems play an important role in the transport of iron,nickel,vitamin B12,zinc and carbohydrates.It is of great significance to further explore the nutrient metabolism and pathogenic mechanism of pathogenic bacteria.Whole-genome sequence analysis showed that there were three TonB systems in A.hydrophila NJ-35,and the functions of the three TonB systems were comprehensively analyzed.We conducted a comprehensive analysis of the functions of this three TonB systems.The role of TonB system in iron utilization,pathogenicity,drug resistance and environmental adaptability was discussed preliminary,and the mechanism of TonB system affecting flagellum formation was also analyzed,which is helpful for a deeper understanding of the role of TonB system in the life activities of A.hydrophila.1.Roles of three TonB systems in the iron utilization and virulence of the A.hydrophilaA homology search using the genome sequence of A.hydrophila NJ-35 displayed the presence of three sets of TonB systems in the chromosome of this strain.To determine the physiological function of the three TonB systems in A.hydrophila NJ-35,the single-,double-and triple-deletion mutants of tonB were constructed by homologous replacement in the wild-type strain.We found that tonBl and tonB2 were preferentially transcribed in iron-chelated conditions,where gene expression levels were approximately 8-and 68-fold higher compared with iron-rich conditions,respectively;tonB3 was consistently transcribed at a low level under iron-repleted and-depleted conditions.Only the TonB2 system was required to utilize iron-binding proteins.In addition,all three tonB genes were involved in A.hydrophila virulence in zebrafish,and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant.TonB2 plays a relatively major role in adhesion,motility and biofilm formation,while TonB3 is more involved in the anti-phagocytosis of A.hydrophila.In each observed phenotype,no significant difference was found between the single-and double-deletion mutants,whereas the triple-deletion mutant exhibited the most serious defects,indicating that all three TonB systems of A.hydrophila coordinately complement one another.In conclusion,this study elucidates the importance of TonB in iron acquisition and virulence of A.hydrophila,which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments.2.Catecholamine-stimulated growth of A.hydrophila requires the TonB2 energy transduction systemSome studies have found that bacteria can use the stress hormones secreted by the host to improve the absorption efficiency of iron ions in the host,which is conducive to the growth of bacteria.The purpose of this study was to investigate whether TonB system was involved in the stress hormone-mediated growth promotion of A.hydrophila.The present study demonstrated that norepinephrine(NE),epinephrine(Epi),dopamine(Dopa),and L-dopa stimulate the growth of A.hydrophila in iron-restricted media containing serum.NE exhibited the strongest growth stimulation,which could be blocked by adrenergic antagonists.Furthermore,it was demonstrated that NE could sequester iron from transferrin,thereby providing a more accessible iron source for utilization by A.hydrophila.The deletion of the TonB2 energy transduction system resulted in the loss of growth promotion by stress hormone,indicating that a specific TonB-dependent outer membrane receptor might be involved in the transport of iron from transferrin.Collectively,our data showed that catecholamine sensing promotes the growth of A.hydrophila in a manner that is dependent on the TonB2 energy transduction system.3.The influencing mechanism of TonB system on A.hydrophila flagella formation In order to explore the mechanism of TonB energy system influencing the motility of A.hydrophila,the effect of this system on bacterial flagellum formation was investigated.Electron microscopy showed that the △tonB123 strain totally lacked flagellation.The transcriptional levels of genes related to flagellum formation were significantly increased in△tonB1,△tonB2,△tonB3,△tonB12,△tonB13,△tonB23 and △tonB123.Lable-free quantitative proteomics techniques were used to investigate the differentily expressed proteins between the △tonB123 and wild-type strain.Among the differentially expressed proteins,24 were up-regulated and 30 were down-regulated.In total,the comparative proteomic analysis revealed that the identifed differentially expressed proteins are involved in transportation,signal transduction,cell division,glycometabolism and cell motility.Especially,the expression of polar flagellin structural proteins,FlaA and FlaB,have a 2.6-and 4.6-fold increase compared with wild-type strain.Meanwhile,qRT-PCR showed that the transcription levels of flaA and flaB were increased compared with the wild-type strain.These results indicated that both of the transcription and translation levels of flaA and flaB were increased when all of the three TonB systems were deleted.Western blot analysis showed that both FlaA and FlaB of the △tonB123 strain displayed a significantly smaller in size compared to that of the wild-type strain.The result suggest that it may be because flagellin has not been glycosylated that resulted in the absence of flagella formation in the△tonB123 strain.Far-western blot analysis showed that GST-FliS binds FlaA/B in the△tonB123 with a weaker affinity than that of in wild-type strain.The results indicated that all three tonB deletions may result in flagellin accumulated intracellularly by obstructing glycosylation.4.Effect of TonB energy system on the antimicrobial susceptibility of A.hydrophilaIn order to investigate whether TonB energy system is related to the drug sensitivity of A.hydrophila,the minimal inhibitory concentration(MIC)of TonB deleted strains against different antibiotics was determined.It was found that the sensitivity to macrolide antibiotics were decreased in △tonB12 and △tonB123.△tonB12 showed a 4-fold increased in MIC of tylosin and a 8-fold increase in MICs of erythromycin,roxithromycin,timicoxin.△tonB 123 showed a 8-fold increase in MIC of tylosin and a 16-fold increase in MICs of erythromycin,roxithromycin and timicoxin.PAβN and reserpine could remove the difference in sensitivity among △tonB12,△tonB123 and wild type strain,but CCCP had no effect on the antibiotic resistance.In addition,it was found that the deletion of three tonB genes could enhance the susceptibility of A.hydrophila NJ-35 to macrolide antibiotics in the presence of macB2,but there was no difference in the sensitivity to macrolide antibiotics between △tonB123 and wild type strain when deletion of macB2.These results suggest that the effect of TonB energy system on the antimicrobial susceptibility of A.hydrophila NJ-35 may be related to the MacA2-MacB2-TolC2 efflux pump.In summary,this study confirmed that the TonB energy system plays an important role in iron acquisition,pathogenicity and drug resistance of A.hydrophila,which laid a theoretical foundation for further study on the pathogenic mechanism of this bacteria. |
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