| The outer membrane proteins(OMPs)of Gram-negative bacteria are integral for maintaining cellular structure stability,controlling nutrient and ion transport,and contributing to bacterial virulence and defense mechanisms.Among these OMPs,Cir A is a specific siderophore channel and colicin Ia receptor,plays a significant role in maintaining bacterial iron homeostasis,biofilm formation,and bacterial drug resistance.However,the biological function and intrinsic mechanism of action of Cir A protein have yet to be fully understood.In this study,the physiological phenotype of the Ah Cir A protein of Aeromonas hydrophila was evaluated after ahcirA deletion.Quantitative proteomics was used to study protein expression differences between the ΔahcirA and wild-type(WT)strains.Molecular biology techniques were used to verify the role of Ah Cir A in nutrient transport.Quantitative proteomics was also used to study protein expression differences between the ΔahcirA and WT strains under different antibiotic stresses.These studies provide a theoretical understanding of the molecular mechanism of Ah Cir A as a multifunctional protein affecting the important physiological functions of bacteria.The main study results are presented as follows:The laboratory-preserved ΔahcirA strain was first used to construct the rescued strain ΔahcirA:: ahcirA and the negative control strainΔahcirA:: vector,and the physiological phenotypic changes were determined.The ΔahcirA strain had significantly enhanced swimming and swamming ability,reduced biofilm formation ability,decreased resistance to norfloxacin and enrofloxacin,and increased resistance to kanamycin and streptomycin compared to the WT strain.The rescued strain could restore its wild-type phenotype,suggesting that Ah Cir A protein plays an important role in the physiological functions of A.hydrophila.A DIA based quantitative proteomics method were used to compare differentially expressed proteins between the WT and ΔahcirA strains.The results showed that 113 differentially expressed proteins were identified in the ΔahcirA strain compared to the WT strain including 106 increasing and7 decreasing abundances.Gene ontology(GO)analysis of these differentially expressed proteins revealed that ΔahcirA enriched 51 biological process-related proteins,including cellular amino acid catabolism,small molecule metabolism carboxylic acid catabolism,organic acid catabolism,and oxidative metabolism.KEGG analysis found that the deletion of ahcirA involves 14 metabolic pathways,including protein changes in several metabolic pathways such as valine,leucine,and isoleucine degradation,glyoxylate and dicarboxylate metabolism,and arginine and proline.Further analysis revealed that among the upregulated proteins,there were 14 proteins related to the transport of nutrients such as amino acids and dipeptides,accounting for 13.20% of the total upregulated proteins.This suggests that Ah Cir A may be involved in the process of nutrient transportTo verify the hypothesis,this paper examined the growth curve of ahcirA strains under specific nitrogen source culture conditions.The growth rate of the ΔahcirA strain was significantly slower than that of the WT strain when using histidine,arginine,and dipeptide(Ala-Glu)as the sole nitrogen source,but there was no significant difference in nutrient-rich culture medium.Far-western assays confirmed that Ah Cir A could interact with histidine,arginine,and dipeptide/oligopeptide,as well as with periplasmic binding proteins Ah Dpp A and Ah His J.These results confirmed that Ah Cir A protein is involved in the transport of nutrients other than iron carriers.DIA based quantitative proteomics was used to compare differential protein expression between WT and ΔahcirA strains under different antibiotic stresses.Compared to the wild-type under the same antibiotic stress conditions,479 differential proteins were identified in ΔahcirA under enrofloxacin stress,including 124 up-regulated proteins and 355 downregulated proteins.612 differential proteins were identified under norfloxacin stress,including 233 up-regulated proteins and 379 downregulated proteins.677 differential proteins were identified under kanamycin stress,including 250 up-regulated proteins and 427 downregulated proteins.522 differential proteins were identified under streptomycin stress,including 209 up-regulated proteins and 313 downregulated proteins.Further bioinformatics analysis found that the types of differential proteins were similar under the same classification of antibiotic stress.Common differential proteins were involved in histidine metabolism,carbon metabolism,and pyruvate metabolism under enrofloxacin and norfloxacin stress,while microbial reactions under different environments,secondary metabolite biosynthesis,amino acid biosynthesis,and histidine metabolism were involved under kanamycin and streptomycin stress.These results suggest that Ah Cir A protein plays an important role in the multidrug resistance of A.hydrophila.Additionally,q-PCR was used to partially verify the expression of differential proteins.In summary,this paper systematically evaluated the physiological function of Ah Cir A protein in A.hydrophila,and compared changes in protein expression between ahcirA deletion strains and wild-type strains under normal culture conditions and different types of antibiotic stresses using quantitative proteomics technology.It confirmed that Ah Cir A protein is involved in the transport of amino acids,dipeptides,and other nutrients,and plays a role in bacterial resistance.The above research has important scientific significance for the prevention and treatment of pathogenic A.hydrophila and the development of new antibacterial targets in the future. |
