Effects And Regulatory Mechanisms Of Tetrahymena Thermophila Predation On The Virulence And Environmental Adaptation Of Aeromonas Hydrophila

Aeromonas hydrophila is a gram-negative bacterium that is ubiquitously found in various aquatic environments.A.hydrophila is responsible for hemorrhagic sepsis in freshwater fish,which leads to huge economic losses in the global aquaculture industry.A.hydrophila is also associated with various severe diseases in other cold-and warm-blooded animals,including humans.The survival and pathogenicity of A.hydrophila is influenced by multiple factors.At present,most studies focused on the virulence factors or pathogenic effects on infected host,but rarely consider the roles of ecological factors on the evolution of pathogenic bacteria.Protozoa predation is the main cause affecting the survival and evolution of bacteria in the environment.To date,the effects and regulatory effects of protozoan predation on the virulence and environmental adaptability of A.hydrophila have not been reported.After coculturing A.hydrophila and Tetrahymena thermophila,in this study,we analyzed the effects on the anti-predatory adaptive characteristics and virulence of A.hydrophila.Proteomics analysis was carried out to elucidate the mechanism of predator-driven virulence evolution.On the basis of transcriptomics analysis,relevant genes involved in the evolution of adaptive characteristics of bacteria were identified.1.Effects of T.thermophila predation on environmental adaptation and virulence of A.hydrophilaThe antipredation evolved strains were selected after a long-term coculture of Aeromonas hydrophila NJ-35,a carp pathogenic strain,and T.thermophila.After subculturing with Tetrahymena,over 70%of A.hydrophila colonies were small colony variants（SCVs）,suggesting that A.hydrophila is able to make adaptive defenses in response to T.thermophila predation.Biological characteristics analysis indicated the SC Vs displayed enhanced biofilm formation,adhesion,fitness,and resistance to bacteriophage infection and oxidative stress as compared to the non-Tetrahymena-exposed strains.In contrast,the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and increased LD₅₀ in zebrafish.It is suggested that T.thermophila predation increased the environmental adaptability of A.hydrophila,but decreased the virulence.2.Effects of nitric oxide reductase NorV on anti-Tetrahymena predation and virulence of A.hydrophilaNorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide（NO）detoxification.Previously,we showed that the norV gene of A.hydrophila was highly upregulated after co-culturing with T.thermophila.In this study,qPCR showed that the norV gene was significantly up-regulated in the Tetrahymena exposed SCV strains.To investigate the roles of norV in resisting predatory protozoa and virulence of A.hydrophila,we constructed the norV gene-deletion mutant（ΔnorV）based on SCV1.After co-culture with T.thermophila,the anti-predation test showed that the anti-predation ability of ΔnorV was significantly reduced.Furthermore,the survival rate of ΔnorV in the food vesicles of T.thermophila decreased,suggesting that NorV is involved in the anti-Tetrahymena predatory defense of A.hydrophila.Considering that the bacteria changed from aerobic metabolism outside Tetrahymena to hypoxic metabolism after being engulfed by Tetrahymena,and the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions.By examining the biological characteristics of ΔnorV under diverse conditions,the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions compared to the wild type.The deletion of norV exhibited markedly decreased cytotoxicity,hemolytic and protease activities under aerobic and anaerobic conditions.Also,the hemolysin co-regulated protein（Hcp）in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type.Moreover,the inactivation of norV led to reduced resistance to predation by T.thermophila,decreased survival within macrophages and highly attenuated virulence in zebrafish.It is suggested that norV is diversely involved in important virulence-associated traits of A.hydrophila that are not completely dependent on nitric-oxide reductase activity,and the reduced virulence of SCV strains was not due to the upregulation of norV expression.3.Activation effects of the up-regulated lipoproteins of A.hydrophila in response to T.thermophila predation on host innate immunity Considering the outer membrane proteins（OMPs）are directly involved in bacterial interaction with the external surroundings,we detected the differentially expressed proteins in the outer membrane proteins of SCV1 strain（exposed to Tetrahymena）and B1 strain（not exposed to Tetrahymena）through quantitative proteomic analysis to investigate the molecular mechanisms involved in the environmental adaptability and virulence evolution of SCVs.A total of 38 differentially expressed proteins were identified in the SCVs,including 14 up-regulated and 24 down-regulated proteins,which are mainly involved in bacterial metabolism,transport,signal transduction,and flagella synthesis.In addition,SurA,Slp,and LpoB encoding gene-deficient strains were constructed by homologous recombination.Characterization analysis showed that they were involved in the biofilm formation,adhesion,antioxidant stress,and resistance to Tetrahymena predation of SCVs.That is,the up-regulated expressions of these three lipoproteins are related to the enhanced environmental adaptation of SCVs.Importantly,the absence of three lipoproteins in SCVs leads to increased virulence in zebrafish;Macrophages infection analysis found that the SCV strains caused an enhancement of mRNA levels of TLR2,TNF-α,IL-1β and IL-6 in RAW264.7 cells compared to those of the ancestor and B strains,while infection of macrophages with the three ΔsurA/SCV1,Δs/p/SCV1,and ΔlpoB/SCV1 mutants led to significantly decreased expression of TLR2,TNF-α,IL-1β and IL-6 compared to those detected in the SCVs.The results suggeated that SurA,Slp,and LpoB up-regulated in SCVs can be recognized by the cell’s TLR2,and then activate the cell’s pro-inflammatory immune response,thereby improving the resistance of the infected host.4.The downregulation of flagellar synthesis associated gene flhF affects the phage resistance of A.hydrophila in response to T.thermophila predationTranscriptomic analysis was performed between SCV1（Tetrahymena-exposed）and the control strain B1（non-Tetrahymena-exposed）.The data revealed 695 significantly differentially expressed genes（DEGs）,including 379 significantly down-regulated genes and 316 significantly up-regulated genes,in SCV1 compared with B1.A large proportion of these genes play roles in motility,metabolism,tansporter and signal transduction.To further elucidate the potential mechanism of phage resistence of SCVs,we characterized the roles of a regulatory gene flhF encoding a SRP-like GTPase that was significantly downregulated in SCV1.To investigate the relationship between flhF gene and phage resistance of SCV strains,a flhF gene-deletion mutant（ΔflhF）,a point mutation(flhF^T340A)and their corresponding complementary strains were constructed with B1（nonTetrahymena exposed）as the parent strain.In addition,a flhF overexpressing strain was constructed in SCV1.ΔflhF and flhF^T340Aappeared nonmotile and lack of polar flagellum,suggesting that the flhF gene is involved in flagella synthesis of A.However,no obvious alteration was obversed in motility and flagella synthesis of flhF-overexpressing strains in SCV1,suggesting that downregulation of flhF in SCV strains was not the key of the repressed motility and flagellum.Interestingly,similar to SCV1,compared to the parental strain B1,the ability to adsorb ΔflhF and flhF^T340Aof phage G65 is reduced;while the complementation or overproduction of flhF lead to an increased phage infection efficiency.Transmission electron microscopy（TEM）observations revealed that both the flhF mutant and SCV1 cells were covered with a large amount of secretions and displayed increased secretion of extracellular proteins,and the complementation or overexpression of flhF could reduce the level of protein secretion,suggesting that the main reason of phage resistance is that a large amount of protein secretion masks the phage receptor,which leads to adsorption inhibition.In summary,a variety of genes are involved in the adaptive evolution of Aeromonas hydrophila against Tetrahymena predation.Especially,flhF is involved in the phage resistance and protein secretion of A.hydrophila in response to protozoan predation,and the T340 of FlhF G3 domain is a key site of function;however,the regulation mechanism of flhF is not due to its roles in flagellar synthesis but protein secretion.In conclusion,this study investigated the influence of Tetrahymena predation on the virulence and environmental adaptability of A.hydrophila from the perspective of evolutionary biology,providing a new perspective for the control of diseases related to the infection of this bacterium.