Enhanced Survival And Gene Transcription Of Aeromonas Hydrophila In Response To Grazing Tetrahymena

Aeromonas hydrophila is distributed widely in aquatic environments.Virulence in A.hydrophila is multifactorial,and the pathogenesis of A,hydrophila still needs further research.The bactivorous ciliate protozoa is commonly found in the same aquatic environments.Predation by protozoa can influence bacterial populations,while the protozoa play an important role as reservoirs for bacteria.Once preyed by protozoa,most microbes are digested as food,but some microbes appear to be resistant to protozoa digestion and can even replicate within protozoa.Their co-evolutionary history suggests that adaptations ensuring bacterial survival should have emerged.Mechanisms that improve resistance to digestion by predators may well express themselves as virulence factors.In this study,we explored the fate of A.hydrophila when co-cultured with the bacteriovorous ciliate T.thermophila and investigated bacterial gene expression associated with the co-culture.Virulent A.hydrophila strains were reported to have ability to evade digestion in the vacuoles.We characterized genes expression in A.hydrophila under phagocytosis by Tetrahymena.116 genes were identified as up-regulated,including genes associated with metabolism,cellular process and signaling,information storage and processing,virulence factors,as well as some genes whose functions are currently unknown.This study will be a starting point for investigating the co-evolution of bacteria and protozoa.1.Effect of co-culture on Aeromonas and Tetrahymena growthIn this study,twelve A.hydrophila strains with differential virulence were co-cultured with T.thermophila for 12 h in poor medium.we observed that T.thermophila fed readily on all A.hydrophila strains,but high virulence Aeromonas strains could grow in the presence of T.thermophila,while avirulent strains were largely phagocytozed.T.thermophila grew well and cells were morphologically complete when co-cultivated with most davirulent Aeromonas strains.2.The fate of A.hydrophila after phagocytozed by Tetrahymena thermophilaTo investigate the fate of A.hydrophila in response to phagocytosis by T.thermophila,12 A.hydrophila strains of different virulence were intrinsically labeled with GFP by transformation with a plasmid.Then,LSCM was used to examine the predation of A.hydrophila by T.thermophila.After the addition of bacteria to the T.thermophila suspensions,green food vacuoles could be observed in nearly all T.thermophila cells.After co-culture for 12 h,a high proportion of the cells of virulent strains maintained their integrity and exhibited bright green fluorescent,while avirulent strains presented dispersed green fluorescent.To further analyze bacterial survival in vacuoles,GFP fluorescence in combination with PI viability staining was used in this study.Compared to avirulent strains,more viable bacterial cells of virulent strains were observed when co-cultured with T.thermophila.In addition,TEM observations also revealed that the intracellular virulent strains remained morphologically intact,whereas most of the intracellular avirulent strains exhibited an irregular shape.To support the speculation that virulent A.hydrophila strains may be able to evade digestion in the vacuoles of T.thermophila,the survival rates of twelve strains in vacuoles were calculated.After 12 h of co-culture,the survival rates of bacterial cells per vacuole in the virulent A.hydrophila groups were all higher than those of avirulent A.hydrophila groups.3.Transcriptome of A.hydrophila exposed to T.thermophila revealed by the selective capture of transcribed sequencesIn this study,we characterized genes expression in A.hydrophila under phagocytosis by Tetrahymena.116 genes were identified as up-regulated,including genes associated with metabolism,cellular process and signaling,information storage and processing,virulence factors,as well as some genes whose functions are currently unknown.Because protozoa share many features with mammalian phagocytes,particularly macrophages,a better understanding of protozoa-bacteria interactions will provide fascinating glimpses into host-pathogen relationships.In conclusion,our findings indicated that virulent A.hydrophila strains may have a better ability to evade digestion in T.thermophila vacuoles.This study will be a starting point for investigating the co-evolution of bacteria and protozoa.