Differential DNA methylation associated with virulence attenuation in Edwardsiella piscicida

A marked difference in virulence was observed between two subpopulations of the Edwardsiella piscicida FSW910410 strain, originally isolated from olive flounder (Paralichthys olivaceus). While the strain maintained under routine laboratory culture conditions retained high virulence, the same strain stored long-term at -80 °C lost its ability to induce disease in olive flounder. Comparative in vivo assays demonstrated complete avirulence in the frozen-stored subpopulation, contrasting with high mortality rates induced by the virulent counterpart. Despite over 99 % genomic sequence identity and identical virulence gene repertoires, the two subpopulations differed significantly in their ability to evade host immune responses. Whole-genome methylation profiling revealed comparable levels of N6-methyladenine (m⁶A) but notably reduced levels of cytosine methylation (m⁴C/m⁵C) in the avirulent subpopulation, particularly in genes associated with type III and type VI secretion systems (T3SS and T6SS). Quantitative RT-PCR analyses confirmed substantially lower expression of T3SS and T6SS genes in the avirulent strain. These results suggest that epigenetic modifications, especially cytosine methylation, may contribute to the observed differences in virulence, although additional regulatory mechanisms cannot be excluded. Collectively, the data highlight the complex interplay of genomic and epigenetic factors that may influence bacterial pathogenicity during prolonged storage or passage.