Virulence plasmid of Aeromonas hydrophila induces macrophage apoptosis and helps in developing systemic infection in mice

Pathogenic Aeromonas hydrophila Strain AO1 bears a 21 kb plasmid encoding several virulence determinants. Infection studies revealed that this isolate induced cytotoxicity in BALB/c mice splenic macrophages involving reactive oxygen species generation. DNA gel, Hoechst 33342, annexin-V and TUNEL assay documented macrophage death induced by 21 kb plasmid bearing isolates to be apoptotic in nature. Apoptosis induced by the plasmid bearing isolates involved initiator caspase-8 and caspase-9 and executed by effector caspase-3. ELISA revealed the wild-type isolate as weak inducer of pro-inflammatory cytokine IL-1β. Oral infection with wild-type isolates caused systemic infection in BALB/c mice. With plasmid curing the isolate looses several virulence attributes including cytotoxic potential. The cured isolate induced significant amounts of IL-1β from infected macrophages, disseminated into Peyer's patches, spleen and liver but never attained the bacterial loads recorded with wild-type isolates and were rapidly cleared. Transformation of 21 kb plasmid helped the cured bacteria regain wild-type virulence attributes, apoptotic potential and ability to cause systemic infection in mice. Thus the 21 kb plasmid is a virulence factor in mice. It helps in suppressing the production of pro-inflammatory cytokine IL-1β and induced apoptosis of host macrophages enabling A. hydrophila to evade host immune responses and establish systemic infection in mice.