BackgroundLegionellaspp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth,Legionellais released from the host cells as free legionellae orLegionella-filled vesicles. The vesicles supportLegionellato survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes ofAcanthamoebainfected byLegionella(ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape ofLegionellafrom theAcanthamoeba.MethodsAfter ingestion ofEscherichia coliandLegionella pneumophila, expression levels of target genes inAcanthamoebawere measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation ofLegionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain.ResultsACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion ofLegionellainAcanthamoeba. ACA1_114460- and ACA1_091500-silencedAcanthamoebafailed to form theLegionella-containing excreted vesicles.Legionellawas released as free legionellae from theAcanthamoeba. When the ACA1_362260 ofAcanthamoebawas silenced,Legionella-containing excreted vesicles were fused with the lysosome.ConclusionsThese results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 ofAcanthamoebaplayed important roles in the formation ofLegionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.Graphical Abstract