Combined action of the type IV secretion effector proteins BepC and BepF promotes invasome formation of Bartonella henselae on endothelial and epithelial cells

Bartonella henselae (Bhe) can invade human endothelial cells (ECs) by two distinguishable entry routes: either individually by endocytosis or as large bacterial aggregates by invasome‐mediated internalization. Only the latter process is dependent on a functional VirB/VirD4 type IV secretion system (T4SS) and the thereby translocated Bep effector proteins. Here, we introduce HeLa cells as a new cell system suitable to study invasome formation. We describe a novel route to trigger invasome formation by the combined action of the effectors BepC and BepF. Co‐infections of either HUVEC or HeLa cells with the Bep‐deficient ΔbepA‐G mutant expressing either BepC or BepF restores invasome formation. Likewise, ectopic expression of a combination of BepC and BepF in HeLa cells enables invasome‐mediated uptake of the Bhe ΔbepA‐G mutant strain. Further, eGFP–BepC and eGFP–BepF fusion proteins localize to the cell membrane and, upon invasome formation, to the invasome. Furthermore, the combined action of BepC and BepF inhibits endocytic uptake of inert microspheres. Finally, we show that BepC and BepF‐triggered invasome formation differs from BepG‐triggered invasome formation in its requirement for cofilin1, while the Rac1/Scar1/WAVE/Arp2/3 and Cdc42/WASP/Arp2/3 signalling pathways are required in both cases.