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

Abstract

Summary 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.

Bartonella henselae engages inside-out and outside-in signaling by integrin β1 and talin1 during invasome-mediated bacterial uptake

“…1B,C). Among these positively validated genes of the secondary screen were the small GTPases Cdc42 and Rac1, which were previously shown to contribute to invasome formation and therefore served as internal positive controls Truttmann et al, 2011). Furthermore, we identified the two tyrosine kinases Src and FAK, as well as the transmembrane receptor integrin b1, and the adaptor proteins paxillin, talin1 and vinculin as novel components essential for invasome formation.…”

“…The Bep proteins trigger all known VirB/D4-associated phenotypic changes of infected endothelial cells (ECs), including (1) inhibition of apoptosis, (2) activation of an NF-kB-dependent pro-inflammatory response, (3) capillary-like sprout formation of ECs embedded in a 3D matrix, and (4) bacterial invasion of ECs as well as epithelial cells by a unique cellular structure termed the invasome (Schmid et al, 2004;Schmid et al, 2006;Rhomberg et al, 2009;Scheidegger et al, 2009;Selbach et al, 2009). Invasome formation has been shown to depend on the translocation of BepG or the combination of BepC and BepF into the recipient cells Truttmann et al, 2011). Bhe internalization by the invasome route is a tightly controlled process, consisting of initial adherence, effector protein translocation, the formation of bacterial clusters and engulfment by plasma-membrane-derived membrane protrusions, eventually resulting in the complete internalization of the bacterial aggregates Rhomberg et al, 2009).…”

Strategies of exploitation of mammalian reservoirs by Bartonella species

“…Indeed, in vitro experiments have indicated that VirB/D4 and its effector proteins mediate a range of profound changes to parasitised endothelial cells [50,51], including (i) massive rearrangements of the actin cytoskeleton, resulting in the formation and internalisation of large bacterial aggregates by a unique “invasome” structure, (ii) NF-κβ-dependent pro-inflammatory activation leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death resulting in enhanced endothelial cell survival. Internalisation of bartonellae via invasomes (characterised by the formation of a bacterial aggregate on the cell surface, which is subsequently engulfed and internalised by an actin-dependant mechanisms [26]) is dependent on three VirB/D4 effectors, BepC, BepG, and BepF [52,53]. BepA has been shown to inhibit endothelial cell apoptosis through upregulation of cAMP levels in the cytosol [49] and it also promotes capillary sprout formation in an endothelial spheroid infection model, whereas BepG inhibits such sprouting [54].…”

Bartonella and Brucella--Weapons and Strategies for Stealth Attack

“…2) Truttmann et al 2011b). This process is dependent on integrin-b1 (ITGB1) binding and activation via Talin-1 inside-out signaling and on integrin-b1-mediated outside-in signaling by focal adhesion kinase (FAK), Src, vinculin, and paxilin (Truttmann et al 2011b). Invasome rearrangement of the F-actin cytoskeleton involves Arp2/3-dependent cortical F-actin polymerization by Rac1/Scar1/WAVE, Cdc42/ WASP pathways, and additionally by cofilin-1 in the case of BepC and BepF Truttmann et al 2011b,c).…”

“…In each case, this results in the formation of a compact F-actin ring composed of condensed stress fibers, and membrane protrusions on the host-cell surface (Fig. 2) Truttmann et al 2011b). This process is dependent on integrin-b1 (ITGB1) binding and activation via Talin-1 inside-out signaling and on integrin-b1-mediated outside-in signaling by focal adhesion kinase (FAK), Src, vinculin, and paxilin (Truttmann et al 2011b).…”