A translocated protein ofBartonella henselaeinterferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome

Abstract

SummaryBartonella henselae enters human endothelial cells (ECs) by two alternative routes: either by endocytosis, giving rise to Bartonella-containing vacuoles or by invasome-mediated internalization. Only the latter process depends on the type IV secretion system VirB/VirD4 and involves the formation of cell surface-associated bacterial aggregates, which get engulfed by EC membranes in an F-actin-dependent manner, eventually resulting in their complete internalization. Here, we report that among the VirB/VirD4-translocated effector proteins BepA-BepG only BepG is required for triggering invasome-mediated internalization. Expression of BepG in the Bep-deficient DbepA-G mutant restored invasome-mediated internalization. Likewise, ectopic expression of BepG in ECs also restored invasome-mediated internalization of the DbepA-G mutant, while no discernable cytoskeletal rearrangements were triggered in uninfected cells. Rather, BepG inhibited endocytic uptake of B. henselae into Bartonella-containing vacuoles and other endocytic processes, that is, invasin-mediated uptake of Yersinia enterocolitica and uptake of inert microspheres. BepG thus triggers invasome-mediated internalization primarily by inhibiting bacterial endocytosis. Bacteria accumulating on the cell surface then induce locally the F-actin rearrangements characteristic for the invasome. These cytoskeletal changes encompass both the rearrangement of pre-existing F-actin fibres and the de novo polymerization of cortical F-actin in the periphery of the invasome by Rac1/ Scar1/WAVE-and Cdc42/WASP-dependent pathways that involve the recruitment of the Arp2/3 complex.

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response ofBartonella henselaeduring Human Endothelial Cell Infection

“…2C), the mutant should be deficient for known T4SS-mediated phenotypes of infected HEC. Indeed, the batR mutant did not provoke the formation of the ring-like F-actin rearrangements that are a characteristic of invasome-mediated uptake of B. henselae (17,41,46), while the complemented ⌬batR-pbatR mutant strain triggered these cytoskeletal rearrangements as efficiently as wild-type bacteria (Fig. 4C).…”

“…Specific attention has been paid to the BatR/BatS TCS and its role in the regulation of the VirB T4SS and its translocated effector proteins. The interaction of B. henselae with HEC has been extensively studied (14,15,17,19,46), and substantial progress has been achieved toward the understanding of the role of the VirB T4SS and its secreted Beps in the mediation of this interaction (41,44,45,49). However, the regulation mechanism(s) controlling the expression of these virulence factors and the global response of the bacteria during HEC interaction remain elusive.…”

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

“…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). This entry process is accompanied by the inhibition of endocytic uptake of single bacteria into Bartonellacontaining vacuoles (BCVs) ).…”

“…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).…”