The role of viruses such as hepatitis B virus, Epstein-Barr virus, and human papillomavirus in carcinogenesis is accepted, given that cell transformation can often be the effect of a single gene. Although it is generally accepted that certain bacterial infections contribute to carcinogenesis, the underlying molecular mechanisms are less well established. Helicobacter pylori is the first bacterial pathogen to be clearly associated with carcinogenesis. However, the formation of cancerous lesions is typically a protracted, multistage process that can take decades to reach its culmination. Consequently, there is currently no clear agreement about the molecular mechanisms(s) by which H. pylori might promote cancer development. The formation of noncancerous tumor lesions by pathogenic bacteria such as Bartonella spp. and Citrobacter rodentium occurs much faster and is therefore easier to assess experimentally. Nevertheless, the molecular mechanism(s) of tumorigenesis by these pathogens remains elusive.
We are studying tumorigenesis by Bartonella henselae for two reasons. First, vascular proliferation (that is, the process of pathological angiogenesis) triggered by this emerging pathogen not only is relevant as a clinical entity of infectious origin but also represents in more general terms a crucial step in the formation of any solid cancerous tumor as well as in the hematogenous spread of cancerous cells resulting in the formation of metastases in distant organs. Consequently, the angioproliferative process triggered by B. henselae has attracted considerable interest as a basic and clinically important biological system. Second, the establishment of genetic methods for B. henselae and the development of an in vitro infection model with human umbilical vein endothelial cells (HUVECs) resulted in the identification of several bacterial virulence factors that are likely involved in vascular tumor formation.
Over the past several years, my laboratory has been studying the role of the type IV secretion (T4S) system VirB/VirD4 of B. henselae in subverting endothelial cell functions that are essential for establishing chronic infection of the vasculature in association with vascular proliferation and remodeling. The T4S systems of pathogenic bacteria have evolved from bacterial conjugation systems that mediate the efficient spread of genetic traits, such as antibiotic-resistance genes, among bacteria. Bacterial pathogens targeting eukaryotic host cells have adapted these DNA transfer systems for the delivery of virulence factors. T4S systems represent supramolecular assemblies of 11 proteins that span both the membranes of Gram-negative bacteria and those of the host cell, allowing translocation of virulence factors from the bacterial cytoplasm directly into the host cell cytoplasm. We have shown that the VirB/VirD4 T4S of B. henselae mediates most of the prominent physiological changes associated with HUVEC infection. These changes include (1) massive rearrangement of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the so-called invasome structure; (2) nuclear factor kappaB–dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion; (3) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival; and (4) modulation of a VirB/VirD4–independent mitogenic signal derived from the bacterial outer membrane.
More recently, we identified seven bacterial effector proteins (BepA–BepG) that are translocated by this VirB/VirD4 T4S system into HUVECs. Intracellular delivery of BepA–BepG by this T4S system depends on a bipartite signal that consists of a conserved domain of 140 amino acids in the proximity of the C terminus and a short positively charged C-terminal tail sequence. In contrast, the N-terminal regions of BepA–BepG are composed of diverse domains that may mediate effector functions within endothelial cells. Notably, deletion of a chromosomal region encompassing all genes encoding BepA–BepG resulted in complete abrogation of the endothelial cell phenotypes associated with a functional VirB/VirD4 T4S system, indicating that the identified T4S effector proteins are important players in B. henselae–triggered vascular proliferation and remodeling.
Our knowledge of how these T4S effector proteins subvert endothelial cell functions is most advanced for BepA. We recently demonstrated that BepA is sufficient to protect HUVECs from apoptotic cell death triggered either artificially by actinomycin D or physiologically by cytotoxic T lymphocytes. The anti-apoptotic activity of BepA depends on proper localization to the plasma membrane and is mediated by augmentation of the intracellular pool of cyclic adenosine monophosphate (cAMP). The mechanism by which BepA augments cAMP levels (either directly by activating adenylate cyclases in the membrane or indirectly by interaction with upstream signaling molecules, such as G proteins or G protein–coupled receptors) represents an active area of research in our laboratory.
BepG was found to represent the only T4S effector that is required to trigger bacterial internalization via the invasome structure. This unique process of uptake of a large bacterial aggregate depends on the capacity of BepG to inhibit the internalization of individual bacteria via conventional phagocytosis. Invasome-mediated uptake is an F-actin–dependent process that requires the small G proteins Rac1 and Cdc42, the adaptor proteins Scar1/WAVE and WASP, and the Arp2/3 complex. The molecular link between the activation of F-actin remodeling through this signaling pathway, resulting in invasome formation, and the inhibition of phagocytosis of individual bacteria by BepG is unknown.
Interestingly, both BepA and BepG effectors modulate the angiogenic response. We have recently established a three-dimensional in vitro angiogenesis assay, using collagen gel–embedded HUVEC spheroids, that allows us to study the angiogenic properties of B. henselae. Upon angiogenic stimulation, the collagen-embedded HUVEC spheroids trigger the formation of capillary-like outgrowths that are a measure of angiogenic activity. Strikingly, BepA demonstrated a marked pro-angiogenic effect, while BepG interfered with in vitro angiogenesis. These opposing activities suggest that both effectors contribute to the fine-tuning of the vasoproliferative response to B. henselae infection.
In addition to the T4S effectors BepA–BepG, other pathogenicity factors of B. henselae have been implicated in the process of vascular proliferation and remodeling. These include a fibronectin- and lamin-binding surface adhesin (BadA) as well as an unidentified outer membrane component that exhibits mitogenic activity toward HUVECs. The established in vitro model of vascular tumor formation by B. henselae should help in the functional analysis of these pathogenicity factors and should further allow us to dissect the molecular and cellular processes involved in pathogen-triggered vascular tumorigenesis.
Our studies are also supported by a grant from the Swiss National Science Foundation.
Last updated September 2008
