Vascular dysfunction has been reported in human cases of anthrax, in mammalian models of Bacillus anthracis, and in animals injected with anthrax toxin proteins. To examine anthrax lethal toxin effects on intact blood vessels, we developed a zebrafish model that permits in vivo imaging and evaluation of vasculature and cardiovascular function. Vascular defects monitored in hundreds of embryos enabled us to define four stages of phenotypic progression leading to circulatory dysfunction. We demonstrated increased endothelial permeability as an early consequence of toxin action by tracking the extravasation of fluorescent microspheres in toxin-injected embryos. Lethal toxin did not induce a significant amount of cell death in embryonic tissues or blood vessels, as shown by staining with acridine orange, and endothelial cells in lethal toxin-injected embryos continued to divide at the normal rate. Vascular permeability is strongly affected by the VEGF/ vascular permeability factor (VPF) signaling pathway, and we were able to attenuate anthrax lethal toxin effects with chemical inhibitors of VEGFR function. Our study demonstrates the importance of vascular permeability in anthrax lethal toxin action and the need for further investigation of the cardiovascular component of human anthrax disease.endothelial ͉ vascular permeability ͉ VEGF

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).

Anthrax lethal toxin (LT) increases vascular leakage in a number of mammalian models and in human anthrax disease. Using a zebrafish model, we determined that vascular delivery of LT increased permeability, which was phenocopied by treatment with a selective chemical inhibitor of MEK1 and MEK2 (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK). Here we investigate further the role of MEK1/phospho-ERK (pERK) in the action of LT. Overexpression of wild-type zebrafish MEK1 at high levels did not induce detrimental effects. However, a constitutively activated version, MEK1 S219D,S223D (MEK1DD), induced early defects in embryonic development that correlated with increased ERK/MAPK phosphorylation. To bypass these early developmental defects and to provide a genetic tool for examining the action of lethal factor (LF), we generated inducible transgenic zebrafish lines expressing either wild-type or activated MEK1 under the control of a heat shock promoter. Remarkably, induction of MEK1DD transgene expression prior to LT delivery prevented vascular damage, while the wild-type MEK1 line did not. In the presence of both LT and MEK1DD transgene expression, cardiovascular development and function proceeded normally in most embryos. The resistance to microsphere leakage in transgenic animals demonstrated a protective role against LT-induced vascular permeability. A consistent increase in ERK phosphorylation among LT-resistant MEK1DD transgenic animals provided additional confirmation of transgene activation. These findings provide a novel genetic approach to examine mechanism of action of LT in vivo through one of its known targets. This approach may be generally applied to investigate additional pathogen-host interactions and to provide mechanistic insights into host signaling pathways affected by pathogen entry.Studies on anthrax pathogenesis have defined three toxin proteins secreted by Bacillus anthracis that can induce severe vascular and organ damage prior to lethality in experimental models (21, 52). Anthrax toxin proteins consist of an internalization subunit, protective antigen (PA), that can couple with either of two catalytic subunits: edema factor (EF) and lethal factor (LF) (11). PA has the ability to bind host receptors with high affinity and is responsible for the internalization of EF and/or LF into the cytoplasm. Biochemical studies have revealed that EF and LF have distinct enzymatic activities. EF increases cellular cyclic AMP (cAMP) levels, and LF is a metalloprotease that can cleave and inactivate MEKs (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK) (52). EF and PA constitute edema toxin (ET), while LF and PA function as lethal toxin (LT). Over the years, studies on LT have generated potent and consistent phenotypes in rodent models, including vascular leakage, lung edema, pleural effusions, and hemorrhage, before lethality (15,21,35,42). Thus, we became interested in further investigating the vascular actions of LT. To do this, we developed a zebrafis...

The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF N , the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF N retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20-to 50-fold more potent than monomeric LF N ) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.Bacillus anthracis causes pathology in infected human or animal hosts in part through the concerted action of three proteins, collectively termed anthrax toxin. The toxin consists of two enzymatic moieties, termed lethal factor (LF) and edema factor (EF), and a transport protein, termed protective antigen (PA), that delivers both LF and EF to the cytosol. LF is a 90-kDa zinc-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (6,20,24), and EF is an 89-kDa calmodulin-dependent adenylate cyclase (12). The intracellular actions of these enzymes impair the functions of various cells and can lead to the death of infected hosts.Delivery of LF and EF to the cytosol begins with binding of PA (83 kDa) to a receptor. Two receptors have been identified: ANTXR1 (for anthrax toxin receptor 1; also known as ATR/ TEM8) and ANTXR2 (for anthrax toxin receptor 2; also known as CMG2) (3, 23). Receptor-bound PA is proteolytically processed by furin or a furin-like protease (19), resulting in the removal of a 20-kDa fragment (PA20) from the N terminus. The remaining, receptor-bound fragment (PA63, 63 kDa) spontaneously oligomerizes, forming a ring-shaped heptamer, called the prepore, which is capable of binding up to three molecules of LF and/or EF with high affinity (17, 18). The resulting toxic complexes are internalized, and the low pH within the endosome promotes a conformational change in the prepore moiety that allows it to insert into endosomal membranes and form a pore. The conformational transition of the prepore to the pore depends on the association of the 2␤2-2␤3