Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells

Abstract

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

Figure 1. Interaction of C2I with PA₆₃ channels.

A: Titration of PA₆₃ induced membrane conductance with C2I. The membrane was formed from diphytanoyl phosphatidylcholine/n-decane, containing about 5,500 channels. C2I was added at the concentrations shown at the top of the panel. Finally, about 40% of the PA₆₃ channels were blocked. The aqueous phase contained 1 ng/ml activated PA₆₃ protein (added only to the cis-side of the membrane), 150 mM KCl, 10 mM MES pH 6. The temperature was 20°C and the applied voltage was 20 mV. Note that C2I only blocks PA₆₃ channels when it is added to the cis-side of the membrane (data not shown). B: Lineweaver-Burke plot of the inhibition of the PA₆₃-induced membrane conductance by C2I. The fit was obtained by linear regression of the data points taken from Figure 1A (r² = 0.996654) and corresponds to a stability constant K for C2I binding to PA₆₃ of (3.98±0.063) ×10⁶ M⁻¹ for C2I binding to PA₆₃ (half saturation constant K_S = 251 nM).

Figure 2. Specificity of HUVECs intoxication by C2I using PA₆₃ in comparison to C2II.

HUVECs (5×10⁵ cells/100 mm well) were intoxicated with the indicated concentration of polypeptides during 48 (A) or 24 hours (B). A: Cells were intoxicated as indicated and levels of cellular ADP-ribosylated actin (ADPr-actin) were determined by in vitro ADP-ribosylation of cell lysates with C2I and radiolabeled [³²P]-NAD. Under these conditions ADP-ribosylated actin formed during the intoxication process is no longer labeled by in vitro ADP-ribosylation, which is indicated by decrease of radioactivity. Immunoblotting anti-beta-actin was performed in parallel on cell lysates to show actin protein levels engaged in the ADP-ribosylation experiments. ADP-ribosylation signals were normalized to actin immunoblot signals. B: Efficiency of cell intoxication. Cells were intoxicated and the number of round cells was directly assessed by counting floating cells. The columns show mean values of 5 independent counting for the individual conditions ± SEM (ns: non significant; * p<0.05 versus control).

Figure 3. HUVECs intoxication by EF and LF using C2II in comparison to PA.

All intoxication experiments were performed on HUVEC monolayers. Cells were treated overnight with either 1 µg/ml of LF or EF in the presence or absence of different amounts of PA or C2II, as indicated. A: Immunoblot anti-MEK2 showing the effect of MEK2 proteolysis by LF. 30 µg of total protein lysate were resolved on 12% SDS-PAGE. Anti-beta-actin immunoblot shows protein loading. MEK2 signals were normalized to actin. B: Graph shows measure of cyclic AMP (cAMP) cellular concentrations, expressed as pmol/ml. Mean values of two independent experiments ± SEM (ns: non significant and * p<0.05).

Figure 4. EF-mediated block of C2I-C2II intoxication in vivo (cell-based assay.

EF-mediated inhibition of C2II-promoted C2I uptake into HUVEC cells. Cells were intoxicated with different concentrations of the binding component C2II and the effectors EF and C2I, as indicated, and the number of intoxicated cells was directly assessed by counting round cells. One representative experiment showing mean values of 5 independent counting for each condition.