The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes

Abstract

The binary C2 toxin of Clostridium (C.) botulinum consists of two non-linked proteins, the enzyme subunit C2I and the separate binding/transport subunit C2II. To exhibit toxic effects on mammalian cells, proteolytically activated C2II (C2IIa) forms barrel-shaped heptamers that bind to carbohydrate receptors which are present on all mammalian cell types. C2I binds to C2IIa and the toxin complexes are internalized via receptor-mediated endocytosis. In acidified endosomal vesicles, C2IIa heptamers change their conformation and insert as pores into endosomal membranes. These pores serve as translocation-channels for the subsequent transport of C2I from the endosomal lumen into the cytosol. There, C2I mono-ADP-ribosylates G-actin, which results in depolymerization of F-actin and cell rounding. Noteworthy, so far morphological changes in cells were only observed after incubation with the complete C2 toxin, i.e., C2IIa plus C2I, but not with the single subunits. Unexpectedly, we observed that the non-catalytic transport subunit C2IIa (but not C2II) alone induced morphological changes and actin alterations in primary human polymorphonuclear leukocytes (PMNs, alias neutrophils) from healthy donors ex vivo, but not macrophages, epithelial and endothelial cells, as detected by phase contrast microscopy and fluorescent microscopy of the actin cytoskeleton. This suggests a PMN selective mode of action for C2IIa. The cytotoxicity of C2IIa on PMNs was prevented by C2IIa pore blockers and treatment with C2IIa (but not C2II) rapidly induced Ca2+ influx in PMNs, suggesting that pore-formation by C2IIa in cell membranes of PMNs is crucial for this effect. In addition, incubation of primary human PMNs with C2IIa decreased their chemotaxis ex vivo through porous culture inserts and in co-culture with human endothelial cells which is closer to the physiological extravasation process. In conclusion, the results suggest that C2IIa is a PMN-selective inhibitor of chemotaxis. This provides new knowledge for a pathophysiological role of C2 toxin as a modulator of innate immune cells and makes C2IIa an attractive candidate for the development of novel pharmacological strategies to selectively down-modulate the excessive and detrimental PMN recruitment into organs after traumatic injuries.

Clostridium botulinum C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages

“…Cell rounding also occurred when U-DCS cells were treated with C2I in combination with its activated B-component C2IIa, providing a robust and established control for successful cytosolic release of cargo ( Figure 7 a). In accordance with the literature [ 44 , 45 ], only minimal effects on cell viability/proliferation were detected for the delivered C2I ( Figure S6 ).…”

“…The proteins His_ eGFP_C3bot, His_ eGFP_C3bot E174Q , and His_ eGFP were produced as described previously [ 23 ]. C2II was produced as a His-tagged protein, as described in [ 45 ]. In general, filtered cell lysate containing the His-tagged proteins was incubated with preequilibrated (in NPI-20, see Section 5.3 ) PureCube 100 INDIGO Ni-agarose (Cube Biotech, Monheim am Rhein, Germany) overnight at 4 °C.…”

The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells

“…In the next step, the cellular uptake of His-tagged cargo and C2IIa was investigated in more detail. Hence, we treated HeLa cells with the established C2IIa pore-blocker quinacrine (Quina) [ 46 , 47 ] and the combination His_DTA/C2IIa ( Figure 2 a–c). Quina treatment blocked the cytosolic release of His_DTA as indicated by hampered cell rounding ( Figure 2 a,b) and diminished loss of cell viability ( Figure 2 c) compared to cells treated only with the combination His_DTA/C2IIa.…”

Cellular Uptake and Cytotoxicity of Clostridium perfringens Iota-Toxin

“…C2IIa induces the extracellular efflux of radioactive rubidium-86 ions, a radioactive analogue of K + , from living cells, indicating that C2IIa forms pores in cytoplasmic membranes [ 52 , 64 , 65 ]. CIIa alone induces morphological alterations and cytotoxicity in primary human polymorphonuclear leukocytes (PMNs) [ 66 ]. C2IIa rapidly causes Ca 2+ influx in PMNs, indicating that the pore formation of the C2IIa oligomer in cell membranes is important for membrane disruption and reduces PMNs chemotaxis [ 66 ].…”

“…CIIa alone induces morphological alterations and cytotoxicity in primary human polymorphonuclear leukocytes (PMNs) [ 66 ]. C2IIa rapidly causes Ca 2+ influx in PMNs, indicating that the pore formation of the C2IIa oligomer in cell membranes is important for membrane disruption and reduces PMNs chemotaxis [ 66 ]. On the basis of these investigations, the binding components of clostridial binary actin-ADP-ribosylating toxins induce cytotoxicity by pore formation in the cytoplasmic membranes of host cells.…”