The C2-streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and T-leukemia cells

Abstract

Macrophages are tightly associated with inflammatory diseases as well as carcinogenesis, and therefore represent promising targets for drug delivery and gene transfer. We have recently established a novel protein delivery system based on the binary C2 toxin of Clostridium botulinum and streptavidin, allowing the uptake of exogenous biotinylated molecules into mammalian cells. Here, we applied this C2-streptavidin delivery system to macrophages and other leukocytes. First, the effect of wild-type C2 toxin on different leukocyte cell lines was tested, indicating no differences in sensitivity. Next, the uptake and stability of the engineered C2-streptavidin was analyzed in macrophages and Jurkat T-cells, showing internalization into the cytosol of both cell types with similar kinetics. The transporter did not exhibit any cytotoxic effect and did not interfere with phagocytosis in primary human macrophages. The C2-streptavidin system promoted specific uptake of biotinylated fluorophores into the cytosol of macrophages as revealed by confocal microscopy. In addition, flow cytometry analysis showed a significantly enhanced uptake of biotinylated fluorescent tracers in Jurkat leukemia cells mediated by the C2-streptavidin transporter. Our results demonstrate that C2-streptavidin is a functional delivery system for transport of biotinylated molecules into macrophages and other leukocytes without compromising cell viability and intrinsic functions such as phagocytosis.

A Cell-Permeable Fusion Protein Based on Clostridium botulinum C2 Toxin for Delivery of p53 Tumorsuppressor into Cancer Cells

“…GST-C2IN-p53 accumulated in the cytosol after 5 h, but was no longer detectable after 24 h (data not shown). In consistency, a cytosolic degradation has also been reported for other C2IN-based fusion proteins, such as C2IN-C3 [36], C2IN-SpvB [37] and C2IN-streptavidin [38]. Upon internalization into HeLa and A549 cells GST-C2IN-p53 was primarily detected in the cytoplasm and less prominently in the nucleus as observed by confocal microscopy.…”

Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines

“…Mutants of our study may not able to catalyze ADP-ribosylation reaction owing to their poor ligand efficiency and binding affinity of NAD + to the binding core. Enzyme deficient mutants of these toxins have been used as drug delivery systems and molecular tools for studying many pathophysiological mechanisms, but no one has been evaluated them as vaccine candidates (Dmochewitz et al, 2013;Fahrer et al, 2010aFahrer et al, , 2010bRohrbeck et al, 2012). A single amino acid substitution/mutation reported as an evolutionary constraint that can change antigenicity of many bacterial and viral antigens (Foley, 2015;Koide et al, 2005;Liang et al, 2010;Meyer and Wilke, 2015;Yoo and Deregt, 2001).…”

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

“…Many of the reported examples, which mainly consist of the chimeric toxins constructed on the basis of the active component of C2 toxin, C2I are intensively reviewed elsewhere [9,318]. More recent reports describe genetically engineered chimeric C2IN-streptavidin complexes, which were designed to delivered biotin-labeled molecules into the cytosol of diverse eukaryotic cell lines by the binding/translocation subunit of the toxin [361,362,363]. The C2-streptavidin delivery system was used to internalize biotin-labeled p53 tumor suppressor into different mammalian cell lines, including human tumor cells [364].…”