EngineeringPhotorhabdus luminescenstoxin complex (PTC) into a recombinant injection nanomachine

Abstract

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

“…Utilizing bacterial toxin-based systems to deliver cargoes into the cytosol of cells has been explored with various toxins such as DT ( Arnold et al, 2020 ; Auger et al, 2015 ; Vidimar et al, 2020 ), BoNTs ( Bade et al, 2004 ; McNutt et al, 2021 ; Miyashita et al, 2021 ), anthrax toxin ( Milne et al, 1995 ; Young and Collier, 2007 ), Pseudomonas exotoxin A ( Ryou et al, 2016 ; Verdurmen et al, 2015 ), heat-labile enterotoxin ( Chen et al, 2015 ), Shiga toxins ( Ryou et al, 2016 ; Schmit et al, 2019 ), Clostridium botulinum C2 toxin ( Barth et al, 2002 ; Fahrer et al, 2013 ), and Photorhabdus luminescens Tc toxins ( Ng Ang et al, 2019 ; Roderer et al, 2019 ). The mechanisms of membrane translocation differ among these toxins.…”

“…Many of these toxins have previously been explored for delivery of protein cargoes into cells ( Auger et al, 2015 ; Bade et al, 2004 ; Barth et al, 2002 ; Beilhartz et al, 2017 ; Chen et al, 2015 ; Fahrer et al, 2013 ; Ho et al, 2011 ; McNutt et al, 2021 ; Miyashita et al, 2021 ; Ng Ang et al, 2019 ; Roderer et al, 2019 ; Ryou et al, 2016 ; Schmit et al, 2019 ; Vidimar et al, 2020 ). In general, the cargo proteins are fused with either the whole or a fragment of the A domain.…”

Report of the 24th Meeting on Signal Transduction 2021

“…The native TccC5 subunit is known to ADP-ribosylate RhoA at glutamine 63, causing the persistent activation of the GTPase [22]. By substituting the C-terminus of TccC5 with the C3bot from Clostridium botulinum or YopT toxin from Yersinia enterocolitica, the translocation machinery of Tc proteins could be re-designed to introduce a RhoA-inactivating toxin [23]. As another element in the PTC toolbox, Schmidt introduced the affibody technology for the modulation of Rho signaling.…”

The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins

“…Considering their excellent bioluminescent and biochemical properties and despite a few examples of applying Gaussia and Metridia luciferases as fusion proteins in in vivo assays [ 30 , 31 , 32 , 33 ], the number of reports on their use as labels in binding assays is still very limited [ 4 ]. This is mainly due to the difficulty of obtaining protein in E. coli cells because the correctly folded copepod luciferases must contain five intramolecular disulfide bonds [ 4 ].…”