Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity

Abstract

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-β4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.

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

“…As a third cargo molecule, we tested the enzyme domain of the Photorhabdus luminescens toxin complex (amino acids 680–960). TccC3hvr is well suited as a cargo protein, since it specifically ADP-ribosylates F-actin, which causes characteristic membrane blebbing, breakdown of the cytoskeleton, and cell rounding [ 36 , 53 ]. Untagged (TccC3hvr), N-terminally His-tagged (His_TccC3hvr), and C-terminally His-tagged (TccC3hvr_His) versions of TccC3hvr were generated (for primary structures, see Supplementary Figure S1c ).…”

Stabilization of F-actin bySalmonellaeffector SipA resembles the structural effects of inorganic phosphate and phalloidin

“…Sequences were verified by Sanger DNA sequencing [Genomics Shared Resource, The Ohio State University Comprehensive Cancer Center (GSR OSUCCC)]. Recombinant proteins were purified by immobilized metal affinity chromatography (IMAC) as previously described 30 using HisPur cobalt resin (Thermo Scientific). Before elution, proteins were subjected to on-column cleavage using TEV protease at a 1:20 mole ratio to 6xHis-tagged protein (estimated from resin binding capacity) overnight at 4°C.…”

“…His-tagged mouse CapZ heterodimer construct consisting of α1 and β2 CapZ subunits in pRSFDuet plasmid was a gift from Dr. J. Cooper. 6xHis-tagged CapZ was purified as previously described 30,49 using HisPur Cobalt resin (Thermo Scientific), and separated from high-molecular weight contaminants by size-exclusion chromatography using a Sephacryl S-200 HR column (Cytiva/GE Healthcare). The purified CapZ was flash-frozen in liquid N 2 and stored at −80 °C in a buffer containing 20 mM Tris-HCl, pH 8.0, 80 mM KCl, 10 mM DTT, and 20% glycerol.…”

“…Pyrenyl-actin depolymerization assays were performed as described 30 . Depolymerization curves were fit to single exponentials, and the decay rates were plotted against SipA C construct concentrations and fitted with a double exponential equation using Origin 2023 v10.0 (OriginLab Corporation) (Fig.…”

“…It is known that the stoichiometric doses of SipA protect F-actin against depolymerization by cofilin 14 . Yet, given the abundance of actin in the cell, stoichiometric toxin concentrations cannot be considered physiological and are insufficient for understanding the pathogenesis [30][31][32] . We found that the SipAC constructs containing Core but lacking Arm2 caused no stabilization of actin in thermal denaturation experiments (Fig.…”