In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells

Abstract

DNase colicins E2 and E7, both of which appropriate the BtuB/Tol translocation machinery to cross the outer membrane, undergo a processing step as they enter the cytoplasm. This endoproteolytic cleavage is essential for their killing action. A processed form of the same size, 18.5 kDa, which corresponds to the C-terminal catalytic domain, was detected in the cytoplasm of bacteria treated with either of the two DNase colicins. The inner-membrane protease FtsH is necessary for the processing that allows the translocation of the colicin DNase domain into the cytoplasm. The processing occurs near residue D420, at the same position as the FtsH-dependent cleavage in RNase colicins E3 and D. The cleavage site is located 30 amino acids upstream of the DNase domain. In contrast, the previously reported periplasm-dependent colicin cleavage, located at R452 in colicin E2, was shown to be generated by the outer-membrane protease OmpT and we show that this cleavage is not physiologically relevant for colicin import. Residue R452, whose mutated derivatives led to toxicity defect, was shown to have no role in colicin processing and translocation, but it plays a key role in the catalytic activity, as previously reported for other DNase colicins. Membrane associated forms of colicins E2 and E7 were detected on target cells as proteinase K resistant peptides, which include both the receptor-binding and DNase domains. A similar, but much less proteinase K-resistant form was also detected with RNase colicin E3. These colicin forms are not relevant for colicin import, but their detection on the cell surface indicates that whole nuclease-colicin molecules are found in a stable association with the outer-membrane receptor BtuB of the target cells.

The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm

“…The inner-membrane protease FtsH (18) had previously been shown to be necessary for the toxicity of nuclease colicins (19), and we showed this requirement was at the level of colicin translocation across the inner membrane and that it is presumably responsible for processing of RNase or DNase colicins (9,20). This hijacked function of FtsH during colicin import is coherent with its usual biological role of degrading misassembled or damaged membrane proteins (21,22).…”

“…Function of LepB in Colicin D Import and Cell Killing-The loss of the interaction of mutated LepB(N274K) with colicin D showed above in vitro and the fact that this mutation specifically prevented both the colicin processing and the sensitivity of target cells to colicin D supported the structural role of LepB in colicin D cytotoxicity (9,20). We attempted to analyze whether (i) the recognition of LepB may be specifically dependent on conserved amino acids belonging to the LBS of colicin D and (ii) the direct interaction between colicin D LBS and LepB is an indispensable prerequisite for the FtsH-dependent colicin processing and subsequent cell killing.…”

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways

“…Two models have been proposed for the role of FtsH in colicin import. De Zamaroczy and coworkers have shown that FtsH is required for the release of colicin nucleases into the cell, and they hypothesize that the protease directly cleaves the domain (23,32). Kleanthous and coworkers have proposed that the ATP-dependent unfoldase activity of FtsH is used to pull the nuclease domain into the cell (24).…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…E-type colicins share the BtuB receptor to enter the target cell [103,104]. The Cterminal nuclease domains of the Colicin E2, E7, E8 and E9 (NColEs) serve as non-specific metallonucleases.…”