FtsH-dependent Processing of RNase Colicins D and E3 Means That Only the Cytotoxic Domains Are Imported into the Cytoplasm

Abstract

It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.Colicins are antibacterial toxins of Escherichia coli that are released into the extracellular medium in response to environmental stress conditions. Colicin D is an RNase that cleaves the anticodon loop of all four isoaccepting tRNA Arg (1). Colicin E3 cleaves 16 S ribosomal RNA (2). Both colicins provoke cell death by inactivating the protein biosynthetic machinery. Colicin producer cells are protected against both endogenous and exogenous toxin molecules by the constitutive expression of a cognate immunity (Imm) 3 protein, which forms a tight heterodimer complex with the nuclease domain of the colicin (3, 4).Colicin E3, like most colicins, has structurally identifiable N-terminal, central, and C-terminal domains. The first two domains are required for translocation and receptor binding, and they "hijack" certain functions of the target cell (i.e. the BtuB receptor and the Tol system) during colicin import. The C-terminal domain carries the cell-killing RNase function (5, 6). The colicin D protein has an unusual tripartite organization. The N-terminal domain is required for both the binding of the colicin to the high affinity, iron siderophore receptor FepA and for its subsequent translocation across the outer membrane. The 280-residue central domain is essential for uptake (and thus for cell killing), and it is also involved in the formation of the colicin D-ImmD protein complex (7). The passage of colicin D through th...

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

“…We have shown that only the cytotoxic C-terminal, catalytic domains are detected in the cytoplasm of cells exposed to DNase colicins, as in the case of RNase colicins [10]. The same 18.5 kDa processed form was detected in OmpT-deficient cells treated with colicins E2 or E7 (Figure 1).…”

“…As a consequence, only the cytotoxic C-terminal domains were detected in the cytoplasm of colicin D or E3-exposed cells [10], [11]. During the last few years, the essential, inner-membrane signal-peptidase LepB was shown to be required specifically for the toxicity of colicin D, whereas the inner-membrane protease FtsH is required for the toxicity of all nuclease colicins [12], [13].…”

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).…”

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI)

“…In fact, toxin activation provides an additional opportunity for target cells to evolve resistance to CDI UPEC536 because cysK is not an essential gene. In contrast, soluble bacteriocins also deliver toxic domains into bacteria to inhibit growth, but typically only require cell envelope proteins for recognition and translocation of the toxin (Cascales et al 2007;Chauleau et al 2011). One exception appears to be colicin M (an inhibitor of peptidoglycan synthesis), which is remodeled into its active conformation by a peptidylprolyl cis-trans isomerase (FkpA) in the periplasm of target cells (Schaller et al 1982;El Ghachi et al 2006;Hullmann et al 2008;Helbig et al 2011).…”