REGLAMENTO DE RÉGIMEN INTERIOR
CAPÍTULO SEGUNDO. ALTERACIONES DE LA CONVIVENCIA POR LOS ALUMNOS
In order to deliver the cytotoxic domain to its site of action in the target cell, colicin-like bacteriocins must first penetrate the Gram-negative cell envelope. To achieve this, these proteins possess additional domains, termed receptor binding and translocation domains, which deliver the cytotoxic domain to its site of action (6).
As the name suggests, the receptor binding domain of colicin-like bacteriocins mediates a high affinity interaction between the bacteriocin and its outer membrane receptor. For most bacteriocins studied thus far this is a TonB-dependent nutrient receptor, however two bacteriocins (colicin N and pyocin L1; pyocin L1 discussed in chapter 6) have recently been shown to utilise lipopolysaccharide (LPS) as a cell surface receptor (12,15). TonB- dependent receptors have a physiological role in the uptake of valuable nutrient substrates (for example iron siderophores or cobalamin) too large to enter the cell by diffusion through outer membrane pores (113). The specific receptor utilised varies between different groups of bacteriocins, with for example, the E colicins utilising BtuB, colicin IA utilising Cir, colicin M utilising FhuA and the S-type pyocins, S2, S3 and S4, which use receptors involved in uptake of the siderophore pyoverdine (Figure 1-10) (4,114). Despite the variety of receptors used, what is invariably true is that the interaction between the colicin-like bacteriocin and its receptor is highly specific, with disassociation constants of low µM to nM reported (6). Binding to its cell surface receptor serves to remove the bacteriocin from the three dimensional milieu, fixing it to the surface of the outer membrane (115). The bacteriocins then needs to enter the target cell, the
mechanism by which colicins-like bacteriocins achieve this crossing or ‘translocation’ can be used to divide these proteins into different groups, based on the domains utilised and the interacting partners required to gain entry to the cell (66). Early genetic screens were able to broadly classify colicins into two groups, A and B based on their requirement for components of either Tol or Ton complexes (116). The Ton and Tol systems are
evolutionarily related protein complexes, with divergent functions, that both play a physiological role in transducing stored energy from the proton gradient across the inner membrane to performed functions at the outer membrane (117,118). While the exact function of the Tol system in unknown, it has been is shown to be recruited to the septation apparatus during cell division, where it plays a role in stabilizing the outer
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membrane (113,119). More generally, mutants in genes encoding its components or its interacting partner Pal have been found to display defects in outer membrane
permeability and stability (120). The Ton complex provides the energy required for the import of iron containing siderophores and related substrates through TonB-dependent receptors. This occurs through interaction with a specific binding epitope (the TonB box) at the N-terminus of the receptor plug domain subsequent to binding of the substrate on the outer surface of the receptor (113). Subsequent to binding, it has been postulated though not conclusively proven that energy provided by the Ton complex through TonB causes removal or rearrangement of the receptor plug domain allowing ingress of the substrate into the cell (121).
It was subsequently discovered that colicins (and potentially colicin-like bacteriocins more generally) possess an intrinsically unstructured translocation domain at their N-terminus (IUTD). Removal of this domain, leads to a bacteriocin unable to kill cells, but still active in receptor binding and cytotoxic domain activity in vitro (66). The IUTD of both A and B group bacteriocins has been shown to deliver a binding epitope to the periplasm, which directly mediates binding to the Tol or Ton complexes. While the specific details of the process are still poorly understood, this interaction presumably allows the bacteriocin to access the energy transduced by these complexes to cross the outer membrane (122). In group B colicins this binding epitope has been shown to be analogous to the epitope which mediates interaction between TonB and the TonB-dependent receptors, suggesting molecular mimicry is involved in colicin-TonB interactions (123).
Colicin-like bacteriocins studied structurally adopt both highly elongated and more compact structures. The elongated colicins (E colicins and colicin Ia) bind to their TonB- dependent receptor via an elongated helical receptor binding domain, while the IUTD searches for a secondary receptor/translocator, the abundant outer membrane porin OmpF for the nuclease colicins (E2, E7, E8 and E9), and in the case of colicin E1 and Ia, TolC and Cir, respectively (4). The IUTD of the nuclease E-group colicins has been shown experimentally to thread through the pore of OmpF to deliver its binding epitope to the periplasm (122). Compact colicins like colicins M, B, D and pesticin have only been shown to bind to a single TonB-dependent receptor, which likely serves both receptor binding and translocator function (6,96). Interestingly, while different colicins recruit different receptor and translocation proteins, Tol/Ton dependence for importation can for some
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colicins be switched by changing the epitope on the IUTD from a Ton to a Tol specific sequence (124). This suggests colicins bind to cell surface receptors regardless of their endogenous function in order to capture transmembrane systems that transduce the PMF to the outer membrane. Upon entering the periplasm pore forming colicins, colicin M and pesticin, can exert their toxic effects, however the nuclease colicins still need to cross the inner membrane and enter cytoplasm of the cell. Limited experimental evidence on this process is available, however work by Walker et al in 2007, showed that both the net positive charge on the colicin E9 nuclease domain and the presence of anionic phospholipids in the outer membrane were important for colicin killing. Additionally, the authors demonstrated that FtsH, an inner membrane ABC transporter responsible for the retro-translocation of misfolded membrane proteins was necessary for colicin toxicity. The model proposed by the authors to explain this data suggests that interactions between the colicin DNAse domain and the inner membrane lead to destabilisation and partial unfolding of the protein, which facilitates its translocation into the cytoplasm via FtsH (125). A schematic of the colicin translocation process is presented in (Figure 1-11).
29 Figure 1-11 Cell entry and cytotoxicity of A- and B-group colicins
A-group colicins bind to their cell surface receptor, before recruiting the porin OmpF as a translocator. The IUTD of the colicins threads through the lumen of OmpF interacting with the Tol complex in the periplasm. This interaction with Tol facilitates entry of the bacteriocin, with energy provided by the proton motive force (4).
B-group also interact with a cell surface receptor, however with the exception of colicin IA which utilises a second copy of its receptor Cir as it’s translocator, a secondary translocator has not been identified. B- group colicins interact with TonB via a TonB-binding epitope to gain entry to the target cell (18). Figure 1-10 Binding of colicin E3 to its cell surface receptor BtuB
The crystal structure of BtuB in complex with the colicin E3 receptor binding domain show in atomic detail were the colicin interacts with BtuB. An overlay of the crystal structure of full length colicin E3, with the receptor binding domain from this complex shows the position and conformation of colicin E3 (and other E colicins which share its domain architecture) on the bacterial cell surface (8).
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