The bacterial protein toxin cytolethal distending toxin CDT is the only known bacterial protein toxin that acts as a DNase in the nucleus. Chapter 2 and Appendix 1 describe the discovery of host proteins that are essential for bringing the enzymatically active CDT subunit CDTB to its target location. With a newly developed haploid genetic screen, we identify for the first time cellular host factors that are essential for intracellular CDT trafficking, and reveal that CDTs from different bacterial species exploit different subsets of the human proteome to achieve intoxication.
Chaper 3 comprises the development of CDT as a tool via sortase-mediated trans-peptidation, to further explore CDT biology and the accompanying host cell biology. TMEM, not only an essential factor for E. In addition, we identify GRB2 as a novel CDT interactor, not uncovered in the haploid genetic screens possibly because of its essential nature for eukaryotic cell survival. In Chapter 4, bacterial protein toxin engineering using sortase is expanded to aerolysin, a model for pore-forming toxins. This is the first description of chemo-enzymatic engineering of aerolysin.
This enzymatic means to install labels at precise predefined locations and the generation of single and double-labeled aerolysin monomers enables investigation of the fate of individual N and C-terminal domains, while preserving the functionality of the toxin. Chapter 5 describes the generation of mouse monoclonal antibodies against the fluorophore Alexa Fluor AF by microengraving screening.
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As an accessory tool, it complements the bacterial protein toxin toolkit as it transforms the single purpose AF into a multi-functional handle that can be used not only in fluorescence detection applications, but also in biochemical approaches such as immunoprecipitation and immunoblotting. Bacterial protein toxins are very precise modifiers of specific host factors, they are exact instruments to dissect basic host cell physiology. Recent advances in molecular engineering allow us now to tweak, calibrate and target those tools better. For both diagrams, the CPD catalytic Cys and His are marked, as are processing site Leu residues see Table 1 found in unstructured segments between effectors indicated by arrows.
TOXINS TARGETING ACTIN AND SMALL RHO GTPASES
These repeats are postulated to form the translocation structure for transfer of centrally located effector domains to the cytosol  Figure 1B. Among the various MARTX toxins, a total of ten potential effectors have been identified, although each independent toxin has an assortment of only one to five . The best-characterized MARTX effector is the actin crosslinking domain ACD , which introduces a GluLys50 isopeptide linkage between actin monomers by a mechanism similar to that for glutamine synthetases  , . Early studies of the CGTs postulated that they would undergo enzymatic processing after exposure to low pH  , .
Subsequent in vivo studies demonstrated that only the kDa GT effector of TcdB is delivered to the cytosol, while a larger C-terminal fragment remains in the membrane fraction . This processing of TcdB also occurred in vitro after residue Leu, with a strict dependence upon addition of eukaryotic cell lysate  Figure 1A. These studies of TcdB were initially interpreted as indicating processing by a host cell—encoded protease, similar to the mechanism for maturation of diphtheria toxin and other bacteria toxins .
However, protein-free extracts also stimulated TcdB processing, indicating autocataytic cleavage .
In fact, it has been demonstrated that MARTX Vc is autoprocessed at four positions located before and after its three effector domains, resulting in the release of these domains from the holotoxin  ,  Figure 1B. This cytotoxicity was disrupted by mutation of a single Cys or His residue, and analysis of protein expression patterns revealed that the mutant proteins were the predicted size, while the wild-type protein was cleaved of its N-terminus. Studies with recombinant protein confirmed autoprocessing after Leu and, similar to TcdB, processing was strictly dependent upon addition of protein-free cell cytosol extracts .
Mutation of the critical Cys in the full-length toxin significantly reduced the ability of the toxin to induce actin crosslinking, confirming autoproteolysis due to this cysteine protease domain CPD enhanced toxin function . Furthermore, for TcdB, mutation of the analogous Cys and His residues reduced cytotoxicity of the full-length toxin, and disrupted processing of recombinant CPD protein as well  , .
Thus, it was recognized that both the CGTs and MARTX toxins share a common mechanism for autocatalytic processing inducible by protein-free eukaryotic cell cytosol and that autoprocessing is essential for optimal cytotoxicity. To identify the molecule in cell cytosol required to induce CPD for autoprocessing, cell extracts that stimulated processing of TcdB were fractionated and analysis of active fractions by mass spectrometry supplied spectra with similarities to inositol phosphates .
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Incubation of TcdB with several inositol phosphates indicated that InsP 6 induced the most efficient autoprocessing activity . As a signal molecule for the eukaryotic intracellular environment, InsP 6 also known as phytic acid is an excellent selection for bacterial toxins, since the molecule is ubiquitous in eukaryotes but absent in bacteria. Within mammalian cells, InsP 6 may function as a high concentration storage molecule for phosphate as it does in plant seeds or as a highly charged buffer for cation- or protein-dependent processes. More recent studies have linked InsP 6 to numerous cellular processes, including vesicle recycling, mRNA transport out of the nucleus, and as a co-factor for a DNA-dependent protein kinase .
Regardless of its normal function, InsP 6 is a molecule constantly present in high concentrations in the eukaryotic cytosol, assuring that induction of CGT and MARTX CPDs occurs only after completion of translocation of effector domains to the cytosol, regardless of whether translocation requires endocytosis or transfer directly across the plasma membrane. Mutational studies  ,  —  and analysis of four independent crystal structures  ,  ,  ,  revealed that binding of InsP 6 to the CPD involves contact of the six negatively charged phosphate groups within a positively charged pocket of the CPD Figure 2.
However, this difference in the structure of the binding pocket may in part account for variances revealed in studies of InsP 6 binding and CPD activation for the different CPDs. Interestingly, despite strong conservation of the critical Lys residues in the primary amino acid sequence, contacts with InsP 6 and the orientation of InsP 6 differ in the two structures.
Although the dissociation constant has not as yet been determined for TcdA, processing studies indicate that its ability to bind InsP 6 may be less efficient than TcdB.carlrareadjuda.ml
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The distance between the catalytic residues indicates that the Cys is not activated by protonation from His, but rather suggests that the Cys is substrate-activated by close alignment of the scissile bond, while the His functions solely to protonate the leaving group  , . The catalytic Cys and His residues are shown in pink with InsP 6 present at the backside of each structure in red.
The P1 Leu turquoise is found only in the unprocessed structure A with the scissile bond oriented between the catalytic residues. In addition, Asp and Glu residues play an essential function in proteolysis. Analysis of the structural models indicates that this conserved Asp residue functions in both proteins to properly orient the catalytic His residue  ,  Figure 3A—3D. The closest known cysteine proteases that share this structural arrangement of the catalytic site are caspase-1 and gingipain R  , .
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Similar to these other proteases  ,  , the CPDs are resistant to cysteine protease inhibitor E64  , but sensitive to N -ethylmaleimide, iodoacetamide, or chloromethyl ketones  ,  , . The CPD proteases have also been incorporated into a larger CPD adh family of putative bacterial and eukaryotic peptidase that are proposed to share a similar fold in the catalytic site . As the binding site for InsP 6 occurs on the opposite side of the protein from the catalytic site Figure 3E—3H , it was recognized that there must be a mechanism to transduce the binding signal across the entire protein structure .
Translocation of effector domains of both CGTs and MARTX toxins is predicted to involve transit through a pore for entry into the cytosol, and thus the CPD is likely partially unfolded when it is first presented to the InsP 6 -rich environment of the cytosol  , . In both proteins, the N-terminus is an unstructured strand wrapped around the outside of the protein and attached to the core structure by embedding of large hydrophobic residues  , .
Mutagenesis studies and structural analysis  ,  have demonstrated that Leu is the only residue that can be accommodated at this position.
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On either side of the Leu, any residue can occur but there is a preference for small residues, creating a consensus sequence of small-Leu-small  ,  and Table 1. The net effect is to orient the scissile bond between the catalytic Cys and His residues, resulting in substrate-activated autoprocessing  Figure 4. Apo-CPD without InsP 6 is an unstable protein susceptible to thermal denaturation at physiological temperature.
Upon binding InsP 6 , the structure rearranges such that the N-terminus yellow becomes locked within the active site between the catalytic Cys C and His H in a rigid alignment amenable to substrate-activated autoprocessing. By contrast, MARTX toxins must undergo processing at multiple sites to release each of the effectors independently  ,  Figure 1B. Since this concentration is above the upper limit of the in vivo concentration of InsP 6  , only a small fraction of processed CPD would bind InsP 6 in vivo, limiting the likelihood of multisite processing.
Both binding studies  and crystallography  Figure 3G have shown that chloromethyl ketone and epoxide inhibitors bound to Leu can substitute for a new substrate to restore the protein to an active enzyme-substrate complex. Upon reactivation, the protein is able to process any other available processing sites  ,  , although there is a preference for processing within the same molecule of MARTX Vc , indicating there may be a physical association of the CPD with the effector domains .
The discovery of InsP 6 -induced autoproteolysis as a critical stage for activation and effector delivery for large bacterial toxins raises the potential for anti-toxin small molecules to be developed as therapeutics. TcdB is the most significant virulence factor of C. By contrast, clinical intervention against any domain of MARTX Vc during cholera disease is impractical since animal studies suggest that MARTX Vc functions only during the earliest stage of infection, prior to the onset of symptoms  , .
Indeed, classical V. These include peptidyl acyloxy methyl ketone epoxide  and chloromethyl ketone  inhibitors in which the amino acid leucine is linked to the functional group independently or as part of a tripeptide. Both classes of inhibitor are cysteine reactive and become covalently linked to the catalytic cysteine Figure 3D.
Thus, inactivation of the CPD with Cys reactive inhibitors requires long incubation times of up to 30 minutes . Yet, upon initial intramolecular processing immediately upstream of the CPD, the catalytic Cys is exposed, facilitating rapid inhibition of subsequent processing events that release effectors  , . Consistent with these in vitro findings, exogenous addition of the membrane permeant z -Leu-Leu-azaLeu-epoxide inhibitor JCP to culture cells reduced actin crosslinking in vivo  , suggesting inhibitors could be useful at a critical point after CPD translocation.
Similar inhibition studies using the more clinically relevant TcdB remain to be performed. Since the CGTs are only processed one time Figure 1A , there is a concern that cysteine reactive inhibitors would be ineffective if the N-terminus bound in the active site blocks access to the catalytic Cys. A structure of the enzyme-substrate complex of TcdA or TcdB CPD is not yet available and inhibition by N -ethylmaleimide has been performed only with 30 minutes of incubation .
As described above, binding of InsP 6 to the recombinant TcdB CPD protein with the P1 leucine removed has been measured and shown to be similar to that with the Leu attached . Hence, the potential for inhibition of InsP 6 -induced autoprocessing by CPDs as a therapeutic intervention against TcdB merits further exploration.
We thank Jessica Queen and Brett Geissler for reading the manuscript. We also thank the reviewers of the initial draft for their insightful contributions. Abstract Large bacterial protein toxins autotranslocate functional effector domains to the eukaryotic cell cytosol, resulting in alterations to cellular functions that ultimately benefit the infecting pathogen. Introduction Pathogenic bacteria frequently export protein toxins that target eukaryotic intracellular proteins to alter host cell function to the benefit of the infectious pathogen.
Overview of Clostridial Glucosylating Toxins Clostridial glucosylating toxins CGTs , also known as large clostridial cytotoxins, are structurally and functionally related toxins produced by different Clostridium sp.