Alireza S Abyaneh

Simon Bauer

Christoph M Kirsch

Philipp Mayer

Clément Christian Mösl

Clément Poncelet

Sara Seidl

Ana Sokolova

Manuel Widmoser

Selfie: Towards Minimal Symbolic Execution

Keywords: • Applied computing → Education, • Software and its engineering → Compilers, Interpreters, Virtual machines, Symbolic Execution, RISC-V, Self-Referentiality

Selfie 1 is a fully self-contained 64-bit implementation of (1) a selfcompiling compiler written in a tiny subset of C called C* targeting a tiny subset of 64-bit RISC-V called RISC-U, (2) a self-executing RISC-U emulator, and (3) a self-hosting hypervisor that virtualizes the emulated RISC-U machine. Selfie is implemented in a single 10k-line file and can compile, execute, and virtualize itself any number of times in a single invocation of the system given adequate resources. There is also a simple in-memory linker, a RISC-U disassembler and debugger with replay, and a profiler. Selfie has originally been developed just for educational purposes but has recently become a research platform as well. C* supports only two data types, uint64_t and uint64_t*, and RISC-U features just 14 instructions, in particular for unsigned arithmetic only, which significantly simplifies reasoning about correctness. In this paper, we report on an ongoing effort in designing and implementing a symbolic execution engine for RISC-U within selfie that is supposed to explore non-trivial parts of the system including itself. The idea is to identify a minimal set of ingredients that are necessary to do this such as the data structures for representing traces, path conditions, and symbolic states as well as algorithms for SAT and SMT solving.

The key difference to related projects is that we are interested in reasoning just about selfie, for now, and are able to change selfie if necessary, as reasoning target but also as integrated platform for compilation, (symbolic) execution, and virtualization. Since selfie generates unoptimized code we are also exploring ways to leverage our symbolic execution engine in RISC-U code optimization.

PROBLEM

We are interested in identifying a minimal set of ingredients necessary for performing fast symbolic execution of systems code. Our target is selfie, a minimal but still realistic implementation of a compiler [START_REF] Wirth | Compiler Construction[END_REF], emulator, and hypervisor [START_REF] Liedtke | Toward Real Microkernels[END_REF]. The key observation is that systems code may not require many reasoning capabilities usually supported by state-of-the-art symbolic execution engines. There are at least two sides to our project, creating an adequate platform for symbolic execution (next section) and developing the actual execution engine (last section).

INFRASTRUCTURE

The original version of selfie was 32-bit, used signed integers and pointers to signed integers, and targeted a MIPS subset with up to 64MB of memory per machine instance [START_REF] Kirsch | Selfie and the Basics[END_REF]. C* even in that version has already been shown to support efficient implementations of non-trivial C programs such as the C* port of a state-of-the-art SAT solver written in C [START_REF] Abyaneh | You can program what you want but you cannot compute what you want[END_REF]. Yet to facilitate symbolic execution, we decided to port selfie to 64-bit, replace signed integers with unsigned integers, support RISC-V rather than MIPS, and increase memory per machine instance to 4GB. All that happened in three steps. First we went from int and int* to uint64_t and uint64_t*, and from MIPS32 to MIPS64. The advantage is twofold. Unsigned integers are significantly easier to reason about, and actually lead to simpler and cleaner code, and 64-bit helps with scaling up memory. We took advantage of that in the second step going up from 64MB to 4GB. This can also be done with 32-bit of course but, if necessary, we are now easily able to go up even further. The third step was to move from MIPS to RISC-V. While RISC-V instruction encoding and MoreVMs'18, April 2018, Nice, France Alireza S. Abyaneh, Simon Bauer, Christoph M. Kirsch, Philipp Mayer, Christian Mösl, Clément Poncelet, Sara Seidl, Ana Sokolova, and Manuel Widmoser decoding is more difficult, everything else is simpler. In particular, the machine state is smaller (no hi and lo registers) and there are fewer instructions, just 14, down from 17 instructions. Moreover, all RISC-U instructions but system calls may only have a side effect (other than on the program counter) on at most one 64-bit machine word in either a register or in memory. The five system calls necessary for bootstrapping selfie (malloc, exit, open, read, and write) only required minor modifications.

The current version of selfie2 generates proper 64-bit RISC-V ELF binaries that are compatible with the official RISC-V toolchain. However, only an earlier 32-bit RISC-V prototype of selfie actually executes on the official RISC-V spike emulator and pk kernel 3 . We are working on getting this done for the current 64-bit version which mostly requires interfacing memory allocation properly.

Other ongoing work on infrastructural level is multicore support. We are exploring simple ways to have selfie fork itself to run in parallel on multicore hardware. This would allow us to execute RISC-U code symbolically in parallel.

ENGINE

Inspired by the success of symbolic execution engines [START_REF] Cadar | KLEE: Unassisted and Automatic Generation of High-coverage Tests for Complex Systems Programs[END_REF][START_REF] Godefroid | Automated Whitebox Fuzz Testing[END_REF], we have recently started developing a symbolic execution engine for RISC-U within selfie. Here, the difference to regular concrete execution of RISC-U code, as performed by the emulator in selfie, is that data read from a file is assumed to be symbolic. For example, if symbolically executed code reads one byte from a file the value of that byte is kept unspecified such that it could be any value between 0 and 255. Any subsequent computation involving that symbolic value needs to keep track of the concrete values it still represents. Arithmetic and logical operations and in particular comparisons change or even constrain the choice of concrete values possibly to the point that no concrete value remains at which point the engine backtracks. The information which values still remain on a path through the control-flow graph is represented by a so-called path condition. As long as a path condition is satisfiable there are concrete values that will drive the code down that path.

Another reason for backtracking is lack of memory in which case the engine is unable to explore the control-flow graph any further. In other words, symbolic execution attempts to execute code for all possible inputs up to the available memory and CPU time. If a runtime error such as division by zero is detected the engine may output a witness of that bug, that is, a file that will, when used as input, drive the code into that bug.

We are currently able to execute RISC-U code symbolically up to a fixed depth into the control-flow graph. We are also able to check satisfiability of path conditions, that is, whether code is actually reachable for some input, but only for a subset of all possible RISC-U programs. The restriction is due to the simplicity of the checker which is based on integer interval constraints [START_REF] Larson | High Coverage Detection of Input-related Security Faults[END_REF]. However, the engine is able to detect if it is executing code within the limitations of the checker. Preliminary results show that at least the selfie compiler is within the supported fragment.

In the current version of selfie, only data read from a file by a read system call can be symbolic. We adapted the read implementation in selfie accordingly. Besides read, selfie uses and implements three other system calls, namely open, write, and exit, which we have not modified. Also, malloc works as before using a simple bump pointer and there is of course no free in selfie.

The key data structure for symbolic execution in selfie is a trace of machine state transitions. Essentially, we record for each executed instruction the current program counter value, the concrete or symbolic side effect of the instruction, and the state transition containing the previous concrete or symbolic value of the affected register or memory location. A trace therefore contains the whole execution history.

An interesting prerequiste of symbolic execution in selfie is replay. We noticed that recording the last, say, hundred instructions during regular concrete execution is a special case of symbolic execution. In the current version of selfie, the system is able to do that and, upon exceptions such as division by zero, undo the side effects of the last hundred instructions and then redo them but this time printing assembly code (with approximate source code line numbers) and its side effects on the console. Backtracking during symbolic execution corresponds to the undo operations during replay. In other words, there are now four implementations of each RISC-U instruction in selfie: concrete execution, concrete undo (inverse execution), symbolic execution, and symbolic backtracking.

We are now working on generating witnesses of bugs, that is, files that will drive RISC-U code into these bugs. The idea is to generate witnesses during symbolic execution of selfie and then provide the witnesses as input to selfie during concrete executions for validation, all in a single invocation of the system.

Another line of work focuses on simple ways for terminating loops during symbolic execution through sufficiently strong invariants provided by us. Also, we will eventually integrate symbolic execution with selfie's fork in order to explore independent code paths in parallel on multicore machines.

https://github.com/cksystemsteaching/selfie

https://riscv.org

ACKNOWLEDGMENTS

We thank the anonymous reviewers for their constructive feedback. This work has been supported by the National Research Network RiSE on Rigorous Systems Engineering (Austrian Science Fund (FWF) Grant S11404-N23 and S11411-N23), a Google PhD Fellowship, and a Google Research Grant.


Katharina Ernst

Simon Langer

Eva Kaiser

Christian Osseforth

Jens Michaelis

Michel R Popoff

Carsten Schwan

Klaus Aktories

Viktoria Kahlert

Miroslav Malesevic

Gunter Fischer

Cordelia Schiene Fischer

Dr Holger Barth
email: holger.barth@uni-ulm.de

Dr Cordelia Schiene-Fischer

Cyclophilin-Facilitated Membrane Translocation as Pharmacological Target to Prevent Intoxication of Mammalian Cells by Binary Clostridial Actin ADP-Ribosylated Toxins

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Various clostridia produce potent exotoxins, which mono-ADP-ribosylate G-actin at arginine-177 in the cytosol of mammalian target cells (for review see [START_REF] Barth | Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins[END_REF]). This toxin-catalyzed modification turns G-actin into a capping molecule which prevents further polymerization of actin filaments [START_REF] Aktories | Botulinum C2 toxin ADP-ribosylates actin[END_REF][START_REF] Ohishi | ADP-ribosylation of nonmuscle actin with component I of C2 toxin[END_REF][START_REF] Vandekerckhove | Clostridium perfringens iota toxin ADPribosylates skeletal muscle actin in Arg-177[END_REF][START_REF] Vandekerckhove | Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177[END_REF] and results in the breakdown of the actin cytoskeleton. Finally, the mode of action of these toxins results in cell-rounding [START_REF] Aktories | ADP-ribosylation of actin by clostridial toxins[END_REF][START_REF] Wegner | ADP-ribosylated actin caps the barbed ends of actin filaments[END_REF][START_REF] Weigt | Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments[END_REF][START_REF] Wiegers | Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase[END_REF][START_REF] Miyake | Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin[END_REF][START_REF] Ohishi | Purification and characterization of two components of botulinum C2 toxin[END_REF] and cell death [START_REF] Heine | ADP-ribosylation of actin by the Clostridium botulinum C2 toxin in mammalian cells results in delayed caspase-dependent apoptotic cell death[END_REF][START_REF] Hilger | The long-lived nature of clostridium perfringens iota toxin in mammalian cells induces delayed apoptosis[END_REF] associated with severe enteric diseases in humans and animals [START_REF] Ohishi | Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components[END_REF][START_REF] Ohishi | Histopathological effect of botulinum C2 toxin on mouse intestines[END_REF][START_REF] Songer | Clostridial enteric diseases of domestic animals[END_REF][START_REF] Geric | Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital[END_REF][START_REF] Mcdonald | An epidemic, toxin gene-variant strain of Clostridium difficile[END_REF].

Binary actin-ADP-ribosylating toxins are the C2 toxin from C. botulinum, the prototype of this toxin family [START_REF] Ohishi | Purification and characterization of two components of botulinum C2 toxin[END_REF], and the iota-like toxins which comprise the iota toxin from C. perfringens type E strains (Stiles and Wilkins, 1986a;Stiles and Wilkins, 1986b), CDT from hypervirulent strains of C. difficile (Perelle et al, 1997a;Popoff et al, 1988;[START_REF] Stubbs | Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile[END_REF][START_REF] Gülke | Characterization of the ADP-ribosyltransferase CDTa from Clostridium difficile by site directed mutagenesis[END_REF]) and the C. spiroforme transferase (CST) (Popoff and Boquet, 1988). The iota-like toxins are closer related among each other than to C2 toxin regarding their degree of sequence homology (Perelle et al, 1997b), the interchangeability of their components and the modification of actin isoforms which serve as a substrate (for review see [START_REF] Barth | Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins[END_REF]).

Moreover, all iota-like toxins bind to the lipolysis-stimulated receptor (LSR) on particular target cells (Paptheodorou et al, 2012;Papatheodorou et al., 2013) while C2 toxin binds to a carbohydrate receptor which is present on all mammalian cell types tested so far (Eckhardt et al, 2002).

However, C2 toxin and the iota-like toxins share an overall comparable unique structure and mode of action. They are composed of two non-linked proteins, an enzyme (A-) component and a separate binding and translocation (B-) component. Both components must assemble on the surface of target cells where the B-component binds to its cellular receptor, forms a complex with the A-component, and facilitates the receptor-mediated endocytosis and subsequently the transport of the A-component from acidified endosomal vesicles into the cytosol (for review see [START_REF] Barth | Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins[END_REF]). The cellular uptake of C2 and iota toxins was discovered by us and others during recent years. C2 toxin consists of the enzyme component C2I (~49 kDa) [START_REF] Fujii | Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in clostridial species[END_REF][START_REF] Barth | Characterization of the catalytic site of the ADPribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis[END_REF] and the binding/translocation component C2II (~80 or 100 kDa, depending on the strain [START_REF] Barth | Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification[END_REF][START_REF] Sterthoff | Functional characterization of an extended binding component of the actin-ADP-ribosylating C2 toxin detected in Clostridium botulinum strain (C) 2300[END_REF]. Proteolytically activated C2II forms ring-shaped heptamers (C2IIa) that bind to asparagine-linked carbohydrate structures on mammalian cells [START_REF] Barth | Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification[END_REF][START_REF] Blöcker | The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding[END_REF][START_REF] Eckhardt | Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates[END_REF]Stiles et al, 2002;[START_REF] Schleberger | Structure and action of the binary C2 toxin from Clostridium botulinum[END_REF]) and form complexes with C2I. After their receptor-mediated endocytosis, the C2IIa/C2I complexes reach early endosomal vesicles where the C2IIa heptamers change their conformation, insert as pores into endosomal membranes and serve as channels for translocation of unfolded C2I from the endosomal lumen into the cytosol [START_REF] Schmid | Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine[END_REF][START_REF] Barth | Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification[END_REF][START_REF] Bachmeyer | Interaction of C2 toxin with lipid bilayer membranes and Vero cells: inhibition of channel function by chloroquine and related compounds in vitro and intoxification in vivo[END_REF]Blöcker et al, 2003a;[START_REF] Blöcker | Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pHdependent C2I translocation in vivo[END_REF].

The cellular uptake and intracellular membrane transport of iota toxin is widely comparable [START_REF] Blöcker | Cellular uptake of the binary Clostridium perfringens iota-toxin[END_REF]. After receptor-mediated endocytosis of the Ib/Ia complexes, the proteolytically activated binding/translocation component Ib also forms heptameric transmembrane pores, which mediate translocation of the enzyme component Ia (47 kDa) across endosomal membranes into the cytosol (Stiles et al, 2002;[START_REF] Knapp | Interaction of the binding component of Clostridium perfringens iota-toxin with lipid bilayer membranes: demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia[END_REF][START_REF] Blöcker | Cellular uptake of the binary Clostridium perfringens iota-toxin[END_REF][START_REF] Nagahama | Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells[END_REF][START_REF] Hale | Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin[END_REF]. However, there are some differences during cellular uptake of C2 toxin and iota toxin since Ia likely translocates from endosomes, which are in a state between early and late endosomes [START_REF] Gibert | Differential requirement for the translocation of clostridial binary toxins: iota toxin requires a membrane potential gradient[END_REF], while C2I translocates from early endosomes [START_REF] Barth | Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification[END_REF]. Most likely, translocation of Ia requires more acidic conditions and a membrane potential gradient in addition to the pH gradient [START_REF] Gibert | Differential requirement for the translocation of clostridial binary toxins: iota toxin requires a membrane potential gradient[END_REF]. Despite these differences, membrane translocation of C2 toxin and iota toxin is facilitated by the same chaperones including heat shock protein (Hsp) 90 [START_REF] Haug | The host cell chaperone Hsp90 is necessary for cytotoxic action of the binary iota-like toxins[END_REF]Haug et al, 2005) and the peptidyl-prolyl cis/trans isomerases (PPIase) cyclophilin (Cyp) A [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF][START_REF] Kaiser | Membrane translocation of binary actin-ADP-ribosylating toxins from Clostridium difficile and Clostridium perfringens is facilitated by Cyclophilin A and Hsp90[END_REF] and FK506 binding protein (FKBP) 51 [START_REF] Kaiser | FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 blocks membrane translocation of the toxin in mammalian cells[END_REF]. Moreover, these host cell factors are also involved in membrane translocation of C. difficile CDT and we have got evidence that the Hsp90/PPIase-mediated membrane transport might be a common feature of bacterial ADP-ribosylating toxins [START_REF] Kaiser | FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 blocks membrane translocation of the toxin in mammalian cells[END_REF]. PPIases are protein folding helper enzymes which catalyze the cis-trans isomerization of proline-peptide bonds, which often represents a rate-limiting step during protein refolding even after membrane transport of proteins [START_REF] Bang | Slow conformational changes in protein folding can be accelerated by enzymes[END_REF][START_REF] Fischer | Cyclophilin and peptidylprolyl cis-trans isomerase are probably identical proteins[END_REF][START_REF] Schmid | Prolyl isomerase: enzymatic catalysis of slow protein-folding reactions[END_REF][START_REF] Schmid | Prolyl isomerases: role in protein folding[END_REF][START_REF] Galat | Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity--targets--functions[END_REF].

Pharmacological inhibition of the PPIase activity of Cyps in cultured cells by cyclosporine A (CsA) [START_REF] Handschumacher | Cyclophilin: a specific cytosolic binding protein for cyclosporin A[END_REF][START_REF] Fanghänel | Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A[END_REF] protected cells from intoxication with C2, iota and CDT toxins because the intracellular membrane transport of the enzyme components of these toxins was inhibited [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF][START_REF] Kaiser | Membrane translocation of binary actin-ADP-ribosylating toxins from Clostridium difficile and Clostridium perfringens is facilitated by Cyclophilin A and Hsp90[END_REF], indicating that Cyps are crucial for this step. Prompted by this observation we demonstrated earlier that CypA interacts with C2I in vitro and in intact cells [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF] and inhibition of CypA prevented translocation of C2I from endosomal vesicles to the cytosol in vitro [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF].

Here, we report that Cyp40 binds to C2I in vitro and in intact cells and characterize this interaction by dot blot and isothermal titration calorimetric analysis in more detail. Cyp40 also interacts with the enzyme components of iota-toxin and CDT, suggesting a common principle for this toxin family. Since Cyps are crucial for uptake of binary actin ADP-ribosylating toxins into the cytosol of mammalian cells, we tested whether a non-immunosuppressive CsAderivative protects cells from intoxication, which might be an attractive pharmacological option against the members of this toxin family.

Materials & Methods

Materials

MEM (cell culture medium) and fetal calf serum were acquired from Invitrogen (Karlsruhe, Germany). Materials for cell culture were purchased from TPP (Trasadingen, Switzerland).

Complete® protease inhibitor and streptavidin-peroxidase were obtained from Roche (Mannheim, Germany). Protein molecular weight marker Page Ruler prestained protein ladder was acquired from Thermo scientific (Bonn, Germany). Biotin-labeled NAD + was purchased from R&D Systems GmbH (Wiesbaden-Nordenstadt, Germany). Baf A1 was obtained from Calbiochem (Bad Soden, Germany) and CsA from Fluka (Munich, Germany). Enhanced chemiluminescence (ECL) system was acquired from Millipore (Schwalbach, Germany). The EZ-Link sulfo-NHS-Biotin and the streptavidin-sepharose beads were from Pierce (Bonn, Germany). Illustra MicroSpin G-25 Columns were from GE Healthcare (Munich, Germany) and Micro Bio-SpinTM Chromatography Columns were from Bio-Rad (Munich, Germany).

Atto-590-NHS-Ester was from Atto-Tec (Siegen, Germany).

Synthese VK112-2 und MM284?--> Cordelia

Protein expression and purification

Human FKBP12 was expressed using pET28a (Novagen, Darmstadt, Germany) in Escherichia coli Rosetta cells and purified according to published procedures (Edlich et al., 2006). Cyp40

…….-->Cordelia

The recombinant C2I, C2IE387/389Q and C2IIa proteins were purified and activated as described before [START_REF] Barth | Characterization of the catalytic site of the ADPribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis[END_REF]2000). Recombinant C3bot was purified as described earlier [START_REF] Fahrer | Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes[END_REF]. Ia and Ib were prepared as described earlier [START_REF] Perelle | Evidence that Arg-295, Glu-378, and Glu-380 are activesite residues of the ADP-ribosyltransferase activity of iota toxin[END_REF].

Recombinant CDTa (from C. difficile strain 196) was produced and purified as a His-tagged protein in the Bacillus megaterium expression system as described earlier (Papatheodorou et al., 2010).

Cell culture and cytotoxicity assays

Vero (African green monkey kidney) and HeLa cells were cultivated in MEM containing 10% heat-inactivated fetal calf serum (FCS), 1.5 g/l sodium bicarbonate, 1 mM sodium-pyruvate, 2 mM L-glutamine and 0.1 mM non-essential amino acids at 37 °C and 5 % CO 2 . Cells were detached using trypsin and reseeded no more than 20 times. For cytotoxicity assays, cells were seeded in culture dishes and incubated in MEM plus FCS with the respective toxin. To inhibit the PPIase activity of Cyps the cells were pre-incubated with indicated concentrations of CsA or the non-immunosuppressive CsA-derivative VK112-2 for 30 min and these inhibitors were present in the medium during the further incubation of the cells with the toxin. Cells were visualized after the indicated incubation times by using a Zeiss Axiovert 40CFI microscope (Oberkochen, Germany) with a Jenoptik progress C10 CCD camera (Jena, Germany). The toxin-induced changes in cell morphology (i.e. cell-rounding) were analyzed and percentage of round cells was determined from the images as described earlier [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF].

Toxin translocation assay with intact cells

The pH-dependent translocation of C2I through C2IIa pores across the endosomal membrane was experimentally mimicked at the cytoplasmic membrane of intact Vero cells as described earlier [START_REF] Barth | Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification[END_REF]. In brief, Vero cells were cooled to 4 °C and C2 toxin was added.

After a binding period of 15 min, an acidic pulse was given (pH 4.6). Under the acidic conditions C2IIa forms pores into the cytoplasmic membrane and C2I translocates directly into the cytosol. Cell rounding was monitored and visualized by photography.

STED -> Christian Osseforth Biophysik

Interaction analysis between C2I and immobilized Cyp40 using the dot blot system Serial dilutions of the purified proteins Cyp40, FKBP12 and C3bot were vacuum aspirated onto a nitrocellulose membrane using the Bio-Rad dot blot system (Munich, Germany) according to the manufacturer's manual. A Ponceau S staining was performed and then the membrane was blocked with 5 % non-fat dry milk in PBS containing 0.1 % Tween-20 (PBST). Subsequently, the membrane was cut and probed with the respective overlay proteins (200 ng/ml) for 1 h.

After extensive washing, the bound proteins were detected by streptavidin-peroxidase (biotinlabeled overlay proteins) or a specific antibody against C2I and a peroxidase-coupled anti-rabbit antibody from Santa Cruz (Heidelberg, Germany) by using the ECL system. To investigate the binding of unfolded C2I, CDTa and Ia to immobilized Cyp40 the biotin-labeled enzyme components were incubated with 6 M guanidine hydrochloride, 2 mM DTT, 30 mM TrisHCl or with PBS for control for 1 h before they were tested in the dot blot assay as described before.

To confirm that this procedure results in denatured C2I, CDTa and Ia, in vitro ADP-ribosylation of actin was tested as described below.

ITC -> Cordelia

Pull-down experiments with GST-C2I and biotin-C2I

The pull-down experiments were performed as described earlier [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF][START_REF] Kaiser | FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 blocks membrane translocation of the toxin in mammalian cells[END_REF]. In brief, HeLa cells were grown in 10 cm dishes and incubated in serum-free MEM at 37°C for 2 h with biotin-C2I (400 ng/ml) plus C2IIa (800 ng/ml) or for control without toxin. Subsequently, cells were lysed using an 18G needle-syringe, the cytoplasmic fraction was obtained by centrifugation and subsequently incubated with a 60 µl bed volume (1:1 in PBS) of streptavidin-sepharose beads overnight at 4°C. As an alternative approach, GST-C2I was applied instead of biotin-C2I and glutathione-sepharose beads were used in place of streptavidin-sepharose beads for pull-down of GST-C2I. The precipitated proteins were washed, subjected to SDS-PAGE and the proteins were transferred onto a nitrocellulose membrane that was probed with a specific antibody against Cyp40 from Thermo Fisher (Bonn, Germany). After incubation with a secondary peroxidase-coupled anti-rabbit antibody the coprecipitated Cyp40 was visualized using the ECL system.

Reproducibility of the experiments and statistics

All experiments were performed independently at least two times. Results from representative experiments are shown in the figures. In each individual Western blot panel shown in the figures, the protein bands were originally detected on the same membrane and cut out and recombined for presentation only in the figures. Values (n ≥ 3) are calculated as mean ± standard deviation (SD) using the Prism4 Software (GraphPad Software).

Results

CsA alters the intracellular distribution of the enzyme component C2I of C. botulinum C2 toxin

in HeLa cells -Initially, we investigated the effect of the specific pharmacological inhibitor CsA which blocks the activity of PPIases on the intracellular distribution of the enzyme component C2I of the C2 toxin. To this end, we used confocal as well as stimulated emission depletion (STED) microscopy for super resolution imaging. HeLa cells were pre-treated with CsA and subsequently the fluorescence-labeled enzyme component C2I plus the transport component C2IIa were added. After 2 h cells were fixed and bright-field, confocal and STED microscopy images taken. The characteristic morphology of C2 toxin-treated cells is shown in the overview in Fig. 1 in the upper panel. Cells treated with C2 toxin alone rounded up within 2 h due to the destruction of the actin cytoskeleton caused by the enzymatic activity of C2I. In the presence of CsA, the number of rounded cells was significantly reduced as demonstrated earlier [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF]. Next, the distribution of C2I in these cells was analyzed by confocal (Fig. 1 middle panel) and in more detail by STED microscopy (Fig. 1 lower panel). In CsAtreated cells there was a more punctured and less diffuse distribution of C2I compared to cells treated with C2 toxin alone. Pre-treatment of cells with bafilomycin A1 (BafA1) which prevents acidification of early endosomes thereby trapping C2I in these vesicles, resulted in an overall comparable cellular distribution of C2I as found in cells treated with C2 toxin in the presence of CsA. Furthermore, the images demonstrate that CsA and BafA1 did not interfere with early steps of toxin uptake like receptor-binding or receptor-mediated endocytosis since C2I was detectable in the cells. Taken together, the microscopic analysis suggests that less C2I translocates into the cytosol of CsA-treated cells which is in line with our earlier results obtained by biochemical approaches [START_REF] Kaiser | Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells[END_REF].

Cyp40 co-precipitates with C2I in lysates from C2 toxin-treated cells -HeLa cells were incubated with GST-C2I plus C2IIa or left untreated for control. Subsequently, cells were lysed and lysates incubated with glutathion-sepharose beads to pull-down GST-C2I and associated proteins. Co-precipitated Cyp40 was analyzed by Western blotting with a specific antibody. As shown in Fig 2A ,Cyp40 was detected in precipitates from HeLa cells treated with GST-C2I plus C2IIa prior to lysis but not in the precipitates from untreated cells, indicating that Cyp40 interacts with GST-C2I. To ensure that the GST-tag had no effect on this interaction, the experiment was repeated with biotin-C2I instead of GST-C2I. As shown in Fig. 2B,a comparable result was obtained, clearly indicating that Cyp40 specifically interacted with C2I in intact cells.

C2I, CDTa and Ia directly interact with Cyp40 in vitro -From the pull-down experiments it could not be concluded whether C2I directly interacts with Cyp40 or whether other proteins might serve as an adaptor for this interaction. Therefore, dot blot analysis was performed to test whether C2I directly binds to Cy40 in vitro. Purified recombinant Cyp40 was spotted onto a nitrocellulose membrane followed by overlay with biotin-labeled C2I. The result shown in Fig 3A revealed a direct interaction between Cyp40 and C2I. This interaction was specific since biotin-C2I did not bind to the non-relevant C3bot protein or to the membrane and there was no signal when the overlay buffer was applied instead of biotin-C2I (Fig. 3A). Comparable results were obtained when CDTa and Ia where analyzed instead of C2I, clearly indicating that Cyp40 directly interacts with the enzyme components of C2 toxin and of the iota-like toxins in vitro.

Prompted by this finding, we next investigated whether this interaction depends on the enzyme activity of the toxins and analyzed the enzymatically inactive C2IE387/389Q [START_REF] Barth | Characterization of the catalytic site of the ADPribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis[END_REF] in parallel to C2I. The signal of bound protein was comparable for the mutant and the wild-type C2I, indicating that the loss of enzyme activity does not interfere with binding of C2I to Cyp40 (Fig 3 B).

However, pre-treatment of C2I, CDTa and Ia by guanidine hydrochloride dramatically enhanced the binding of these proteins to Cyp40 in vitro (Fig. 3C), strongly suggesting that the interaction with Cyp40 preferentially occurs in the unfolded conformation of the enzyme components. The difference in binding affinity of unfolded CDTa and Ia was so strong that detection of both signals, unfolded and native, during the same exposure time was difficult.

Respective binding of the native CDTa and Ia was confirmed by longer exposure times (not shown). The denaturing effect of guanidine hydrochloride-treatment on the enzyme components of the toxins was confirmed by in vitro ADP-ribosylation of actin (not shown).

Quantitative analysis of the interaction between Cyp40 and C2I by ITC -The interaction between Cyp40 and C2I was analyzed in more detail by ITC à Cordelia

The intoxication of Vero cells with C2 toxin and iota toxin is inhibited by the nonimmunosuppressive CsA derivative VK112-2 -Finally, we tested whether the nonimmunsosuppressive CsA-derivative VK112/2 also protects cells from intoxication with C2 toxin and iota toxin. To this end, Vero cells pre-treated with increasing concentrations of VK112/2 or with CsA for control were challenged with either C2 toxin or iota toxinand cellrounding was analyzed (Fig. 5). Although less efficient compared to CsA, pre-treatment of cells with VK112/2 significantly delayed the toxin-induced cell-rounding in a time-and concentration-dependent manner, indicating that VK112/2 also interferes with mode of action of the toxins. Treatment of cells with VK112/2 alone had no effect on cell morphology (Fig. 5A). Noteworthy, pre-treatment of cells with the non-cellpermeable CsA-derivative MM284 had no inhibiting effect on intoxication with C2 or iota toxin (Fig. 5C). Moreover, pre-treatment with this compound inhibited the pH-driven translocation of cell-bound C2 toxin across the cytoplasmic membrane of intact cells (Fig. 6), implicating that VK112/2 inhibits the translocation of C2I from the lumen of acidic endosomes into the host cell cytosol and thus interferes with exactly the same step during toxin uptake into cells as CsA. This is a wellestablished approach in the field of toxin research to mimic the situation in the lumen of acidified endosomal vesicles directly on the surface of living cells which was used to characterize the molecular mechanisms underlying membrane translocation of various bacterial protein toxins [START_REF] Sandvig | Diphtheria toxin entry into cells is facilitated by low pH[END_REF] including C2, iota and CDT toxins in vitro (Barth et al, 2000[START_REF] Blöcker | Cellular uptake of the binary Clostridium perfringens iota-toxin[END_REF], Kaiser et al, 2011). In summary, prompted by our earlier findings that the PPIase activity of Cyps facilitates membrane translocation of clostridial binary actin ADP-ribosylating toxins, Cyp40 was identified as a new binding partner for the enzyme components of C2, iota and CDT toxins and the protective effects of a non-immunosuppressive CsA-derivative against these toxins established in a cell model.

Discussion

The results described in this article are an extension of our ongoing research on the role of host cell chaperones/PPIases for membrane translocation of bacterial protein toxins. In summary, we discovered that besides Hsp90 the PPIase activity of cyclophilin(s) and FKBP(s) facilitates the membrane translocation of clostridial actin ADP-ribosylating toxins C2, iota and CDT from acidic endosomes into the cytosol, and demonstrated that pharmacological inhibition of Hsp90, Cyps or FKBPs prevents toxin translocation and thereby intoxication of cultured cells with C2 toxin, iota toxin and CDT. In contrast, the membrane translocation of bacterial toxins, which deliver their enzyme moieties also from acidic endosomes into the cytosol but are not ADPribosyltransferases, does not require Hsp90 and the PPIases. In conclusion, the results implicate a common Hsp90/Cyp/FKBP-dependent membrane translocation mechanism, which is selective for ADP-ribosylating toxins.

In vitro, purified recombinant Hsp90, CypA and FKBP51 directly bound to the enzyme components of the binary actin ADP-ribosylating toxins and for the prototype of this family, C2 toxin, the interaction between the enzyme component C2I and these factors was demonstrated in intact cells by pull-down experiments. However, we could not exclude that further Cyps besides CypA also interact with C2I since the pharmacological inhibitor CsA is not selective for CypA. Recently, by using the actin-ADP-ribosylating Photorhabdus luminescence toxin TccC3, we could show for the first time that besides Hsp90, CypA and FKBP51, also Cyp40 interacts with the isolated ADP-ribosyltransferase domain of this toxin in intact cells. This was an interesting finding since the PPIase activity of Cyp40 is inhibited by CsA and Cyp40 is part of a multi-chaperone complex that contains Hsp90 and FKBP51, both binding partners of the enzyme components of the binary actin ADP-ribosylating toxins, C2I, Ia and CDTa. In cells, this multi-chaperone complex mediates the membrane translocation of the steroid hormone receptor across the nuclear membrane. Moreover, another Hsp90containing translocation factor complex facilitates the translocation of diphtheria toxin (DT), another ADP-ribosylating bacterial toxin, from acidified endosomal vesicles into the cytosol in vitro, as discovered earlier by Ratts and co-workers. We found previously that the membrane translocation of DTA, the enzyme domain of DT, also requires the PPIase activity of Cyp(s) and showed the direct interaction between DTA and recombinant CypA in vitro. However, it is not known so far whether PPIases are part of the translocation complex.

Therefore, we investigated whether C2I and the enzyme components of the iota-like toxins interact with Cyp40. Here, we demonstrated by dot blot analysis that C2I, Ia and CDTa directly bind to Cyp40 in vitro and characterized this interaction in more detail for the prototype C2I.

The ADP-ribosyltransferase activity of C2I is not essential for the binding to Cyp40 in vitro, which is in line with our earlier result obtained for DTA and CypA. However, treatment of C2I with guanidine hydrochloride, which results in loss of its enzyme activity indicating the unfolding of C2I, dramatically increased the binding of C2I to Cyp40 in vitro and comparable results were obtained for Ia and CDTa. This finding strongly suggests that Cyp40 shows preferred interaction with the enzyme components of the binary actin ADP-ribosylating toxins in their unfolded conformation, which is plausible since they unfold, at least partially, during translocation across endosomal membranes through the trans-membrane pores formed by the separate binding/translocation components of these toxins. However, from the pull-down experiments performed in this study, it can not be concluded whether the interaction between C2I and Cyp40 in intact cells is direct or whether Cyp40 is part of a protein complex, such as a Hsp90-multi-chaperone complex, which binds to C2I. Therefore, the logical next steps will be the closer investigation of the interaction between the enzyme components of these toxins with Hsp90 and the PPIases during their membrane translocation in living cells and to test whether Cyp40 interacts with the enzyme moieties of other clinically relevant ADP-ribosyltransferases including diphtheria toxin.

The finding that Cyps are crucial for the uptake of the medically relevant group of bacterial ADP-ribosylating exotoxins including CDT, which is associated with severe forms of C. difficile-associated pseudomembranous colitis, should also be of clinical interest. Cyps might serve as novel drug targets to prevent the uptake of the toxins into their human and/or animal target cells, which protects cells from intoxication. Since the toxin-induced cell damage represents the reason for the clinical symptoms of the toxin-associated diseases caused by the pathogenic bacteria, pharmacological inhibitors of PPIases such as CsA, could be considered for treatment and/or prevention of such diseases. This might in particular be attractive for diseases caused by toxin-producing bacteria which became (multi-)resistant against our common antibiotics. However, CsA is an immunosuppressive drug and might not be indicated during bacterial infections, but non-imunosuppressive CsA-derivatives such as VK112/2 used in the cell-based experiments of this study might be an alternative and should be tested in animal models. Cells were incubated for 2 h at 37 °C with C2IIa (800 ng/ml) plus either GST-C2I (400 ng/ml, A), or biotin-C2I (400 ng/ml, B), or without any protein for control (con). Cells were lysed and the cytoplasmic fraction was obtained by centrifugation. For pull-down of GST-C2I or biotin-C2I, the cytoplasmic fractions were incubated with glutathione-sepharose beads (A) or streptavidin-agarose beads (B), respectively over night at 4 °C. The beads were washed and precipitated proteins separated by SDS-PAGE and analyzed by Western blotting with a specific antibody against Cyp40. Comparable protein loading was confirmed by Ponceau S staining of the blot membranes. (starting concentration 1 µg, serial dilution) was spotted onto a nitrocellulose membrane via vacuum-aspiration using the dot blot system. For negative control PBS and purified recombinant C3bot, an Rho-modifying ADP-ribosyltransferase of Clostridium botulinum (starting concentration 1 µg, serial dilution) were spotted. The membrane was blocked and incubated for 1 h with biotin-labeled C2I, CDTa, or Ia (200 ng/ml) or for control with PBST alone. After extensive washing, the bound proteins were detected via streptavidin-peroxidase using the ECL system. B. Enzymatically inactive C2IE387/389Q interacts with Cyp40 in vitro.

Purified Cyp40 and for negative control the proteins FKB12 and C3bot, as well as PBS were spotted onto a nitrocellulose membrane (starting concentration 0.5 µg, serial dilution). Wild type C2I and the enzymatically inactive C2IE387/389Q (each 200 ng/ml) were used for overlay.

Bound C2I and C2IE387/389Q proteins were detected by an antibody against C2I and peroxidase-coupled secondary antibody using the ECL system. C. Binding of denatured versus native C2I, CDTa and Ia to Cyp40. For unfolding, the biotin-labeled enzyme components C2I, CDTa and Ia were incubated with 6 M guanidine hydrochloride, 2 mM DTT, 30 mM TrisHCl for 1 h or with PBS for control. Subsequently, a nitrocellulose membrane with spotted Cyp40 (starting concentration 1 µg, serial dilution) and FKBP12, C3bot and PBS for negative control was probed for 1 h with denatured or native C2I, CDTa and Ia proteins (each 200 ng/ml). The bound enzyme components were detected via streptavidin-peroxidase using the ECL system. Subsequently, C2 toxin (75 ng/ml C2I plus 150 ng/ml C2IIa) was added and cells were further incubated at 37 °C. Morphological changes were monitored at the indicated time points. The pictures shown were taken after 2.25 h. For quantitative analysis, percentage of rounded cells was determined from the pictures taken at the indicated time points. Values are given as mean ± SD (n = 3). Significance was tested by using the student's t-test and values refer to samples treated with toxin only (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001). B.

Effect of VK112/2 on intoxication of cells with iota toxin. Vero cells were treated with iota toxin (40 ng/ml Ia plus 80 ng/ml Ib) in the presence or absence of CsA and VK112/2 exactly as described for C2 toxin in A. The pictures shown were taken after 3 h. C. Effect of the noncellpermeable CsA-derivative MM284 on intoxication of cells with C2 and iota toxin. Vero cells were treated with C2 toxin (50 ng/ml C2I plus 100 ng/ml C2IIa) or iota toxin (30 ng/ml Ia plus 60 ng/ml Ib) in the presence or absence of 500 µM MM284 exactly as described in A. pre-incubated for 30 min at 37 °C with 100 nM BafA1 and VK112/2. For control, cells were treated with the non-cellpermeable CsA-derivative MM284 or left untreated. Subsequently, cells were cooled to 4 °C and C2 toxin (150 ng/ml C2I plus 300 ng/ml C2IIa) was added. After 15 min at 4 °C the medium was adjusted to pH 4.6 and the cells were incubated at 37 °C for 10 min. Afterwards, the acidic medium was removed and cells were further incubated with FCSand BafA1-containing medium at 37 °C. Toxin-induced morphological changes were monitored. The pictures shown were taken after 5.5 h (scale bar = 100 µm). For quantitative analysis, percentage of rounded cells was determined from the pictures taken at the indicated time points. Values are given as mean ± SD (n = 3). Significance was tested by using the student's t-test and values refer to samples treated with toxin only (ns = not significant, *** = p < 0.001).

Fig. 1 .

 1 Fig. 1. Effect of CsA on the intracellular distribution of the enzyme component C2I of

Fig. 3 .

 3 Fig. 3. Interaction of the enzyme components of C2, CDT and iota toxins with Cyp40 in

Fig. 4

 4 Fig. 4 ITC à Cordelia

Fig. 6 .

 6 Fig. 6. The non-immunosuppressive CsA-derivative VK112/2 inhibits the pH-dependent

  Figure 1

  Figure 4

Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft DFG (grant BA 2087/2-2 to H.B.). K.E. is a fellow of the International Graduate School in Molecular Medicine Ulm (IGradU).

We thank Ulrike Binder for excellent technical assistance.

Cyclophilins in membrane transport of actin ADP-ribosylating toxins † This work was financially supported by the Deutsche Forschungsgemeinschaft (grant to H.B. BA 2087/2-2).


Vsevolod Peysakhovich

Frédéric Dehais

Andrew Duchowski

published or not. The documents may come

ArUco/Gaze Tracking in Real Environments

V. Peysakhovich*, F. Dehais*, and A. T. Duchowski** * ISAE-SUPAERO, Université de Toulouse, France, vsevolod.peysakhovich@isae.fr ** Clemson University, SC, USA, duchowski@clemson.edu Abstract. The emergence of affordable mobile eye-trackers has allowed to study gaze behavior in real-world environments. However, the gaze mapping from recorded video to a static reference image is a complex and open problem. Finding a reference image within the video frames, i.e., image matching, can give satisfying results, but occluded or overlapped objects are almost impossible to locate using this technique. We suggest using ArUco fiducial markers (and their associated software library available in OpenCV) to map gaze to dynamic Areas Of Interest (AOIs) within a reference image. Although such markers have been used previously, technical details of marker detection and mapping have been sparse. The current approach consists of three steps: (1) define an AOI using markers, then (2) resolve any conflict among overlapping AOIs, and (3) map the gaze point to the reference image. A dynamic AOI can be defined using one or more corner markers. When camera rotations are limited and the object is relatively orthogonal to the camera, it is possible to define an AOI using only one corner marker. When the camera rotates, its pose estimation is required to project corner points to the camera image plane. An AOI can also be defined with four corner markers, which has the advantage of robustness with respect to camera rotations, and no a priori required knowledge of the physical dimensions of the object. The two approaches can be combined, e.g., when using four corner markers and one of the markers is lost (due to occlusion or view angle), the basis vectors can be used to interpolate the position of the lost marker. When two or more AOIs overlap and all the markers are tracked, gaze should be marked on the AOI closer to the camera. The distance to an object can be defined knowing the length of the object, the number of pixels spanned on the image and the pre-computed camera focal parameter. Once the AOIs are defined and marker overlaps are resolved, the gaze point can be mapped to the coordinates of the reference image using homography.