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J. Biol. Chem., Vol. 279, Issue 30, 31599-31605, July 23, 2004

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NMR Structure of a Type IVb Pilin from Salmonella typhi and Its Assembly into Pilus^*

Xing-Fu Xu, Yih-Wan Tan, Lam Lam, Jim Hackett, Mingjie Zhang, and Yu-Keung Mok¶

From the Department of Biological Sciences, National University of Singapore, Singapore 117543 and the Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Received for publication, April 28, 2004 , and in revised form, May 24, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The structure of the N-terminal-truncated Type IVb structural pilin (t-PilS) from Salmonella typhi was determined by NMR. Although topologically similar to the recently determined x-ray structure of pilin from Vibrio cholerae toxin-coregulated pilus, the only Type IVb pilin with known structure, t-PilS contains many distinct structural features. The protein contains an extra pair of -strands in the N-terminal loop that align with the major -strands to form a continuous 7-stranded antiparallel -sheet. The C-terminal disulfide-bonded region of t-PilS is only half the length of that of toxin-coregulated pilus pilin. A model of S. typhi pilus has been proposed and mutagenesis studies suggested that residues on both the loop and the C-terminal disulfide-bonded region of PilS might be involved in binding specificity of the pilus. This model structure reveals an exposed surface between adjacent subunits of PilS that could be a potential binding site for the cystic fibrosis transmembrane conductance regulator.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Type IV pili have long been recognized as major bacterial virulence-associated adhesins that promote bacterial attachment to host cells (1). The genome of the typhoid fever bacterium, Salmonella typhi, contains a large insertion or "pathogenicity island" that is not found in the chromosome of the non-invasive Salmonella typhimurium (2). DNA sequencing within the novel pathogenicity island (Salmonella pathogenicity island 7) identified a pil operon (11 genes, from PilL to PilV) that contains genes required for biosynthesis of Type IV pili in S. typhi (3). S. typhi uses the cystic fibrosis transmembrane conductance regulator (CFTR)¹ as an epithelial cell receptor for the Type IV pili prior to entry of the gastrointestinal submucosa following oral intake (4). The structural pilin protein (PilS) of the Type IV pili interacts with the first extracellular domain (residues 103–117) of CFTR (3–5). Type IV pilins are further classified into Type IVa and IVb pilins according to the length of their signal peptides and the identities of the first residues of the mature pilins. Type IVa pilin has a much shorter signal peptide than Type Ivb, and the N-terminal residue of the mature pilin is always an N-methylated phenylalanine, whereas the N-terminal residue of Type IVb pilin may be either methionine or leucine (6). Type IVa pili are found in Pseudomonas aeruginosa, Neisseria gonorrhoeaem, and Mycobacterium bovis. The pilus of S. typhi, the Tcp of V. cholerae (7), the bundle-forming pilus of enteropathogenic Escherichia coli (8), the longus pilus of enterotoxigenic E. coli (9), and the R64 thin pilus (10) all belong to Type IVb. Both mature Type IVa and IVb pilins contain an N-terminal hydrophobic region of 24 residues (Fig. 1) that is believed to be the oligomerization domain required for Type IV pilus assembly through the "general assembly pathway" (11).

View larger version (52K): [in this window] [in a new window]   FIG. 1. Sequence alignment and secondary structures of Type IVb pilins. Sequence alignment of Type IVb pilins from S. typhi pilus (S. typhi), R64 thin pilus (R64), bundle-forming pilus of enteropathogenic E. coli (EAF), and toxin-coregulated pilus of V. cholerae (Vch) using the program CLUSTALW (36). Arrows and cylinders above the alignment indicate -strands and -helices of S. typhi t-PilS, respectively. Arrows and cylinders below the alignment indicate secondary structures of Tcp pilin as reported. The signal sequences are bold-faced. The N-terminal hydrophobic domains are underlined. The acidic residues found within the N-terminal hydrophobic domains are boxed. The down arrow indicates the starting boundary of the PilS construct used for NMR structure determination. The N-terminal loop and the D region are shaded.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression and Purification of PilS—The DNA fragment for S. typhi t-PilS (residues 26–181) was subcloned between the BamHI and EcoRI sites of a modified pET-32a (Novagen) vector with S-tag and thioredoxin gene removed. The correct sequence of the construct was confirmed by Big-dye sequencing. The His-tagged protein was expressed in BL21(DE3) E. coli cells as inclusion bodies (induction with 0.3 mM final concentration of isopropyl-1-thio--D-galactopyranoside; cells were grown at 37 °C). Nine extra residues from the vector (GSAMADIGS) remain N-terminal to residue Met-26 of PilS after thrombin cleavage. The pellet of the expressed inclusion bodies was purified with nickel-nitrilotriacetic acid resin (Qiagen) using buffers containing 6 M GdnCl. The purified protein was refolded by rapid dilution (30x) into ice-cold buffer (50 mM Tris, pH 7.9) with stirring. Residual GdnCl was removed by dialysis, and proper folding of the soluble protein was checked by far-UV CD. The refolded protein was concentrated and cleaved with thrombin (3 units/mg protein) before further purification on a Sephacryl S-100 (Amersham Biosciences) gel filtration column (50 mM Tris, pH 7.9 + 0.5 M NaCl) for NMR studies.

The DNA fragments for PilS (residues 1–181) and its mutants were subcloned between BamHI and EcoRI site of the same vector. All mutants were confirmed by DNA sequencing. The proteins were expressed as inclusion bodies in BL21(DE3) by inducing at a cell density of A₆₀₀ = 0.6 and at 30 °C. The inclusion body pellets were resuspended in binding buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM Imidazole) with 2% v/v Triton-X100 and 0.1 mM phenylmethylsulfonyl fluoride. The proteins were bound onto nickel-nitrilotriacetic acid resins, washed, and eluted with buffers that did not contain Triton X-100. The eluted proteins were further purified on a MonoQ ion-exchange column (Amersham Biosciences) before being used for binding assays.

NMR Spectroscopy and Structure Calculation—Purified t-PilS protein was concentrated to 1 mM and buffer-exchanged to 50 mM phosphate, pH 6.0, with 0.5 M sodium sulfate for NMR studies. NMR experiments were performed on a 500-MHz Bruker DRX spectrometer equipped with cryoprobe or a 750-MHz Varian Inova spectrometer at 30 °C. Sequence-specific backbone assignments were done by HNCACB (20), CBCA(CO)NH (21), HNCO, and HN(CA)CO experiments using a ¹⁵N-¹³C uniformly labeled sample. Side chain assignments were obtained from H(CCO)NH, (H)CC(CO)NH (22), and HCCH-TOCSY (23) spectra. The stereo-specific assignment of methyl groups was obtained by analyzing the ¹H-¹³CHSQC spectrum of a 10% ¹³C-labeled sample (24). Hydrogen bond restraints from amide hydrogen exchange were determined by recording the ¹H-¹⁵N HSQC spectrum of a sample left in 99% D₂O buffer for 6 h. Proton distance constraints were derived from three-dimensional ¹⁵N-edited NOESY (mixing time 150 ms) and three-dimensional ¹³C-edited NOESY. NMR data were processed using the MMRPipe/NMRDraw (25) suite and analyzed by NMRView (26).

The initial structures of t-PilS were generated by DYANA (27) using manually assigned unambiguous NOE restraints from ¹⁵N-NOESY and ¹³C-NOESY and dihedral angle restraints predicted by TALOS (28). Many other NOE cross-peaks in the two NOESY spectra were further assigned by CYANA (29) automatically. The unambiguous NOE restraints, automatically assigned NOE restraints, dihedral angle restraints, and hydrogen bond restraints were used for structure calculation. One hundred structures were calculated by DYANA using standard TAD protocol, and 20 conformers with the lowest target function values were selected for further energy refinement in AMBER 7.0 (30). The final ensemble of 10 structures with the lowest amber energies was checked by Procheck-NMR (31) and deposited at the Protein Data Bank.

Model of PilS Pilus—The two subunits of PilS were first docked manually using the program "O" in such a manner that 2 is in close vicinity to 4. The interface was then refined using the program MULTIDOCK with the following parameters. To define the interface, a distance cut-off of 15 Å (cut_iface) was used to include the region for multiple copy side chain rotamer representation. A relatively large distance cut-off of 200 Å (cut_jface) was used to include the whole molecule for fixed side chain representation. For mean field optimization performed at 298.0 K, a residue-residue non-bonded cut-off distance of 15 Å (cut_res_nb) was used. The criterion for convergence in terms of change in energy was set at 0.4 kcal/mol (emax). For rigid body energy minimization, a distance cut-off of 15 Å (cut_lface) across the interface was used for inclusion of residues in the calculation of non-bonded interaction. The maximum rotation step size was 1° and maximum translation step size 0.3 Å. Minimization continued until the energy of the system decreased by less than 1x e^–6 kcal/mol (ftol) for any given step. The energy cap for the number of atom-atom van der Waals clashes was set at 2.5 kcal/mol (eatmax).

Inhibition of Bacterial Entry Experiment—The procedures for inhibition of bacterial entry into human intestinal cells by recombinant PilS proteins were described elsewhere (3). Human embryonic intestinal cells INT407 were grown in tissue culture flasks and seeded in 24-well plates to obtain a monolayer in basal medium Eagle's with 15% calf serum (BME) by cultivating overnight at 37 °CinaCO₂ incubator. The medium was removed, and the cells were washed with 1x PBS. 0.35 ml of fresh BME was added, followed by 100 µl of solutions of PilS or mutant PilS to a final protein concentration of 2 µM (1x PBS was used as a control for 100% invasion) and then 50 µlof S. typhi bacterial cells in saline (3.6 x 10⁷ cells). The mixture was centrifuged for 10 min at 2,164 x g and incubated for 2 h to allow bacterial invasion. The culture medium was removed, and the cells were washed once with 1x PBS. 0.5 ml of fresh BME with 100 µg/ml gentamycin was added, and the cells were incubated for 1 h to kill S. typhi that remained outside the intestinal cells. The medium was removed, and the cells were washed twice with 1x PBS to remove any trace of gentamycin. 1 ml of 0.02% Triton X-100 was added, and the cells were pipetted up and down to ensure that the intestinal cells were detached and lysed to release the S. typhi cells. The mixture was incubated at 37 °C for 1 h before spreading onto agar plates (with kanamycin) for cell counts.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Structure of S. typhi PilS—The construct used for the PilS structural determination is N-terminal-truncated (Met-26-Gly-181, referred as t-PilS), and the truncation of the N-terminal 25 residues is necessary to prevent the pilin from oligomerization. The three-dimensional structure of t-PilS was solved by NMR spectroscopy (Table I). Fig. 2A shows a stereoview of the best fit superposition of the family of 15 final structures of t-PilS. The N-terminal five residues of t-PilS (residues 26–30) are not well defined because of the lack of structural restraints. The other less defined regions in the protein include the loop between helices 1 and -2 (residues 49–60), the loop between strands 1 and -2 (residues 84–86), the loop between strands 3 and -4 (residues 105–112), and the loop between helix 4 and strand 7 (residues 164–171). Residues in these regions have relatively fewer NOE restraints compared with the well defined regions and are likely to be intrinsically more flexible.

View larger version (49K): [in this window] [in a new window]   FIG. 2. NMR structure and topology of t-PilS. A, stereoview showing the best fit superposition of the backbone atoms of the final 15 structures of the S. typhi t-PilS protein. B, ribbon diagrams of the three-dimensional structure of t-PilS. Panels A and B were prepared with the program MOLMOL (37). C, secondary structure topologies of t-PilS and Tcp pilin. The loop and D region are shown with shaded background. Locations of the disulfide bonds are indicated with open circles.

Although topologically similar, t-PilS contains distinct structural features in both the loop and D region when compared with Tcp (Fig. 2C). The loop of t-PilS contains an extra pair of antiparallel -strands (strands 1 and -2) between helix 2 and strand 3. The corresponding region in the Tcp pilin, between helix 2 and strand 1, is replaced with an extended loop of 18 residues (Fig. 1). The extra pair of -strands in t-PilS aligns continuously at the edge of the major -sheet (strands 3, -4, and -7) and allows helix 2 to pack on top at an angle of 124° to helix 1. In the Tcp pilin, helix 2 lies perpendicular to helix 1 (90°) and across on the outside of the major -sheet. Consequently, helix 2, strand 1, and the 1-2 loop of t-PilS are exposed at the edge of the molecule, whereas only helix 2 is exposed at the same edge in the Tcp pilin (Fig. 3, A and B).

View larger version (46K): [in this window] [in a new window]   FIG. 3. Comparison of the structures of t-PilS and Tcp pilin in two different views. In both panels, the central hydrophobic core, the loop, and the D region are red, green, and blue, respectively. The extended 3-4 and 4-4 loops in Tcp pilin and their corresponding counterparts in t-PilS are yellow. A, the extended loops in Tcp pilin are highly exposed and cover most of the -sheet. The much shortened 5-6 and 6-4 loops in t-PilS allow different parts of the protein to be exposed. B, during pilus assembly, helix 4 of PilS is aligned almost parallel to the helical axis of the pilus; helix 4 of Tcp pilin makes an angle of around 50° with the helical axis. The figure was prepared with the program MOLMOL (37).

Model of S. typhi Pilus Assembly—A model of S. typhi pilus was built based on our t-PilS structure and the model of Tcp (19). The postulated association interface (2 against 4) between two adjacent t-PilS subunits was energy-refined (total energy = –34.6 kcal/mol) using the program MULTIDOCK (33). The refined dimeric subunit was used to build a single helical strand that was subsequently intertwined with another two identical helical strands to form the pilus model. The model shows that t-PilS can also be arranged into a left-handed three-start helix. Each turn of the helical strand contains six subunits of t-PilS with their 2 helices interacting with the 4 helices of an adjacent subunit in a perpendicular fashion (Fig. 4A). The helix 4 of t-PilS, unlike that of Tcp, is almost parallel to the helical axis of the pilus. Three such helical strands intertwine to form a cylindrical pilus with a helical pitch of around 30 Å and an outer diameter of around 100 Å (Fig. 4B). This arrangement of the pilus places the hydrophobic N-terminal helix (helix 1) of every subunit at the innermost core of the PilS pilus, where it can pack to form a coiled coil providing mechanical strength and stability.

View larger version (74K): [in this window] [in a new window]   FIG. 4. A model of PilS pilus assembly. A, each left-handed helical strand of the pilus contains six subunits of PilS with their 2 helices interacting with 4 helices of an adjacent subunit. -helices and loops are red; -sheets are cyan. B, the PilS pilus can form a three-start helix with a pitch of 30 Å between each helical strand and an outer diameter of 100 Å. The N-terminal hydrophobic helices of each subunit are packed at the central core of the pilus. The 3 helical strands of the PilS pilus are red, yellow, and blue. The figure was prepared with the program MOLMOL (37).

View larger version (53K): [in this window] [in a new window]   FIG. 5. Peptide binding surface of PilS pilus. A, ribbon diagram showing exposed and charged residues on t-PilS. The two subunits are gray and light blue, respectively. Exposed and charged residues at the subunit interfaces and on the -sheet are shown in wire-frame models. Positively charged residues are blue. Negatively charged residues are red. Note that residues are labeled only on one of the subunits. B, molecular surface of four subunits of PilS on the pilus model showing the distribution of charges and locations of exposed charged residues. Positively and negatively charged surfaces are blue and red, respectively. Exposed charged residues found to be essential for bacterial cell entry into human intestinal cells are underlined.An asterisk next to the label represents functional residues from adjacent subunits on the same helical strand. A hash sign next to the label represents functional residues from adjacent subunits on a different helical strand. The border of one of the subunits is highlighted for easier identification. The figure was prepared with the program MOLMOL (37).

Exposed and charged residues in t-PilS are mainly found on or around the helices 2 (Asp-54, Lys-63, Asp-66, and Asp-82) and 4 (Asp-151, Lys-153, and Glu-162) at the interface between two adjacent subunits and also on the exposed -sheet (Asp-103 and Lys-143) (Fig. 5A). Fig. 5B shows the locations of all the exposed and charged residues found on the surface of PilS pilus. In an attempt to determine the exact binding site of CFTR on PilS, we have synthesized a 10-residue peptide with a sequence derived from residues 108–117 (SYDPDNKEER) of CFTR. The peptide was titrated into a ¹⁵N-labeled t-PilS NMR sample, but to our surprise no binding could be observed (data not shown). The inability of t-PilS to bind CFTR is further confirmed by the loss of its ability to inhibit entry of S. typhi into intestinal cells (Fig. 6). Only full-length PilS protein that contains the N-terminal hydrophobic oligomerization domain retained the ability to bind CFTR and inhibit entry of S. typhi into intestinal cells. To identify the exposed charged residues that could involve direct interaction with CFTR, we have generated eight mutants of the full-length PilS protein and tested their abilities to inhibit entry of S. typhi into intestinal cells (Fig. 6). Asp-66 was not tested because the protein tends to precipitate out of solution. Results of the inhibition of bacterial entry experiment show that most of the "functional" charged residues involved in CFTR binding are located on or around helix 4 in the D region (residues Asp-151, Lys-153, and Glu-162). This agrees with the findings on Tcp pilin in which all of its functional residues are found exclusively on or around 4 (19). Unlike Tcp pilin, the residue on the 1-2 loop of PilS (Asp-54), which is in close proximity to Lys-153 on helix 4 of an adjacent subunit, is also essential for CFTR binding. Other charged residues that are exposed on the loop (Lys-63 and Asp-82) and the -sheet (Asp-103 and Lys-143) of PilS are not essential for CFTR binding.

View larger version (18K): [in this window] [in a new window]   FIG. 6. Inhibition of bacterial invasion by PilS mutants. The percentage of bacterial invasion is an average of four separate experiments. The error bar indicates S.D. For each experiment, the number of bacterial colonies was compared with the control, which used only 1x PBS, set as 100% bacterial invasion. All the mutants were confirmed to have a similar CD spectrum and elution profile on a gel filtration column as did the wild-type PilS protein. t-PilS eluted at a volume corresponding to the monomeric molecular weight of PilS (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here, we have described the structure of the Type IVb pilin from S. typhi, which has an extra pair of -strands in the loop and much shortened loops in the D region compared with Tcp pilin, the only Type IVb pilin with known structure. Sequence alignment shows that there is only 16% sequence identity between S. typhi PilS and Tcp, whereas there is close to 50% identity between PilS and the Type IVb pilin from R64 thin pilus, which is required for bacterial mating (Fig. 1) (10). We propose to classify the Type IVb pilin from S. typhi and R64 plasmid as a distinct subclass of Type IVb pilin having significantly shortened loops in the D region from those in Tcp and bundle-forming pilus. Differences between the loop and D region of the two subclasses of Type IVb pilins affected not just the assembly of the pili but also the availability of exposed residues in the vicinity of the D region for receptor binding.

Although the structures of both Type IVb pilins belong to the same roll class as Type IVa pilins, the addition of extra -helices at both ends of the -sheet in Type IVb pilin generates a unique pattern of pilus assembly. Type IVa pilin forms either a right-handed (MS11 and PAK) or left-handed (K122–4) 1-start helix with five subunits/helical turn. The N-terminal -helices interacting with each other at the middle of the pilus form a coiled coil structure, whereas the interface between adjacent subunits is formed by continuous -strand hydrogen bonding. The Type IVa pilus is relatively thin and fragile with a helical pitch of around 40 Å and a diameter of around 50 Å (K122–4). Type IVb pilin, such as Tcp, adopts a left-handed three-start helix of six subunits/turn with 2 from the loop of one subunit interacting with 4 from the D region of an adjacent subunit. The Type IVb pilus is relatively thick and sturdy with a helical pitch of 45 Å and a diameter of 80 Å (Tcp).

The proposed model shows that S. typhi PilS can also be assembled into a left-handed three-start helix with six subunits/helical strand. Despite the difference in the orientation of 4 relative to the helical axis, the helical pitch (30 Å) and diameter (100 Å) of the PilS pilus model are comparable with those, 37.5 and 100 Å, respectively, obtained for the crystallo-graphic fibers of N-terminal-truncated Tcp. The helical pitch and diameter of the PilS model, however, differ from the intact Tcp filaments, which have a 45-Å pitch and a diameter of 80 Å (19). Addition of extra pulling forces from the N-terminal helices is expected to increase the helical pitch and reduce the diameter of the intact S. typhi pilus. This speculation also agrees with the measured diameter of intact S. typhi pilus by electron microscopy, which is around 6–8 nm (data not shown).

CFTR has long been recognized as the cellular receptor for S. typhi entry. Human cells expressing wild-type CFTR ingested significantly more S. typi than cells expressing F508 CFTR (5). This entry can be inhibited either with a peptide derived from the first extracellular domain of CFTR (5) or with soluble recombinant protein of PilS (3). The CFTR peptide, but not a scrambled one, can effectively neutralize the cell entry inhibition afforded by the PilS protein and vice versa (4). Because the proposed receptor of PilS on CFTR carry mostly charged residues, we have generated mutants for most of the exposed and charged residues on PilS according to the model to pinpoint its receptor binding site. Based on data from inhibition of bacterial entry by full-length PilS protein, the binding site for CFTR on PilS pilus might be at the interface between adjacent subunits with charged residues not only from the D region but also from the loop. Depending on the conformation of the bound CFTR peptide, the binding site could be between adjacent subunits on the same helical strand or even between subunits on different helical strands (Fig. 5B). In Tcp, functional charged residues that extend outward to the surface of the Tcp filament for interaction with other pili, bacteriophage, or host cells are found exclusively on or adjacent to helix 4 in the D region (19).

This finding also explains why the monomeric t-PilS is unable to bind CFTR, because the complete binding site could be comprised of charged residues from different subunits. CFTR binding is strictly dependent on the assembly of PilS pilin into pilus and might be for improved specificity and cooperativity of binding. This is the first report on structural study of PilS from S. typhi. Identification of the structure of t-PilS and its pattern of pilus assembly, as well as candidate residues that are essential for receptor binding, not only provides the basis for other structural studies but also opens the possibility of rational drug or vaccine design against this adhesin that is commonly used by S. typhi for host invasion.

	FOOTNOTES

 
^* This work was supported by the A*Star BMRC Young Investigator Award (to Y.-K. M.) and by the Academic Research Fund, National University of Singapore. Some of the NMR spectra used in this work were acquired at the 750-MHz NMR spectrometer in Hong Kong University of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1Q5F) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The NMR chemical shifts (accession code 5879) have been deposited in the BioMagResBank (BMRB), Department of Biochemistry, University of Wisconsin, Madison (www.bmrb.wisc.edu.).

^¶ To whom correspondence should be addressed: Dept. of Biological Sciences, 14 Science Dr. 4, National University of Singapore, Singapore 117543. Tel.: 65-68742967; Fax: 65-67792486; E-mail: dbsmokh{at}nus.edu.sg.

¹ The abbreviations and trivial terms used are: CFTR, cystic fibrosis transmembrane conductance regulator; t-PilS, N-terminal-truncated structural pilin; D region, C-terminal disulfide-bonded region; NOE, nuclear Overhauser effect; Tcp, toxin-coregulated pilus; NOESY, nuclear Overhauser effect spectroscopy.

	ACKNOWLEDGMENTS

 
We thank Daiwen Yang for providing technical support on NMR experiments setup and K. Swaminathan for advice in using the software "O."

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