Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Abstract

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNF_Y . CNF_Y consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused β-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNF_Y , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.

Keywords: Yersinia; AB-toxin; ADP-ribosyl transferase; CNF; DUF4765.

Figure EV1. Sequence alignment of CNF_Y to other CNFs

Sequences from top to bottom: 1. CNF_Y from Yersinia pseudotuberculosis, 2. CNF1 from E. coli (NCBI accession: AAA85196, 61% sequence identity to CNF_Y), 3. CNF2 from E. coli (NCBI accession: ACT33566, 61% sequence identity), 4. CNF3 from E. coli (NCBI accession: CAK19001, 68% sequence identity), 5. CNF from Salmonella enterica (NCBI accession: WP_079946821, 69% sequence identity), 6. CNF from Shigella boydii (NCBI accession: WP_075330563, 68% sequence identity), 7. CNF from Moritella viscosa (NCBI accession: AHI58923, 58% sequence identity). 8. CNF from Photobacterium damselae (NCBI accession: WP_005306733, 58% sequence identity). Columns with identical residues are highlighted in red. The sequence alignment was generated using ClustalX (Larkin et al, 2007) and formatted using the ESPript 3 webservice (Robert & Gouet, 2014). The alignment has been annotated with the secondary structure extracted from the structural model of full‐length CNF_Y. The domains of CNF_Y are indicated below the sequences using the coloring scheme used in Fig 1. Specific residues that are supposed to be functionally relevant are marked by arrows in the domain annotation.

Figure 1. The crystal structure of CNF_Y from Y. pseudotuberculosis

1. Domain boundaries and sequence motifs mapped to the sequence of CNF_Y.

2. Cartoon representation of CNF_Y, colored according to domain boundaries determined with PiSQRD (Aleksiev et al, 2009). Dark blue: domain D1, cyan: domain D2, dark green: domain D3, yellow: ADP‐ribosyltransferase‐like domain D4, pink: deamidase domain D5. Other colors indicate the position of sequence motifs that have been identified in E. coli CNF1, namely light blue: p37LRP/67LR receptor‐binding motif, red: hydrophobic stretches predicted to form membrane‐inserting α‐helices, orange: cleavage site, magenta: main Lu/BCAM receptor‐binding motif. The positions of N‐ and C‐terminus are indicated by N and C, respectively.

3. Surface representation of CNF_Y as seen from two different orientations with respect to (B). Note that the cleavage site between D3 and D4 (orange) as well as the deamidase active site in D5 are partially blocked in the structure of full‐length CNF_Y. The C‐terminal domain D5 interacts mainly with D3 (610 Å²), which partially hides the catalytic site of D5, but it interacts only weakly with D4 (380 Å²), which itself establishes an extensive interface with D1 (1,390 Å²) by mainly hydrophilic interactions (17 hydrogen bonds and 6 salt bridges).

Figure 2. Synthesis, secretion, and host cell binding of N‐ and C‐terminal deletion variants of CNF_Y

1. Schematic overview of marker‐tagged CNF_Y deletion variants.

2. 3xFlag‐tagged CNF_Y deletion variants were expressed in Y. pseudotuberculosis YP147 (ΔcnfY) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti‐Flag antibody.

3. To test secretion of the CNF_Y variants, full‐length CNF_Y and different N‐ and C‐terminally deleted variants fused to β‐lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (ΔcnfY). β‐lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate. The data represent the mean ± SD of three independent experiments, carried out in triplicates.

4. HEp‐2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full‐length CNF1, CNF_Y or the N‐ or C‐terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti‐Flag antibody.

Source data are available online for this figure.

Figure 3. Translocation of the deletion variants of CNF_Y and their influence on RhoA activation, actin rearrangements and multinucleation of host cells

1. HEp‐2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full‐length CNF_Y or the N‐ or C‐terminally deleted toxin variants fused to β‐lactamase (TEM) at 37°C for 4 h. Cleavage of the reporter dye CCF4‐AM was used to visualize toxin delivery. After cell entry, CCF4‐AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (530 nm). In the presence of translocated β‐lactamase fusion proteins, CCF4‐AM is cleaved, and disruption of FRET results in blue fluorescence (450 nm). Scale bar: 20 µm.

2. Left upper and right panel: HEp‐2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full‐length CNF_Y or the N‐ or C‐terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS–PAGE gels; left lower panel: HEp‐2 cells were lysed and the cell extracts were incubated with full‐length CNF_Y or the N‐terminally deleted toxin variants for 4 h. The deamidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS–PAGE after detection with anti‐RhoA antibodies.

3. HEp‐2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full‐length CNF_Y or the N‐ or C‐terminally deleted toxin variants for 24 h. The cell nuclei were stained with DAPI (blue) and the actin cytoskeleton was stained using FITC‐phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNF_Y‐treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.

Source data are available online for this figure.

Figure 4. Localization of the N‐ and C‐terminal deletion variants of CNF_Y in the late endosome

HEp‐2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full‐length CNF_Y, N‐ or C‐terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes‐RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.

Figure EV2. Exploded view of CNF_Y

Cartoon representation of full‐length CNF_Y (middle) surrounded by enlarged perpendicular views of the individual domains D1–D5, colored according to domain boundaries determined with PiSQRD (Aleksiev et al, 2009). Dark blue: domain D1, cyan: domain D2, dark green: domain D3, yellow: ADP‐ribosyltransferase‐like domain D4, pink: deamidase domain D5. Other colors indicate the position of sequence motifs that have been identified in E. coli CNF1, namely light purple: p37LRP/67LR receptor‐binding motif, red: hydrophobic stretches predicted to form membrane‐inserting α‐helices, orange: cleavage site, magenta: main Lu/BCAM receptor‐binding motif.

Figure 5. Structural homology of the CNF_Y toxin and domain organization of toxins with a CNF‐like translocation apparatus

1. Side‐by‐side comparison of CNF_Y and nigritoxin. Nigritoxin is a toxin of crustaceans and insects. The translocation domain of nigritoxin [PDB entry 5M41, (Labreuche et al, 2017)] and domain D1 of CNF_Y show partial structural similarity (highlighted areas). This similarity was identified with DALI (Holm & Rosenström, 2010) which was also used to align both structures.

2. The ART‐like domain D4 of CNF_Y. Essential residues of canonical ARTs are not conserved in CNF_Y (RSE‐ARTs exemplified by C. perfringens iota toxin, PDB entry 4H03 (Tsurumura et al, 2013); HYE‐ARTs exemplified by P. aeruginosa ExoA, PDB entry 2ZIT (Jørgensen et al, 2008); carbon atoms of NAD⁺ shown in black).

3. The deamidase domain D5 of CNF_Y. C866 and H881 form a conserved catalytic dyad also found in the deamidase domain of E. coli CNF1 [PDB entry 1HQ0, (Buetow et al, 2001)] and Burholderia pseudomallei lethal factor BLF1 [PDB entry 3TU8, (Cruz‐Migoni et al, 2011)].

Figure 6. Characterization of mutants in the linker region connecting domain D3 and D4

1. HEp‐2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing CNF_Y, the toxin variant mut1: CNF_YI535L/P536A/V537G or mut2: CNF_YI535L/P536A/V537G/F539L/D541A/K542A fused to TEM or no CNF_Y protein (empty vector) for 4 h. Cells were lysed and the binding of the different CNF_Y proteins to HEp‐2 cells was analyzed by immunoblotting.

2. HEp‐2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing CNF_Y, the toxin variant mut1: CNF_YI535L/P536A/V537G or mut2: CNF_YI535L/P536A/V537G/F539L/D541A/K542A or no CNF_Y protein (empty vector). The cell nuclei were stained with DAPI (blue) and the actin cytoskeleton was stained using FITC‐phalloidin (green). The results indicated the formation of polynucleated cells and stress fibers only in cells treated with CNF_Y and CNF_YI535L/P536/V537G. The white scale bar is 20 µm.

3. Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNF_Y derivatives to determine β‐lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.

4. The viability of Y. pseudotuberculosis YPIII expressing the indicated CNF_Y derivatives was assessed in equalized bacterial cultures using the BacTiter‐Glo Microbial Cell Viability Assay kit (Promega). The data represent the mean ± SD of three independent experiments, carried out in triplicates.

5. HEp‐2 cells treated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing indicated CNF_Y variants for 4 h were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified RhoA GTPase detected by immunoblotting.

6. The activity of the CNF_Y derivatives was tested by analyzing the deamidation of RhoA in HEp‐2 cell lysates by the mobility shift of the modified GTPase detected by immunoblotting.

Source data are available online for this figure.

Figure EV3. The DUF4765 family and the ART‐like domain of CNF_Y

1. Sequence alignment of representative sequences of the DUF4765 family. Red stars indicate the position of three residues that reside in similar positions as catalytical residues in canonical ART domains (R599, E639, H676 in CNF_Y). The aligned sequences are 1: M. viscosa, CNF1, 55% sequence similarity to domain 4 of CNF_Y (UniProt‐ID: W6AYD9), 2: P. damselae, CNF1, 55% seq. similarity (UniProt‐ID: D0Z517), 3: Bacteriophage Stx2a_WGPS2, EspN, 23% seq. similarity (UniProt‐ID: A0A0P0ZCL8), 4: C. rodentium, EspN2‐2, 23% seq. similarity (UniProt‐ID: D2TQZ7), 5: S. bongori, EspN, 24% seq. similarity (UniProt‐ID: A0A3S5D9E8), 6: S. enterica subsp. arizonae, uncharacterized protein, 23% seq. similarity (UniProt‐ID: A9MRQ7), 7: S. enterica subsp. arizonae, uncharacterized protein, 24% seq. similarity (UniProt‐ID: A9MQT6), 8: S. enterica subsp. arizonae, EspN, 24% seq. similarity (UniProt‐ID: A0A2X4TBS3), 9: E. coli O157:H7, EspN, 26% seq. similarity (UniProt‐ID: A0A0H3JD33), 10: P. temperata, uncharacterized protein, 18% seq. similarity (UniProt‐ID: T0QEQ3), 11: Streptomyces sp. H‐KF8, uncharacterized protein, 20% seq. similarity (UniProt‐ID: A0A1A5P6M0), 12: S. xinghaiensis, uncharacterized protein, 22% seq. similarity (UniProt‐ID: A0A420VA44). The sequence alignment has been performed with PROMALS3D (Pei et al, 2008) and was rendered with ESPript (Gouet et al, 2003).

2. Comparison of the ART‐like domain D4 of CNF_Y with canonical ART domains. Note that the hypothetical NAD⁺ binding site of D4 is shallower than that of the other two domains and that the NAD⁺ molecules found in these examples would clash with residues of D4 when bound in the same conformation.

Figure EV4. C‐terminal domain D3–5 and D4–5 are able to bind to host cells and deaminate RhoA

1. The expression of purified recombinant CNF_Y N‐terminal domains D3–5 (CNF_Y 426–1,014) or D4–5 (CNF_Y 526–1,014).

2. HEp‐2 cells remain untreated or were incubated with 500 nM purified full‐length CNF_Y, domains D3–5 (CNF_Y 426–1,014) or D4–5 (CNF_Y 526–1,014) for 24 h. The formation of large, multinuclear cells was observed by fluorescence microscopy. The cell nuclei were stained with DAPI (blue) and the actin cytoskeleton was stained using FITC‐Phalloidin (green). The white scale bar is 20 µm.

3. Binding of purified full‐length CNF_Y, domains D3–5 (CNF_Y 426–1,014) or D4–5 (CNF_Y 526–1014) to HEp‐2 cells was analyzed by immunoblotting after HEp‐2 cells were incubated with 500 nM of the purified toxin or toxin fragments for 4 h.

4. HEp‐2 cells treated with 500 nM purified full‐length CNF_Y, protein fragments D3–5 (CNF_Y 426–1,014) or D4–5 (CNF_Y 526–1,014) were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified GTPase detected by immunoblotting.

5. The activity of purified CNF_Y and the protein fragments D3–5 (CNF_Y 426–1,014) or D4–5 (CNF_Y 526–1,014) was tested by analyzing the deamidation of RhoA in HEp‐2 cell lysates by the mobility shift of the modified GTPase detected by immunoblotting.

Source data are available online for this figure.

Figure 7. Structure and deamidation activity of the free D4–5 subunit of CNF_Y

1. Crystal structure of the free D4–5 subunit. Note the different relative orientations of domains D4 and D5 with respect to the structure of full‐length CNF_Y (top, thin gray lines). The domain D4 forms a large interface area (1,100 Å²) with the catalytic domain D5 involving several polar interactions (8 hydrogen bonds and 8 salt bridges), whereby the active crevice is extended and fully solvent‐exposed as can be seen in the right surface plot at the bottom of the panel. The hypothetical NAD⁺ binding site of the ART‐like D4 domain is located on the opposite face (left surface plot). Note that the deamidase active site of domain D5, unlike in the full‐length structure (Fig 1), is fully accessible and that its extended shape is also determined by domain D4.

2. Comparative analysis of RhoA activation in HEp‐2 cell lysate by CNF_Y and the recombinant D4–5 protein. Purified CNF_Y or the D4–5 fragment (1 µM) was added to extracts of HEp‐2 cells and incubated for 10, 20, 30, or 60 min. Deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS–PAGE gels after detection with anti‐RhoA antibodies.

3. Comparative analysis of recombinant RhoA deamidation by CNF_Y and the D4–5 fragment. Recombinant RhoA was incubated with purified CNF_Y or the D4–5 fragment and samples were separated by SDS–PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.

Source data are available online for this figure.

Figure 8. Architecture of bacterial toxins with a CNF‐like translocation apparatus

Shown is the architecture of CNF_Y, Pasteurella multocida toxin PMT and two uncharacterized proteins from Pseudomonas syringiae. The released fragment of PMT contains three domains of which C1 is required for membrane binding, the C2 domain has an unknown function, and the C3‐domain activates heterotrimeric G‐proteins by deamidation. The two Pseudomonas syringiae proteins A0A0P9UH04 and A0A0N8SZE6 represent uncharacterized toxins that encode catalytic domains of the indicated type. While sequence alignments unequivocally reveal a CNF‐like imperfect β‐barrel in PMT, the presence of this domain in the P. syringiae toxins is less obvious.