Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.

Keywords: ADP-ribosyltransferase; mycoplasma cytotoxin; reactive airway disease; single-crystal X-ray diffraction; vacuolation.

Fig. 1.

CARDS TX tertiary structure. In all figures, the D1 mART domain is shown in light blue, the steric block of the NAD⁺-binding pocket is violet, the linker connecting D1 to D2+D3 is dark red, D2 is dark blue, D3 is green, Cys-Sγ atoms are yellow spheres, and F591 is bright red. The aromatic patch in D3 is shown as orange sticks.

Fig. 2.

CARDS TX secondary structure. (A) Secondary structure observed crystallographically as a function of amino acid sequence. (B) The D1 mART domain lacking the steric block labeled according to A. The view is ∼90° around the horizontal relative to Fig. 1. (C) Divergent stereo pair in the same orientation as Fig. 1 showing the steric block of the NAD⁺-binding pocket, linker, and D2+D3 β-trefoil domain structural elements labeled as in A.

Fig. 3.

CARDS TX is a member of the CTx subgroup (CTxg) of bacterial mART domains. (A) Structure-based alignment of ADP-ribosylating toxins from the cholera (CTxg) and diphtheria toxin (DTxg) subgroups. CARDS TX secondary structure is indicated at the top. The R–STS/T–E signature motif is shared by CTxg members (yellow). ARTT residues are in cyan. The S6-H5 portion of the H4-S6-H5 motif found only in CARDS TX is boxed. (B) CARDS TX (light blue) and PTx (green) mART domains superimposed on the CTx mART domain (pink) in complex with NAD⁺ (cyan sticks).

Fig. 4.

Steric blocks of CARDS TX and PTx mART active sites. The R–STS/T–E signature motif residues are colored as in Fig. 3A. (A) CARDS TX mART domain. C230 and C247 within the steric block form a disulfide bond (yellow spheres). The view is as in Fig. 2B. (B) PTx mART domain. C41 and C201 form a disulfide bond (yellow spheres) linking the steric block to the N-terminal portion of the mART domain. (C) Surface representation highlighting the steric block of the CARDS TX NAD⁺-binding pocket. (D) Catalytic and substrate recognition residues are buried at the D1/D2D3 interface. D2 and D3 have been removed for clarity. A tunnel into the active site runs adjacent to the ARTT motif. The yellow surface inside the tunnel is E132. (E) 1.9-Å electron density with coefficients 2mF_o − DF_c contoured at 1.2 σ superimposed on the refined CARDS TX structure. The C-terminal residues of the toxin, R590 and F591, project into the active site.

Fig. 5.

CARDS TX binds PC, DPPC, and SM specifically over other membrane lipids. (A) ELISA and (B) dot blot analyses of CARDS TX binding to a panel of diverse membrane lipids and their components as described in Methods. Full-length, D1, and D2+D3 constructs were tested. The 16 lipids/lipid components on each strip are (1) triglyceride, (2) diacylglycerol, (3) phosphatidic acid, (4) phosphatidylserine, (5) phosphatidylethanolamine, (6) PC, (7) phosphatidylglycerol, (8) cardiolipin, (9) phosphatidylinositol, (10) phosphatidylinositol 4-phosphate, (11) phosphatidylinositol 4,5-bisphosphate, (12) phosphatidylinositol 3,4,5-trisphosphate, (13) cholesterol, (14) SM, (15) 3-sulfogalactosylceramide, and (16) blank.