Functional mapping of community‐acquired respiratory distress syndrome (CARDS) toxin of Mycoplasma pneumoniae defines regions with ADP‐ribosyltransferase, vacuolating and receptor‐binding activities

Abstract

SUMMARY Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591 amino acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modeling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.

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

“…The steric blocks of target recognition elements and the NAD + -binding sites suggest that CARDS TX-mediated ADP ribosylation requires a conformational change or proteolytic nicking to permit the separation of D1 from D2+D3. This notion seems plausible, given previous work demonstrating that D1 and D2+D3 in isolation retain their respective mART and binding and internalization activities (27). The solvent-exposed loop between Cys230 and Cys247 is a candidate for a proteolytic cleavage event, because residues 237-241 of this loop are disordered in all seven CARDS TX molecules in the present study ( Fig.…”

“…Importantly, the former truncation abrogates binding and internalization ( Fig. S4A) but the latter truncation does not (27), implicating D3 as the mediator of these activities. The steric blocks of target recognition elements and the NAD + -binding sites suggest that CARDS TX-mediated ADP ribosylation requires a conformational change or proteolytic nicking to permit the separation of D1 from D2+D3.…”

“…5B). CARDS TX D2+D3 domains were previously reported to mediate binding, internalization, and vacuolation activities (19,27). The crystal structure suggests that deletion of residues 571-591 in D3 or residues 274-308 in D2 will severely disrupt the integrity of the β-trefoil domains in which they reside ( Fig.…”

“…These data suggest that entry of CARDS TX into host cells is mediated by D3. Previous work indicated that CARDS TX-mediated vacuolating activity also resides in D2+D3 and is independent of D1 or mART activity, because catalytic mutants Arg10Ala, His36Ala, and Glu132Ala and N-terminal truncation variants 178 CARDS 591 and 264 CARDS 591 all induce vacuolation when incubated with HeLa cells as efficiently as the native toxin (27).…”

“…3B). The N-terminal arginine (R10) and the midregion Ser-Thr-Ser (S49-T50-S51) are predicted to bind and orient NAD + , whereas the invariant catalytic Glu (E132) is responsible for the transferase activity (12,27). The six residues preceding and two residues following E132 form the ADP-ribosylating turn-turn (ARTT) motif ( Fig.…”