A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins

Abstract

The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs. IMPORTANCE The cholesterol-dependent cytolysins’ pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.

Bacterial pore-forming toxins

“…As previously discussed, the ECTGLAWEWWR sequence, which mediates binding to cholesterol, is highly conserved within this family [59]. Furthermore, recent work has highlighted that the motif F/Y-F/Y-Xn-YGR also displays a high degree of conservation within CDCs [114]. In particular, this motif is highly conserved in several proteins that share almost no other sequence similarity with known CDCs and are found in different bacterial species [114].…”

“…Furthermore, recent work has highlighted that the motif F/Y-F/Y-Xn-YGR also displays a high degree of conservation within CDCs [114]. In particular, this motif is highly conserved in several proteins that share almost no other sequence similarity with known CDCs and are found in different bacterial species [114]. The X-ray structure of one of the newly identified proteins has been solved, highlighting that their 3D structure is nearly identical to monomers of different CDCs, defining these proteins as a new class of CDC-like toxins [114].…”

“…In particular, this motif is highly conserved in several proteins that share almost no other sequence similarity with known CDCs and are found in different bacterial species [114]. The X-ray structure of one of the newly identified proteins has been solved, highlighting that their 3D structure is nearly identical to monomers of different CDCs, defining these proteins as a new class of CDC-like toxins [114]. Furthermore, the positioning, orientation and molecular contacts of the F/Y-F/Y-Xn-YGR motif in the Elizabethkingia anophelis CDC-like toxin and PFO are conserved.…”

Structural Basis of the Pore-Forming Toxin/Membrane Interaction

“…However, the factors and mechanisms triggering the transmembrane pores are still not entirely clear. A conserved F/Y-F/Y-Xn-YGR motif with the CDCs is reported critical as the sensor to initiate the prepore-to-pore transition [ 94 ].…”

Diversity of β‐hemolysins produced by the human opportunistic streptococci

“…In recent years, it has been reported that the strains belonging to S. pseudopneumoniae and S. mitis possess ORFs encoding novel molecules, including the partial domain(s) derived from CDCs 63 . In addition, other CDC‐like molecules were reported 104 . Recently, the functional investigations for one of the CDC‐related molecules named mitilectin (MLC) derived from the strains of S. pseudopneumoniae and S. mitis had been conducted and revealed that MLC functions as an adhesion molecule 105 …”