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.
Phocaeicola vulgatus is one of the most abundant and ubiquitous bacterial species of the human gut microbiota, yet a comprehensive analysis of antibacterial toxin production by members of this species has not been reported. Here, we identify and characterize a previously undescribed antibacterial protein. This toxin, designated BcpT, is encoded on a small mobile plasmid that is largely confined to strains of the closely related species Phocaeicola vulgatus and Phocaeicola dorei. BcpT is unusual in that it requires cleavage at two distinct sites for activation, and we identify bacterial proteases that perform this activation. We further identify BcpT’s receptor as the Lipid A-core glycan, allowing BcpT to target species of other Bacteroidales families. Exposure of cells to BcpT induces a response involving an unusual sigma/anti-sigma factor pair that is likely triggered by cell envelope stress, resulting in the expression of genes that partially protect cells from multiple antimicrobial toxins.
Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins that are secreted as soluble monomers and oligomerise into large circular pre-pores on the surface of cholesterol-rich membranes. Various structural changes and transitions results in insertion of β-hairpins into the lipid bilayer, forming a large β-barrel pore that results in cell lysis [1]. We have identified a highly conserved structural motif of CDCs that plays a critical role in the prepore-to-pore transition [2]. Furthermore, this motif is also highly conserved in a large, diverse family of uncharacterised proteins from over 220 species, which we have designated the name "CDC-like" (CDCL) protein family [2]. One partner of the CDCL pair, termed CDCL long, consists of four domains: three similar to CDCs and a unique fourth domain. The other partner, CDCL short, possesses three domains, all similar to CDCs. One CDCL pair, referred to as ALY long (ALY L ) and ALY short (ALY S ), originate from the species Elizabethkingia anophelis; an emerging and opportunistic pathogen of unknown virulence and transmission. X-ray crystallography revealed the structure of monomeric ALY L consists of characteristic CDC domain 1 -3 structure despite only 22% identity with the archetype CDC perfringolysin O; however, domain 4 is completely different to the equivalent domain in CDCs that plays a role in sensing cholesterol. In the presence of lipids, ALY L and ALY S show pore-forming activity and analysis by electron microscopy reveals a large circular oligomeric complex reminiscent of CDC pore complexes. ALY S also forms a non-lytic circular oligomer in the absence of ALY L . Cross-linking mass spectrometry data reveals structural changes between the monomeric and protomeric states, giving insight to the mechanism of pore formation. To determine the atomic structure of ALY pores, cryo-EM single-particle analysis is currently being pursued In summary, we have shown that the ALY toxins share some structural resemblance to CDCs, but in contrast form a two-component pore complex. CDC-like proteins are present in a wide range of bacterial species and are suspected to play key roles in microbial survival and human disease. An understanding of pore formation by ALY may yield new knowledge of Elizabethkingia anophelis virulence, in addition to providing a system that could be applied to biotechnological applications. Our work on ALY provides the first functional and structural insights into this fascinating family of proteins.
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