Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis

Abstract

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.Cholesterol-dependent cytolysins (CDCs) 4 are a family of pore-forming toxins from many organisms, including but not limited to the genera Archanobacterium, Bacillus, Clostridium, Listeria, and Streptococcus. Recently, work in vertebrates has revealed that CDCs and membrane attack complex/perforin superfamily domain-containing proteins share a similar fold, suggesting that vertebrates use a similar mechanism for defense against infection (1, 2). A common feature of the CDC family is the requirement of cholesterol in the membrane to form pores (3). In addition to cholesterol, certain members of the family also require a cellular receptor, such as CD59 for the toxin ILY from Streptococcus intermedius (4). The specific mechanism by which CDCs form pores is not completely resolved; however, what is generally known is that ring-shaped oligomerization at the cellular membrane is followed by large conformational changes in each unit of the oligomer, resulting in the insertion of a ␤-barrel into the cellular membrane (5). Pore formation results in a variety of downstream signaling effects, including but not limited to the influx of Ca 2ϩ into the cell (6).A good deal is known about structures of the prepore conformation of CDCs. The crystal structures of prepore PFO, from Clostridium perfringens, and ILY have previously been elucidated (7,8). Each structure shows a characteristic fourdomain architecture, in which domain 4 (D4) is involved in membrane recognition, domain 3 (D3) is involved in ␤...

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

“…S1). These observations are also consistent with a lack of conservation of the 3D structures of the undecapeptide in the closely related CDCs PFO (17) and Bacillus anthracis anthrolysin O (ALO) (18) (Fig. S1).…”

Development of a Single-Gene, Signature-Tag-Based Approach in Combination with Alanine Mutagenesis To Identify Listeriolysin O Residues Critical for theIn VivoSurvival of Listeria monocytogenes

“…The CDCs act as potent virulence factors by assembling into large, homooligomeric pores upon encountering a mammalian cell (reviewed in references 18, 25, and 80). These toxins share a high degree of sequence identity (40 to 70%) and adopt the same four-domain secondary structure (4,56,65). The structural similarity of the CDCs suggests that these toxins also exhibit similar pore-forming activities.…”

Structural classification of insecticidal proteins – Towards an in silico characterisation of novel toxins

“…These include Cry37 (de Maagd et al, 2003) and the aegerolysin-like Cry34 (Kelker et al, 2014) which both form part of binary toxins (with Cry23 and Cry35 respectively, discussed below). The presence of extended beta sheets also characterises the sphaericolysin/anthrolysin family of toxins that is highly conserved across isolates from (Berry, 2012;Bourdeau et al, 2009) and appear to act as cholesterol dependent cytolysins (From et al, 2008;Nishiwaki et al, 2007). At present, the literature lacks sufficient information to allow structure/function predictions for these toxins.…”