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

Richard F. Rest,Eugene B. Chang,Mitch L. Villereal,Enrico Malito,Brian L. Bishop,Alexandre Chenal,Elise M. Mosser,Wei-Jen Tang,Mark W. Musch,Raymond W. Bourdeau

doi:10.1074/jbc.m807631200

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.

Highlights

A good deal is known about structures of the prepore conformation of cholesterol-dependent cytolysin (CDC)
anthrolysin O (ALO) Decreases the Barrier Function of Human Gut Epithelial Cells—One of the signs of active anthrax infection within the gastrointestinal tract is the presence of primary lesions within the intestinal tissue [29]
Since other CDCs have been reported to act on epithelial cells, we proposed that ALO could potentially function to alter barrier integrity of Caco-2 brush border expressor (C2BBE) monolayers [45]

Summary

Functions and Structure of Anthrolysin O

ALO is secreted by Bacillus anthracis, the etiologic agent for anthrax. ALO is chromosomally encoded by a gene whose regulation is poorly understood, and it is highly homologous to other members of the CDC family [12]. Interplay among anthrax secreted factors on cells relevant to anthrax infection is just beginning to be understood. This network of interactions is vital to the molecular basis of how anthrax bacteria interact with the hosts during anthrax infection. Anthrax infection initiates when B. anthracis spores enter the host through one of three routes: cutaneous, inhalational, or gastrointestinal (GI) [27, 28]. The barrier is produced by a matrix of transmembrane and membrane-associated proteins These cell to cell contacts, or tight junctions, are sometimes altered during bacterial infection to disrupt the barrier function of epithelial cells. We used domain swapping to examine the structural components that confer specificity of ALO to gut epithelial cells

EXPERIMENTAL PROCEDURES

RESULTS

Protein Data Bank accession code

DISCUSSION

The epithelial lining of mucosal