Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay. 1. Introduction Listeriosis is a foodborne illness caused by infection with Listeria monocytogenes, a gram-positive pathogenic bacterium [1]. L. monocytogenes is the only species in the genus Listeria that can infect humans [2]. After the bacteria are phagocytosed, listeriolysin O (LLO), an exotoxin, forms a pore in the membrane of the phagosome that allows the bacteria to escape into the cytosol of the phagocyte, where it is adapted to grow [2]. L. monocytogenes that is incapable of secreting LLO (Δhly) is not pathogenic [3]. Listeriolysin O is a 529 amino acid protein that is a member of the cholesterol-dependent cytolysin (CDC) family of proteins [4]. LLO is a four-domain poreforming protein that is regulated by pH [5]. LLO also contains a 21 amino acid PEST sequence (Figure 1) in the amino terminus that probably helps to control LLO production in the cytosol [6], as well as an undecapeptide sequence that is important for membrane binding [7] (Figure 1). Figure 1: ClustalW alignment of antigenic LLO amino acid sequences from GenBank. Red Letters denote the PEST sequence. Amino acids highlighted in red belong to domain 1, amino acids highlighted in blue belong to domain 2, amino acids highlighted in yellow belong to domain 3, and amino acids highlighted in green belong to domain 4. Amino acids highlighted in magenta are polymorphic. Black triangle denotes the location of signal sequence cleavage site. Purple amino acids denote the location of the undecapeptide sequence, required for the formation of the oligomeric pore complex. The purple α and brown β symbols above the alignments denote the locations of alpha helices and beta-pleated sheets, respectively, predicted
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