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Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogenListeria monocytogenes . LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis ofL. monocytogenes , we developed a screen that combined saturation mutagenesis and signature tags, termedi n v ivo a nalysis bys aturation mutagenesis ands ignature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzedin vivo each conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using commonin vitro techniques suggested that the functional contributions of some residues could only have been revealed throughin vivo analysis.

infection and immunity

Development of a Single-Gene, Signature-Tag-Based Approach in Combination with Alanine Mutagenesis To Identify Listeriolysin O Residues Critical for the<i>In Vivo</i>Survival of Listeria monocytogenes

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