Poster Presentation The 3rd Prato Conference on the Pathogenesis of Bacterial Diseases of Animals 2014

A novel prfA truncation is associated with reduced invasion, replication and cell-to-cell spread of Listeria monocytogenes (#32)

Sebastian Rupp 1 , Claudia Guldimann 2 , Vidhya Jagannathan , Cord Drögemüller 3 , Joachim Frey 4 , Torsten Seuberlich , Anna Oevermann 1
  1. Neurocenter, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University Bern, Bern, Switzerland
  2. Vetsuisse Faculty, University of Bern, Bern, BERN, Switzerland
  3. Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland
  4. Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland

Introduction: L. monocytogenes is an environmental bacterium that may act as an intracellular pathogen upon ingestion. It causes gastroenteritis, septicaemia, abortions and (frequently fatal) CNS infections. During virulence screening of L. monocytogenes field strains, one strain (O/D1387/06, lineage II, PCR-serotype 1/2a; 3a, isolated from a bovine placenta in the context of abortion) failed to replicate in bovine brain-slice cultures and produced severely reduced plaques in cell culture. This study further analyzed O/D1387/06 in order to identify possible mechanism of attenuation.

Material & methods: The whole bacterial genome was sequenced using an Illumina next-generation sequencing system. We analyzed replication, actin polymerization and intercellular spread of O/D1387/06 and complementation mutants in various cell-lines (foetal bovine brain cells, bovine macrophage cells, bovine caruncular epithelial cells, and the widely used human epithelial colorectal adenocarcinoma cells) by quantification of CFUs and immunofluorescence. Phospholipase and hemolysis activity of O/D1387/06 was photometrically quantified. Data were compared to the widely used L. monocytogenes strain EGD-e.

Results: O/D1387/06 showed reduced replication in all tested cell lines and reduced phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased when compared to EGD-e, and actin-polymerization was absent. We detected a frame shift deletion in the major regulator for virulence, prfA, leading to a truncation at the c–terminus (WEN* vs. WGKLN*) and a point mutation leading to a K -> N substitution at aa position 197. Complementation with prfA from EGD-e and with (EGD-e)prfA-K197N increased replication and spread efficiency, while complementation with the truncated version of (EGD-e)prfA had no significant effect.

Conclusion: We identified a novel truncation at the C-terminus of prfA, the regulator responsible for transcription of several virulence factors needed for cell invasion, escape from the phagocytic vacuole and cell to cell spread, which is responsible for a strongly attenuated phenotype in vitro.