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Updated: Apr 15, 2026
RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells
Published on: June 4, 2016
A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and
Sebastian Rupp1, Lisandra Aguilar-Bultet2, Vidhya Jagannathan3
1Division of Neurological Sciences, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern CH-3001, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern CH-3012, Switzerland.
Abstract:
Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.
Insights
A novel Listeria monocytogenes strain (JF5171) shows reduced virulence due to a truncated prfA gene. This attenuated strain offers insights into Listeria pathogenesis and potential control strategies for infections.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Genomics
Background:
- Listeria monocytogenes is a significant foodborne pathogen causing severe infections.
- Understanding virulence factors is crucial for developing control strategies.
Purpose of the Study:
- To characterize a newly isolated, attenuated Listeria monocytogenes strain (JF5171) from bovine abortion.
- To identify the genetic basis for its reduced virulence.
Main Methods:
- Whole genome sequencing of strain JF5171.
- In vitro assays for invasion, replication, cell-to-cell spread, phospholipase, and hemolysis activity.
- Analysis of virulence gene transcription (actA, hly, prfA).
- Genetic complementation studies.
Main Results:
- Strain JF5171 displayed significantly reduced replication, invasion, and cell-to-cell spread compared to the reference strain EGD-e.
- JF5171 showed diminished phospholipase and hemolysis activity, with absent actin polymerization.
- A frame shift deletion in the prfA gene, leading to a truncated protein, was identified as the primary cause of attenuation.
- Complementation with functional prfA restored virulence, while complementation with the truncated version had no effect.
Conclusions:
- The truncated C-terminus of the virulence regulator prfA is a major contributor to the attenuated phenotype of Listeria monocytogenes strain JF5171.
- This finding deepens our understanding of Listeria monocytogenes virulence regulation.
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