Oral Presentation Lorne Infection and Immunity 2020

Inhibition of PrfA activity restricts L. monocytogenes to spacious vacuoles and licenses macrophages to eliminate high intracellular bacterial loads (#26)

Thanh Thao Tran 1 , Marcela Gatica-Andrades 1 , Carmen D. Mathmann 1 , Rachel F. Rollo 1 , Melanie Oelker 2 , Tam T.K. Nguyen 1 , Alina Zamoshnikova 1 , Lalith K. Kummari 3 , Orry K.J. Wyer 1 , Katharine M. Irvine 4 , Javier N. Melo-Bolivar 1 , Annette Gross 1 , Darren Brown 5 , Jeffrey Y.M. Mak 5 , David P. Fairlie 5 , Karl A. Hansford 5 , Matthew A. Cooper 5 , Rabina Giri 4 , Veronika Schreiber 4 , Shannon Joseph 1 , Fiona Simpson 1 , Timothy C. Barnett 6 , Timothy J. Wells 1 7 , Ronan Kapetanovic 5 7 , Matthew J. Sweet 5 7 , Eleanor A. Latomanski 8 , Hayley J. Newton 8 , Romain J.R. Guérillot 8 , Abderrahman Hachani 8 , Timothy P. Stinear 8 , Sze Ying Ong 8 9 , Yogeswari Chandran 8 9 , Elizabeth L. Hartland 8 9 , Bostjan Kobe 3 7 , Jennifer L. Stow 5 7 , A. Elisabeth Sauer-Eriksson 2 , Jakob Begun 4 , Jessica C. Kling 1 , Antje Blumenthal 1 7
  1. The University of Queensland Diamantina Institute, The University of Queensland, Brisbane
  2. Department of Chemistry, Umea University, Umea, Sweden
  3. School of Chemistry and Molecular Bioscience, The University of Queensland, Briabne, QLD, Australia
  4. UQ Mater Research, The University of Queensland, Brisbane, QLD
  5. Institue for Molecular Bioscience, The University of Queensland, Brisbane
  6. Westfarmers Centre for Vaccines and Infectious Diseases, Telethon Kids Institute, University of Western Australia, Perth, WA, Australia
  7. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane
  8. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  9. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research and Department of Molecular and Translational Science, Monash University, Melbourne, VIC, Australia

A hallmark of L. monocytogenes pathogenesis is escape of the bacteria from maturing phagosomes into the cytoplasm where the bacteria replicate rapidly and progress to infect neighbouring cells. Phagosome escape is facilitated by listeriolysin O (LLO) in concert with other virulence factors, whose expression is controlled by the transcription factor PrfA. Control of L. monocytogenes by the infected host requires T-cell mediated adaptive immunity, in particular cytotoxic T cell activity. More recently, L. monocytogenes has been found to reside inside vacuoles when expressing low levels of LLO in mouse macrophages or during low-dose infection of human hepatocytes and trophoblasts. This been associated with slow/non-replication of the bacteria and chronic infection in vivo.

Here we report that functional inhibition of the master regulator of L. monocytogenes virulence gene expression, PrfA, prevents phagosomal escape of L. monocytogenes. Instead, the bacteria rapidly replicated inside LAMP1-/LC3- vacuoles, which required LLO activity and functional host cell mitochondria. In contrast to the non-degradative spacious Listeria-containing phagosomes described previously in macrophages, these novel Listeria replication vacuoles (LisRVs) acquired lysosomal proteases and acidified, which was associated with clearance of L. monocytogenes by infected cells in the absence of T cell contributions. Crystallography of the PrfA:inhibitor complex showed direct binding of the inhibitor to the PrfA activator site. Our data demonstrating that pharmacologic targeting of L. monocytogenes PrfA licences macrophages to eradicate the bacteria might be harnessed for the rational design of adjunct therapies supporting treatment of listeriosis in high-risk patients.