Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo Juan J. Quereda, a,b,c Marie A. Nahori, a,b,c Jazmín Meza-Torres, a,b,c Martin Sachse, d Patricia Titos-Jiménez, e Jaime Gomez-Laguna, e Olivier Dussurget, a,b,c,f Pascale Cossart, a,b,c Javier Pizarro-Cerdá a,b,c Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France a ; Institut National de la Santé et de la Recherche Médicale, U604, Paris, France b ; Institut National de la Recherche Agronomique, USC2020, Paris, France c ; Institut Pasteur, Ultrapole, Paris, France d ; Anatomy and Comparative Pathology Department, University of Cordoba, International Excellence Agrifood Campus CeiA3, Cordoba, Spain e ; Cellule Pasteur, Université Paris Diderot, Sorbonne Paris Cité, Paris, France f ABSTRACT Streptolysin S (SLS)-like virulence factors from clinically relevant Gram- positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin pres- ent among Listeria monocytogenes strains responsible for human listeriosis out- breaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers re- sistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes. Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal sys- tem, and only in this environment LLS expression is detected in vivo. Finally, we confirm that LLS production is associated with destruction of target bacteria. Our re- sults demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we de- scribe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demon- strate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections. IMPORTANCE The most severe human listeriosis outbreaks are caused by L. mono- cytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacte- rial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttrans- lational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity. KEYWORDS Listeria, listeriolysin S, streptolysin S, cytotoxin, epidemics, infection Received 15 February 2017 Accepted 7 March 2017 Published 4 April 2017 Citation Quereda JJ, Nahori MA, Meza-Torres J, Sachse M, Titos-Jiménez P, Gomez-Laguna J, Dussurget O, Cossart P, Pizarro-Cerdá J. 2017. Listeriolysin S is a streptolysin S-like virulence factor that targets exclusively prokaryotic cells in vivo. mBio 8:e00259-17. https://doi.org/10 .1128/mBio.00259-17. Editor Julian E. Davies, University of British Columbia Copyright © 2017 Quereda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Javier Pizarro- Cerdá, javier.pizarro-cerda@pasteur.fr. RESEARCH ARTICLE crossm March/April 2017 Volume 8 Issue 2 e00259-17 ® mbio.asm.org 1