Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine Aurelie Hanin 1 , Irina Sava 2 , YinYin Bao 2 , Johannes Huebner 2 , Axel Hartke 1 , Yanick Auffray 1 , Nicolas Sauvageot 1 * 1 Laboratoire de Microbiologie de l’Environnement, EA956 USC INRA2017, Universite ´ de Caen, Caen, France, 2 Division of Infection Diseases, Department of Medicine, University Medical Center, Freiburg, Germany Abstract Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a Recombination-based In Vivo Expression Technology for E. faecalis. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, during growth in urine, in a mouse bacteremia and in a mouse peritonitis model. Our combined results led to the identification of 81 in vivo activated genes. Among them, the ef_3196/7 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system was essential to the E. faecalis pathogenic potential in Galleria. Gene ef_0377, induced in insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Thus these R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen. Citation: Hanin A, Sava I, Bao Y, Huebner J, Hartke A, et al. (2010) Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine. PLoS ONE 5(7): e11879. doi:10.1371/journal.pone.0011879 Editor: Niyaz Ahmed, University of Hyderabad, India Received April 12, 2010; Accepted July 5, 2010; Published July 29, 2010 Copyright: ß 2010 Hanin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Auralie Hanin is a PhD student whose thesis was granted by the French Ministre de l’Enseignement suparieur et de la Recherche. This work was partly supported by grants from the Agence Nationale de la Recherche in the frame of a transnational ERA-NET PathoGenoMics program (ANR-06-PATHO-008-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nicolas.sauvageot@unicaen.fr Introduction Enterococcus faecalis is a ubiquitous lactic acid bacterium and a core constituent of the intestinal flora of humans and many animals. The intrinsic ability of this bacterium to resist strongly against stressing environments may allow the bacterium to persist in hospital environments and to survive host defences [1,2]. In the last decades, Enterococci have been recognized as one of the most common bacteria involved in hospital-acquired infections [3]. Indeed, these microorganisms can trigger serious infections such as sepsis, urinary tract infections, peritonitis and endocarditis and the species E. faecalis is still responsible for the majority of human enterococcal infections [3,4]. For this reason, clinical strains have been studied for their virulence-associated factors: the Cytolysin CylL [5], the Aggregation substance Agg [6], the metallo- endopeptidase GelE [7], the Extracellular Surface Protein Esp [8], and the cell surface protein EfaA [9]. Despite studies characterizing these proteins, our knowledge of the mechanisms involved in infections, especially transcriptional modulation occurring in living hosts, remains incomplete. Thus, in order to further increase our understanding of enterococcal pathogenesis, it is necessary to identify genes that are specific to infection. In an infected host, microorganisms are subjected to combined stresses. Although these conditions could be simulated in vitro by the study of each of these stresses independently, it is not possible to reproduce an exact mimic of the complex and dynamic environment encountered by bacteria in infected hosts. In a different approach, several In Vivo expression technologies (IVET) have been developed [10,11]. With this technology, the living infected animal is the inducing-signal of virulence-associated genes expression. An IVET strategy was developed for the first time in Salmonella enterica serovar Typhimurium to identify in vivo highly expressed genes as compared to expression under laboratory conditions. Five in vivo-induced (ivi) operons have been identified using this strategy, originally based on the use of an auxotrophic marker. Three of them, corresponding to the carAB, pheST-himA and rfb operons, have been confirmed to play essential roles in virulence [12]. Other IVET approaches, using antibiotic resis- tance markers [13] or dual reporters [14–16], have also been employed. However, these strategies present common drawbacks due to the fact that weakly or transiently expressed ivi genes are difficult or impossible to detect. These disadvantages have led some authors to choose a fourth IVET approach, called R-IVET for Recombination-based IVET [17]. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11879