Avant-propos

L’étude bibliographique présentée dans le chapitre précédent, a permis de réaliser une synthèse des principales informations relatives à l’écologie de L. monocytogenes dans le sol. Les populations de L. monocytogenes sont soumises dans l’environnement à de nombreuses contraintes. Pour y faire face et survivre dans l’environnement tellurique, L. monocytogenes

doit être capable d’intégrer les signaux environnementaux et de répondre de façon concertée afin d’ajuster sa physiologie.

Comme décrit dans l’introduction bibliographie, le système Agr de L. monocytogenes

est impliqué dans l’adhésion aux surfaces abiotiques (Rieu et al., 2007), dans les premières étapes de formation des biofilms (Rieu et al., 2007, Riedel et al., 2009) et pendant l’infection des hôtes (Autret et al., 2003, Riedel et al., 2009). Cependant, le rôle du système Agr dans l’adaptation des populations de L. monocytogenes dans l’environnement naturel et spécifiquement dans l’environnement tellurique n’est pas décrit. Ce chapitre sera donc un volet mécanistique consacré au régulateur transcriptionnel AgrA et aux gènes qu’il régule au cours de l’adaptation de L. monocytogenes dans le sol.

Ainsi, afin de mieux comprendre le rôle du système Agr et du régulateur AgrA durant l’adaptation de L. monocytogenes au sol, nous avons étudié l’évolution de la souche parentale et du mutant de délétion ∆agrA, inoculés dans des microcosmes de sol, en présence ou non du microbiote indigène du sol. Nous avons par la suite utilisé une approche transcriptomique différentielle pour évaluer l’impact de la délétion du gène agrA sur le transcriptome de

L. monocytogenes dans le sol.

Survival of Listeria monocytogenes in soil requires AgrA-mediated regulation

Anne-Laure Vivant^1,2, Dominique Garmyn^1,2 and Pascal Piveteau^1,2* 1

Université de Bourgogne, UMR1347 Agroécologie,BP 86510, F-21000 Dijon, France 2

INRA, UMR1347 Agroécologie, BP 86510, F-21000 Dijon, France

*Corresponding author. INRA, UMR1347 Agroécologie, 17 rue Sully, 21000 Dijon, France. E-mail: piveteau@u-bourgogne.fr

Running title: AgrA promotes L. monocytogenes soil adaptation

2. Abstract

In this study, we investigated the role of the response regulator AgrA during adaptation of Listeria monocytogenes in soil. The fate of the parental strain L. monocytogenes

L9 (Rifampicin resistant mutant of L. monocytogenes EGD-e) and a ∆agrA deletion mutant were compared in soil microcosms either in the presence (biotic soil) or absence (sterilised soil) of the soil microbiota. The ∆agrA mutant displayed significantly reduced survival in biotic soil microcosms and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional. Indeed, in biotic soil environments, 578 coding genes and an extensive repertoire of ncRNA were differentially transcribed. Transcription of genes coding proteins involved in cell envelope and cellular processes, including PTS system and ABC transporter, and proteins involved in resistance to antimicrobial peptides was affected. These results suggest that the response regulator AgrA of the Agr communication system play important roles during the saprophytic life of L. monocytogenes in soil.

3. Introduction

L. monocytogenes is the causative agent of listeriosis, a serious food-borne infection affecting essentially immuno-compromised individuals, the elderly and pregnant women (Cossart & Toledo-Arana, 2008). The pathogen is largely spread in the environment. It has been isolated from water systems (De Luca et al., 1998, Garrec et al., 2003, Lyautey et al., 2007b), vegetation (Beuchat, 1996), soil (Welshimer, 1960, Weis & Seeliger, 1975), farms (Nightingale et al., 2004, Latorre et al., 2009, Moshtaghi et al., 2009, Latorre et al., 2010), food industries (Goulet et al., 1998, Garrido et al., 2009, van Houdt & Michiels, 2010) and faeces of animals (Fenlon, 1985, Iida et al., 1991, Ho et al., 2007).

Environmental adaptation requires the ability to integrate environmental cues in order to adapt the cell’s physiology to the surrounding conditions through regulation of gene expression. Genomics showed that an important part of L. monocytogenes genome (7.3%) is dedicated to regulation including 209 transcriptional regulators, 15 histidine kinases and 16 response regulators constituting two-component systems (Glaser et al., 2001). Two-component systems participate to the ability of prokaryotes to sense and respond to fluctuating environmental conditions. AgrC/AgrA is a two-component regulatory system part of the Agr communication system. Initially described in Staphylococcus aureus, this communication system is organized as a four genes operon agrBDCA. AgrB is a membrane-bound protein that processes the propeptide AgrD into a mature autoinducing peptide (AIP).

Detection of AIP by the histidine kinase AgrC induces transcriptional regulation through activation of the regulator AgrA. Detailed data concerning the role of the Agr system on the physiology of S. aureus are available (Yarwood et al., 2004, Novick & Geisinger, 2008, Queck et al., 2008). So far, its role in the adaptation of L. monocytogenes to its environment is only partially understood (Garmyn et al., 2009). Reports suggest that the Agr communication system of L. monocytogenes is involved in adhesion to abiotic surfaces (Rieu et al., 2007), in the early stages of biofilm formation (Rieu et al., 2007, Riedel et al., 2009) and during infection of the mammalian host (Autret et al., 2003, Riedel et al., 2009). Indeed, ∆agrA and

∆agrD in-frame deletion mutants showed defects in adherence and early biofilm development. Virulence of a ∆agrA mutant was attenuated in mice but not in vitro in cell lines (Autret et al., 2003) while virulence in mice and invasion of Caco-2 intestinal cells was reduced in an agrD mutant (Riedel et al., 2009). This was correlated with a lower expression of Internalin. However, the role of the Agr communication system in adaptation of

L. monocytogenes populations in the natural environment and especially in soil has not been described.

In order to gain a better understanding of the role of the transcriptional response regulator AgrA and the Agr communication system during adaptation of L. monocytogenes to the telluric environment, we monitored the fate of inoculated L. monocytogenes parental and in-frame ∆agrA deletion mutant in soil microcosms in the presence and absence of indigenous soil microflora. We further investigated the consequences of inactivation of AgrA on the transcriptome of L. monocytogenes during adaptation to the soil environment through a differential transcriptomic analysis approach.