Javier Pizarro-Cerda

Pascale Cossart

Listeria monocytogenes: cell biology of invasion and intracellular growth

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The Cell Biology of Invasion and Intracellular Growth by Listeria monocytogenes

Javier PIZARRO-CERDA and Pascale COSSART Listeria monocytogenes (Lm) is a facultative intracellular pathogen that has the capacity to actively invade and multiply within mammalian cells. Intracellular replication of Lm within mononuclear cells was already noted in the 1926 publication from E.G.D. Murray and colleagues, reporting on this bacterial pathogen for the first time [START_REF] Murray | A disease of rabbits characterized by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n.sp.)[END_REF]. In the 1960s, the seminal work of Mackaness identifying the main actors of cellular immunity against bacterial intracellular pathogens took advantage of the Lm intracellular lifestyle as a model ((2). In the late 1980s and early 1990s, major Lm virulence factors involved in the bacterial adaptation to the intracellular life were molecularly characterized [START_REF] Mengaud | Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin[END_REF][START_REF] Sun | Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread[END_REF][START_REF] Domann | Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene[END_REF][START_REF] Gaillard | Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from grampositive cocci[END_REF][START_REF] Kocks | L. monocytogenesinduced actin assembly requires the actA gene product, a surface protein[END_REF], the precise stages of the Lm intracellular life-cycle were morphologically identified [START_REF] Mounier | Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2[END_REF][START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF] and since, cellular effectors involved in this infection process have been identified [START_REF] Cossart | Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes[END_REF][START_REF] Rolhion | How the study of Listeria monocytogeneshas led to new concepts in biology[END_REF][START_REF] Radoshevich | Listeria monocytogenes: towardsa complete picture of its physiologyand pathogenesis[END_REF]. In this chapter, we will review the molecular mechanisms driving Lm adaptation to the mammalian host cell intracellular environment.

ADAPTATION TO THE INTRACELLULAR LIFE: GENERALITIES

Lm is able to invade and proliferate within macrophages and epithelial non-phagocytic cells. For entry in the latter, bacterial surface molecules (InlA, InlB) interact with cellular ligands, activating signaling cascades that lead to internalization of the pathogen within a membranebound compartment (Fig. 1). Residency in the internalization vacuole is prevented by secretion of a pore-forming toxin (LLO) and two phospholipases (PlcA, PlcB) that disrupt the vacuolar membrane, promoting Lm translocation to the host cell cytoplasm. In this intracellular location, Lm activates several bacterial metabolic pathways that favor the uptake of cellular resources sustaining bacterial proliferation. The pathogen also displays several strategies to escape cytoplasmic innate immune responses, which include the polymerization of actin by a bacterial surface protein (ActA) allowing Lm spread to neighboring cells. In secondary infected cells, Lm is located in a double-membrane compartment that is disrupted by the same set of secreted enzymes that favor lysis of the primary internalization vacuole. Lm translocation to the cytoplasm of secondary infected cells leads to a new bacterial replication cycle and further spread to other cells in infected tissues. The intracellular lifestyle is therefore critical for Lm virulence: it allows escape from extracellular host defense mechanisms including the complement or antibodies, and hinders detection by patrolling cell populations, e.g. neutrophils; in macrophages, cytoplasmic translocation allows escape from degradative components of the phagocytic cascade, while it provides access to a 'Trojan horse' host cell population that can safely transport bacteria to distant locations within the infected organism.

THE CELLULAR INVASION PROCESS

Upon Lm contact with host target tissues, cellular invasion is morphologically characterized by a localized extension of the plasma membrane around invading bacteria, triggering bacterial internalization within a tight vacuolar compartment. Mechanistically, bacterial surface proteins interact with host cell receptors that are post-translationally modified (phosphorylation, ubiquitylation), favoring the recruitment of protein adaptors and enzymes that contribute to actin polymerization, the key molecular event required for plasma membrane reorganization.

Depending on the invaded cell type, bacterial modulation of the phosphoinositide metabolism is also critical to trigger cortical actin polymerization.

InlA is the archetype member of a family of Lm surface proteins named internalins, characterized by the presence of N-terminal leucine rich repeats (LRR) which mediate interaction with host cell ligands [START_REF] Gaillard | Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from grampositive cocci[END_REF][START_REF] Bierne | Internalins: a complex family of leucinerich repeat-containing proteins in Listeria monocytogenes[END_REF]. The internalins InlA and InlB, encoded within a single locus in the Lm genome, are the two major surface molecules driving bacterial entry into host cells. More than 20 other internalins have been identified, but they do not necessarily participate in the cell invasion process, contributing instead to diverse functions including cell-to-cell spread and escape from innate immune responses (i.e. InlC [START_REF] Rajabian | The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-tocell spread of Listeria[END_REF][START_REF] Gouin | The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the I{kappa}B kinase subunit IKK{alpha}[END_REF]) or escape from autophagy (i.e. InlK, [START_REF] Dortet | Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy[END_REF]. The poreforming toxin listeriolysin O (LLO), the actin polymerizing factor ActA and other bacterial surface proteins have been described as supporting bacterial entry independently of the InlA-and InlBinvasion pathways (see below).

InlA/E-cadherin mediated entry

InlA displays a C-terminal LPXTG domain that favors covalent binding to the Lm cell wall [START_REF] Gaillard | Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from grampositive cocci[END_REF].

The LRR domain of InlA interacts with the cellular receptor E-cadherin [START_REF] Mengaud | E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells[END_REF][START_REF] Lecuit | Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization[END_REF], a transmembrane glycoprotein present in the adherens junctions of polarized tissues (e.g. the intestine and the placenta). E-cadherin normally plays a key role in the maintenance of tissue stability, and while the its ectodomain participates in most cases in homotypic interactions (E-cadherin/E-cadherin intercellular binding), the cytoplasmic domain interacts with the actin cytoskeleton machinery. By subverting the E-cadherin physiological function, Lm promotes cortical actin polymerization and plasma membrane rearrangements, favoring cellular invasion and traversal of the intestinal and the feto-maternal barriers [START_REF] Lecuit | A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier[END_REF][START_REF] Lecuit | Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin[END_REF]. InlA access to intestinal E-cadherin mostly occurs at the level of goblet cells, which expose this cellular receptor to bacteria during mucus secretion [START_REF] Nikitas | Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin[END_REF].

Exposure of E-cadherin to Lm during cellular apoptotic cell extrusion at the tip of intestinal villi has also been documented [START_REF] Pentecost | Listeria monocytogenes Invades the Epithelial Junctions at Sites of Cell Extrusion[END_REF]. The interaction between InlA and E-cadherin is species-specific [START_REF] Lecuit | A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes[END_REF]. A proline at position 16 allows interaction between InlA and human E-cadherin, while a glutamic acid at the same position, as observed in the mouse E-cadherin, does not allow InlA binding. A transgenic mouse model specifically expressing the human E-cadherin in the intestine allows a more efficient animal infection through the oral route, demonstrating the pivotal role of InlA in the crossing of the intestinal barrier [START_REF] Lecuit | A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier[END_REF].

In in vitro polarized cellular systems, lipid rafts are critical for InlA-dependent E-cadherin clustering [START_REF] Seveau | Role of lipid rafts in E-cadherin-and HGF-R/Met-mediated entry of Listeria monocytogenes into host cells[END_REF]. InlA binding promotes two successive post-translational modifications in the cytoplasmic tail of E-cadherin: phosphorylation by the host kinase Src, followed by ubiquitylation by the ubiquitin ligase Hakai [START_REF] Bonazzi | Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes[END_REF]. These events are critical for the recruitment of a clathrin coat via the adaptor Dab2; the coat is stabilized by tyrosine phosphorylation of the clathrin heavy chain, followed by sequential recruitment of the protein adaptor Hip1R, which in turn coordinates recruitment of actin; myosin VI and unconventional myosin VIIa provide the pulling force that finally leads to bacterial internalization [START_REF] Bonazzi | Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization[END_REF][START_REF] Sousa | Unconventional myosin VIIa and vezatin, two proteins crucial for Listeria entry into epithelial cells[END_REF]. Interestingly, the non-muscle myosin heavy chain IIA is specifically phosphorylated by Src upon Lm infection and restricts bacterial entry [START_REF] Almeida | Src-dependent Tyrosine Phosphorylation of Non-muscle Myosin Heavy Chain-IIA Restricts Listeria monocytogenes Cellular Infection[END_REF].

Several other molecules modulate actin association to the E-cadherin cytoplasmic site during Lm

InlA-dependent invasion: β-and ⍺-catenins provide a physical link between E-cadherin and actin filaments during bacterial entry [START_REF] Lecuit | A role for alphaand beta-catenins in bacterial uptake[END_REF][START_REF] Sousa | ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion[END_REF] while cortactin and Src participate in the activation of the Arp2/3 complex, a major actin nucleator [START_REF] Sousa | Src, cortactin and Arp2/3 complex are required for E-cadherin-mediated internalization of Listeria into cells[END_REF], highlighting the exploitation of adherens junctions and classical E-cadherin endocytosis components by Lm during invasion of polarized tissues [START_REF] Bonazzi | A Common Clathrin-Mediated Machinery Co-ordinates Cell-Cell Adhesion and Bacterial Internalization[END_REF][START_REF] Pizarro-Cerdá | Clathrin-mediated endocytosis: What works for small, also works for big[END_REF]. In the intestinal barrier, the constitutive phosphoinositide (PI) 3-kinase activity is required for promoting actin polymerization during Lm cell entry; in the placenta, PI 3-kinase activity is not constitutive and InlB is required for PI 3-kinase activation and InlA-mediated cell invasion [START_REF] Gessain | PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes[END_REF][START_REF] Disson | Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis[END_REF].

InlB/Met mediated entry

InlB was identified as a second Lm invasion protein [START_REF] Gaillard | Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from grampositive cocci[END_REF][START_REF] Dramsi | Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family[END_REF]. InlB allows Lm entry into nonpolarized epithelial cells in vitro [START_REF] Lingnau | Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -independent mechanisms[END_REF], and it cooperates with InlA during placental invasion in vivo [START_REF] Gessain | PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes[END_REF][START_REF] Disson | Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis[END_REF]. In non-pregnant animals, InlB expression is associated to an increase of necrotic foci in livers and spleens [START_REF] Quereda | Accepted Manuscript[END_REF]. The C-terminal region of InlB is characterized by the presence of GW repeats that favor loose binding to bacterial membrane-tethered lipoteichoic acids [START_REF] Braun | InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association[END_REF][START_REF] Jonquières | Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of gram-positive bacteria[END_REF] and pedptidoglycan-bound wall teichoic acids [START_REF] Carvalho | l-Rhamnosylation of wall teichoic acids promotes efficient surface association of Listeria monocytogenesvirulence factors InlB and Ami through interaction with GW domains[END_REF]. At the surface of host cells, the GW repeats mediate binding to the receptor of the globular part of the complement component C1q [START_REF] Braun | gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes[END_REF] and to glycosaminoglycans [START_REF] Jonquières | Synergy between the N-and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes[END_REF]. The N-terminal region displays LRRs that are critical for cell invasion [START_REF] Braun | The InIB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells[END_REF][START_REF] Braun | The 213-amino-acid leucine-rich repeat region of the listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling[END_REF] and bind the hepatocyte growth factor receptor Met [START_REF] Shen | InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase[END_REF] in a species-specific manner [START_REF] Khelef | Species specificity of the Listeria monocytogenes InlB protein[END_REF]. Met expression is modulated by epithelial keratins, which promote InlB-mediated Lm infection of epithelial cells [START_REF] Cruz | Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells[END_REF]. Met is a tyrosine kinase receptor, and its interaction with InlB leads to Met auto-phosphorylation and recruitment of the protein adaptors Gab1, Shc, Cbl and CrkII [START_REF] Ireton | The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase[END_REF][START_REF] Sun | Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes[END_REF][START_REF] Basar | Redundant roles for Met docking site tyrosines and the Gab1 pleckstrin homology domain in InlB-mediated entry of Listeria monocytogenes[END_REF], which play a key role in the activation of PI 3-kinase [START_REF] Ireton | A role for phosphoinositide 3-kinase in bacterial invasion[END_REF][START_REF] Dokainish | The carboxyl-terminal SH3 domain of the mammalian adaptor CrkII promotes internalization of Listeria monocytogenes through activation of host phosphoinositide 3-kinase[END_REF][START_REF] Jiwani | Identification of Components of the Host Type IA Phosphoinositide 3-Kinase Pathway That Promote Internalization of Listeria monocytogenes[END_REF]. Production of PI [START_REF] Mengaud | Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin[END_REF][START_REF] Sun | Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread[END_REF][START_REF] Domann | Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene[END_REF]P3 and its accumulation in lipid rafts leads to Rac1 activation [START_REF] Seveau | Role of lipid rafts in E-cadherin-and HGF-R/Met-mediated entry of Listeria monocytogenes into host cells[END_REF][START_REF] Seveau | A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway[END_REF] and recruitment of Ena/VASP, WAVE and N-WASP, which activate the Arp2/3 complex promoting actin polymerization in a tightly regulated manner [START_REF] Bierne | A role for cofilin and LIM kinase in Listeria-induced phagocytosis[END_REF][START_REF] Bierne | WASPrelated proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor[END_REF]. The serine/threonine kinases mTOR and proteine kinase C-⍺ also control actin polymerization downstream of the InlB/Met interaction [START_REF] Bhalla | Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes[END_REF]. The host 5'phosphatase OCRL restricts Lm entry by reducing PI(3,4,5)P3 levels and actin polymerization at bacterial entry foci [START_REF] Kühbacher | Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection[END_REF]. Production of PI4P by type II PI 4-kinases, downstream of the tetraspanin CD81, is also critical for Lm entry into host cells [START_REF] Pizarro-Cerdá | Type II phosphatidylinositol 4-kinases promote Listeria monocytogenes entry into target cells[END_REF][START_REF] Tham | Tetraspanin CD81 is required for Listeria monocytogenes invasion[END_REF]. The PI 3-kinase adaptor Cbl also displays ubiquitin ligase activity and promotes ubiquitylation of Met upon InlB binding, leading to modulation of actin polymerization via clahtrin recruitment [START_REF] Veiga | Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells[END_REF][START_REF] Veiga | Invasive and adherent bacterial pathogens co-Opt host clathrin for infection[END_REF][START_REF] Pizarro-Cerdá | Listeria monocytogenes membrane trafficking and lifestyle: the exception or the rule?[END_REF]. InlB also modulates exocytosis and favors the delivery of the endocytic GTPase dynamin 2 to bacterial entry sites [START_REF] Van Ngo | A role for host cell exocytosis in InlBmediated internalisation of Listeria monocytogenes[END_REF]. Finally, the septin cytoskeleton is recruited during cell invasion by Lm in an InlB-dependent manner [START_REF] Pizarro-Cerdá | Distinct protein patterns associated with Listeria monocytogenes InlA-or InlB-phagosomes[END_REF] and it controls the anchorage of Met to the cortical actin cytoskeleton [START_REF] Mostowy | Septins Regulate Bacterial Entry into Host Cells[END_REF][START_REF] Mostowy | Septin 11 restricts InlB-mediated invasion by Listeria[END_REF][START_REF] Mostowy | A Role for Septins in the Interaction between the Listeria monocytogenes Invasion Protein InlB and the Met Receptor[END_REF].

Additional adhesion/entry effectors

Lm displays other surface and secreted molecules that can modulate adhesion and entry into host cells by indirectly affecting the surface exposure of InlA or InlB, by behaving as adhesins, by directly binding putative cellular receptors, and/or by activating cellular pathways that lead to actin rearrangements and bacterial engulfment. For example, the internalins InlE, InlG and InlH support the InlA-dependent-invasion pathway in Caco-2 cells and might modulate the bacterial cell wall organization, consequently affecting InlA exposure [START_REF] Bergmann | InlA-but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins[END_REF]. On the other hand, InlJ favors bacterial adhesion but no invasion in a specific subset of polarized epithelial cells [START_REF] Sabet | LPXTG protein InlJ, a newly identified internalin involved in Listeria monocytogenes virulence[END_REF][START_REF] Sabet | The Listeria monocytogenes virulence factor InlJ is specifically expressed in vivo and behaves as an adhesin[END_REF]. A role for InlF in cell adhesion and invasion has been detected only upon inhibition of the RhoA/Rho kinase pathway [START_REF] Dramsi | Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD[END_REF][START_REF] Kirchner | Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes[END_REF].

Several Lm autolysins modulate cell adhesion and/or entry processes: Ami is involved in cell adhesion [START_REF] Milohanic | Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium[END_REF][START_REF] Milohanic | The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor[END_REF], Auto has been implicated in entry [START_REF] Cabanes | Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence[END_REF] while IspC is required for adhesion ando/or invasion in a cell line-dependent manner [START_REF] Wang | A novel cell wall-anchored peptidoglycan hydrolase (autolysin), IspC, essential for Listeria monocytogenes virulence: genetic and proteomic analysis[END_REF]. The lipoteichoic acid modifiers GtcA and DltA [START_REF] Promadej | Cell wall teichoic acid glycosylation in Listeria monocytogenes serotype 4b requires gtcA, a novel, serogroupspecific gene[END_REF][START_REF] Abachin | Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes[END_REF], the lipoprotein LpeA (81), the prolipoprotein transferase Lgt (82) and the lysylphosphatidylglycerol modifier MprF (83) play roles in host cell adhesion or invasion probably by modulating the bacterial surface charge and/or by altering the organization of the bacterial surface proteins. The surface protein ActA, involved in cytoplasmic actin-based motility (see below), has been proposed to favor host cell invasion through interaction with heparan sulfate [START_REF] Suárez | A role for ActA in epithelial cell invasion by Listeria monocytogenes[END_REF][START_REF] Alvarez-Dominguez | Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition[END_REF]. Additional Lm surface adhesins or invasins include Vip [START_REF] Cabanes | Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein[END_REF][START_REF] Martins | Listeria monocytogenesTriggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection[END_REF], Lap [START_REF] Jagadeesan | N-terminal Gly(224)-Gly(411) domain in Listeria adhesion protein interacts with host receptor Hsp60[END_REF], LapB [START_REF] Reis | LapB, a novel Listeria monocytogenes LPXTG surface adhesin, required for entry into eukaryotic cells and virulence[END_REF] and FbpA [START_REF] Dramsi | FbpA, a novel multifunctional Listeria monocytogenes virulence factor[END_REF].

LLO, a secreted cholesterol-dependent pore-forming toxin that is required for Lm vacuolar escape (see below), is also secreted by extracellular bacteria and induces a transient influx of extracellular calcium within host cells that correlates with increased cell invasion [START_REF] Dramsi | Listeriolysin O-mediated calcium influx potentiates entry of Listeria monocytogenes into the human Hep-2 epithelial cell line[END_REF].

Mitochondrial fragmentation also correlates with the LLO-dependent calcium influx and it has been proposed that Lm modulates the bioenergetic state of resting cells to trigger cell invasion [START_REF] Stavru | Listeria infection modulates mitochondrial dynamics[END_REF]. LLO has been recently proposed to mediate Lm entry into epithelial cells in a Ca2 + /K + -, cholesterol-, dynamin-, tyrosine kinase-and actin-dependent but InlA/InlB-and clathrinindependent manner [START_REF] Vadia | The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes[END_REF][START_REF] Vadia | Fluxes of Ca2+ and K+ Are Required for the Listeriolysin O-Dependent Internalization Pathway of Listeria monocytogenes[END_REF]. A broad-range phospholipase C of Lm, PlcB (see below) has been reported to induce a calcium influx required for efficient bacterial internalization in macrophages [START_REF] Wadsworth | Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells[END_REF].

THE VACUOLAR STAGE

The modulation of the actin cytoskeleton and the rearrangements of the plasma membrane upon Lm interaction with its host cell receptors lead to bacterial engulfment and internalization in a membrane-bound vacuole. In the intestinal epithelium, and particularly in the goblet cells, Lm does not escape from this compartment and is directly transcytosed to the lamina propria, where the bacteria disseminate systemically [START_REF] Nikitas | Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin[END_REF]. In other cell types, Lm is able to disrupt its containing compartment and translocates to the host cell cytoplasm. The cholesteroldependent pore-forming toxin LLO, together with two bacterial phospholipases, are the major bacterial effectors controlling Lm vacuolar escape. Lm residency and persistence in vacuolar compartments has also been described (96, 97) (see below).

LLO and vacuolar disruption

LLO is able to induce hemolysis in vitro and this activity was early on correlated with Lm virulence [START_REF] Seastone | Pathogenic organisms of the genus Listerella[END_REF]. This toxin is encoded by the hly gene, located within a pathogenicity island that also encodes other important virulence factors including two phospholipases, a metalloprotease, the actin polymerizing factor ActA and the transcriptional activator PrfA [START_REF] Mengaud | Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated[END_REF]. Inactivation of the hly gene coupled with electron microscopy observations subsequently demonstrated that LLO is required for bacterial escape from internalization vacuoles (100). LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), which includes also perfringolysin O (PFO) from

Additional mechanisms regulating vacuolar disruption

Several other bacterial-and host-related mechanisms have been proposed to modulate the Lm vacuolar stage. A recent study indicates that Lm secretes a pheromone, pPplA, that triggers the production of an unknown factor that cooperates with LLO in facilitating vacuolar disruption (139): pPplA is processed from the N-terminal secretion signal sequence of the lipoprotein PplA; pPplA is secreted, accumulates in the space of the Lm vacuole, and is then exported by the CtaP peptide transporter; cytoplasmic pPplA induces the production of a factor that accelerates vacuolar disruption mediated by LLO in a still non-identified manner(139).

Modulation of bacterial gene expression by «reversible lysogeny» has also been proposed to modulate Lm vacuolar escape (140). The prophage A118 is inserted within the coding region of the gene comK, a master regulator of competence genes that are normally not expressed by Lm; interestingly, during the bacterial vacuolar stage, A118 is excised and this event allows reactivation of comK and expression of the competence machinery by Lm; by a still unknown mechanism, the competence system promotes efficient bacterial translocation to the host cell cytoplasm. In this environment, the phage reinserts into comK (140).

Additional host factors have been reported to control Lm vacuolar residency. Rab5a was shown to control the accelerated maturation of Lm-containing vacuoles (141, 142); the product of the gene Lmo2459 was subsequently shown to induce the specific ADP ribosylation of Rab5a, inhibiting its activation and reverting its bactericidal functions (143). The activity of the cytosolic cysteine protease calpain has been shown to be required for efficient Lm vacuolar escape, but the targets of this protease remain to be identified (144).

THE CYTOPLASMIC STAGE

By translocating from the vacuolar stage towards the cytoplasm, Lm escapes cellular degradative mechanisms associated to phagosomal pathways. On the other hand, Lm must adapt its metabolism to nutrients and metabolites found in this novel intracellular compartment, and must also escape from additional innate immunity defenses including autophagy. The hexose phosphate transporter Hpt and the actin-polymerizing surface protein ActA play key roles in the survival of Lm in the host cell cytoplasm.

Utilization of host metabolites

Glucose-1-phosphate (G1P) is the primary degradation product of glycogen, and is broadly available within mammalian cells. The observation that Lm uses G1P as a carbon source in a PrfAdependent manner suggested that related hexose phosphates could be important growth substrates for intracellular bacteria (145). In silico analysis of the Lm genome identified the gene hpt as encoding an hexose phosphate transporter responsible for the uptake of glucose-6phosphate in the cytoplasm of host cells, playing a key role in the Lm in vivo virulence (146). A subsequent screen for identification of additional genes required for bacterial intracellular replication recognized lplA1 as a lipoate protein ligase that could potentially use host-derived lipoic acid to modify bacterial substrates (147). LplA1 was afterwards confirmed to be essential for intracellular growth of Lm under limiting concentrations of available small mammalian lipoylated peptides (148). A more recent genetic screen led to the discovery that the menaquinone synthesis intermediate 1,4-dihydroxy-2-naphtoate is required for Lm cytosolic survival, but not full-length menaquinone (149).

Cytoplasmic innate immune responses

Autophagy is a cellular mechanism responsible for protein turnover and removal of sequestering by ActA may also act as a protective physical barrier preventing the accumulation of signaling molecules (i.e. ubiquitin) that are required for autophagy activation (152, 153). PlcA and PlcB have been also implicated in autophagosomal avoidance (151,154,155) and recent studies suggest that these PLCs decrease cytoplasmic levels of PI3P, causing stalling of preautophagosomal structures and preventing efficient targeting of cytosolic bacteria (156). The surface internalin InlK has also been proposed to recruit the major vault protein and to protect cytoplasmic Lm from autophagic recognition ( 16) but these results have been recently challenged using a different Lm strain (157).

Cytoplasmic Lm secret small molecules leading to activation of an IRF3-dependent cytosolic pathway, resulting in type I interferon activation (158). One of these small molecules, cyclic-di-AMP, is sufficient to activate production of interferon β in macrophages (159). Sensing of triphosphorylated RNA via RIG-I and a MAVS-dependent pathways triggers type I interferon production in epithelial cells (160, 161). Lm cytoplasmic DNA is recognized through STING, TBK1, IRF3 and IRF7, leading to the up-regulation of the di-ubiquitin-like protein ISG15 and ISGylation of ER and Golgi proteins, which correlate with increased secretion of cytokines that counteract infection (162). Lm also activates the type III interferon pathway (163).

Persistence

It is increasingly recognized that bacterial pathogens may persist within host tissues in a dormant state that allows resistance to antibiotics and subsequent reinfection. In macrophages of severe combined immunodeficient (SCID) mice, Lm can persist in large compartments termed spacious Listeria-containing phagosomes (SLAPS) which are formed in an LLO-dependent manner [START_REF] Birmingham | Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles[END_REF]. LC3-associated phagocytosis (LAP) has been proposed to precede the formation of SLAPS (164). A recent study indicates that in epithelial cells, cytoplasmic Lm in which ActA production is halted are trapped in acidic vacuoles that are not associated with classical autophagosomal markers and in which bacteria enter a viable but non-culturable state [START_REF] Kortebi | Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells[END_REF]. These studies indicate that Lm may persist in different host cellular populations, favoring the asymtomatic carriage of this pathogen.

CELL-TO-CELL SPREAD

Actin-based motility allows Lm not only to escape autophagy but also to reach neighboring cells within an infected tissue, favoring the cell-to-cell spread and bacteria dissemination in organs avoiding exposure to humoral immunity. Motile Lm first induce the formation of a membrane protrusion in the primary infected cell that is accompanied by membrane internalization in the neighboring by-stander cell, leading to bacterial entrapment in a double-membrane vacuole that is then disrupted [START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF]. Several bacterial virulence factors including ActA, the internalin InlC, the poreforming toxin LLO and the phospholipases PlcA and PlcB participate at different stages of the Lm cell-to-cell spread.

Cytoplasmic actin-based motility

The surface protein ActA is sufficient to trigger actin polymerization at the surface of Lm [START_REF] Kocks | L. monocytogenesinduced actin assembly requires the actA gene product, a surface protein[END_REF]. The central region of ActA contains four short proline-rich repeats that bind members of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family; these molecules contribute to the persistence of speed/directionality of bacterial movement by recruiting profilin, which provides polymerization-competent actin monomers (165). The N-terminal region of ActA recruits the Arp2/3 complex which drives actin nucleation (166, 167). The Arp2/3 complex is formed of seven subunits and it has been traditionally considered as a single molecular entity (168). A recent genome-wide small interfering RNA screen demonstrated that different Arp2/3 complexes are required to control Lm cell invasion and actin-based motility: the Arp2, Arp3, ARPC2 and ARPC3 subunits are conserved, but the ARPC1B subunit is only involved in cell invasion while the ARPC1A subunit is required for actin-based motility, and the ARPC4 subunit is dispensable for cell invasion while the ARPC5 subunit is dispensable both for cell invasion and actin-based motility (169).

Multiple actin cross-linking proteins, actin filament capping or severing proteins and protein scaffolds are recruited to the Lm actin tail (170). Recent cryo-electron tomography of actin tails has demonstrated that actin bundling is critical for ensuing actin-based motiliy (171).

Cortical actin rearrangements and protrusion formation

In mammalian tissues, cortical membrane tension represents a physical barrier for motile Lm, inhibiting the deformation of the plasma membrane into protrusions. InlC, a secreted member of the internalin family devoid of a cell wall anchoring motif [START_REF] Bierne | Internalins: a complex family of leucinerich repeat-containing proteins in Listeria monocytogenes[END_REF], perturbs apical cell junctions by interacting with the protein adaptor Tuba, inhibiting the recruitment of the actin regulatory protein N-WASP as well as COPII proteins, relieving therefore cortical membrane tension and favoring Lm protrusion formation [START_REF] Rajabian | The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-tocell spread of Listeria[END_REF]172,173). The down-regulation by Lm of the small GTPase Cdc42, another Tuba interactor, is also required for efficient protrusion formation (174). Within the protrusion, the membrane-cystokeletal linker ezrin has been proposed to support the formation and stabilization of protrusions (175). Arp2/3 drives actin polymerization at the proximal Lm rear-end within protrusions, but at distal locations the recruitment of Rho GTPases activate diaphanous-related formins which promote the formation of unbranched actin filaments (176). Inhibition of actin polymerization by components of the AIP1-dependent actin disassembly machinery (177) as well as ActA processing by the metalloprotease Mpl (178) are proposed mechanisms for the resolution of membrane protrusions into double membrane vacuoles.

Efficient cell-to-cell spread can be facilitated by the exofacial exposure of phosphatidylserine at the tip of protrusions promoted by the pore-forming activity of LLO, which leads to phosphatidylserine binding by the TIM-4 receptor in macrophages and protrusion internalization (179).

Lysis of Secondary Vacuoles

Internalization of Lm-induced protrusions into neighboring by-stander cells leads to bacterial localization within a double-membrane compartment containing [START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF]. Initial studies suggested that phospholipases PlcA and PlcB, together with LLO, contributed to cell-to-cell spread [START_REF] Sun | Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread[END_REF]125,127), and a more specific contribution of PlcB to double-membrane vacuolar rupture was suggested (128). A current model proposed that PlcA and PlcB contribute to the disruption of the inner membrane of the spreading vacuole, while LLO participates more precisely in the disruption of the outer membrane of this vacuole (180). Bacterial translocation to the cytoplasmic space of neighboring cells allow Lm to start a new infection process.

MODULATION OF CELLULAR, ORGANELLAR AND NUCLEAR FUNCTIONS

Lm is able to modulate a broad range of cellular functions, even before being internalized within host cells. The pore-forming toxin LLO, which plays a major role in vacuolar escape (see above), is able to modulate from the extracellular space the function of mitochondria, the endoplasmic reticulum, lysosomes, protein post-translational modifications and DNA stability.

Several bacterial nucleomodulins have been identified which directly affect the transcription of host genes involved in the control of immune responses.

LLO influence on mitochondria

Mitochondria are critical organelles involved in the generation of chemical energy in eukaryotic cells. As mentioned above, extracellular LLO triggers the influx of calcium, which leads to transient fission of mitochondria, triggering a bioenergetic change of host cells that is beneficial for Lm host cell invasion [START_REF] Stavru | Listeria infection modulates mitochondrial dynamics[END_REF]. Interestingly, atypical mitochondrial fission through a Drp1independent fragmentation process has been associated to Lm cellular infection (181).

LLO influence on the endoplasmic reticulum

The unfolded protein response (UPR) is a signaling cascade that maintains the function of the endoplasmic reticulum (ER) under stress conditions. Lm activates the UPR in an LLOdependent manner prior to bacterial entry into host cells (182). The induction of the ER stress by drugs such as thapsigargin or tunicamycin leads to a decrease in bacterial intracellular numbers, suggesting that UPR represents an innate immune response to bacterial infection (182).

LLO influence on lysosomes

The integrity of lysosomes has been shown to be compromised by extracellular LLO, which induces permeabilization and release of lysosome content, including cathepsins, which remain transiently active in the cytoplasm (183). The functional significance of this finding for Lm infection and survival remains to be identified.

LLO influence on protein post-translational modifications

Post-translational modifications (PTM) allow the rapid modification of the activity of cellular proteins. Sumoylation is an essential PTM that is impaired by Lm through the proteasomeindependent degradation of the E2 enzyme Ubc9, following calcium influx mediated by extracellular LLO (184). The down-regulation of cellular protein sumoylation, together with the proteasome-dependent degradation of some sumoylated proteins, favors bacterial infection in vitro and in vivo (184). Histone modifications are also associated to the Lm infection process (185) (see below).

LLO influence on DNA stability

Lm modulates general DNA stability in host cells in different manners. Lm induce DNA strand breaks and simultaneously blocks the DNA damage response (DDR) through degradation of the sensor Mre11 in an extracellular LLO-dependent manner (186), promoting a cell cycle delay that favors bacterial intracellular replication (187). Interestingly, it has been also reported that LLO-induced calcium influx leads to the proteasomal degradation of the human telomerase reverse transcriptase (hTERT), an event that is detrimental to bacterial replication (188).

Bacterial influence on gene expression

LntA is the first nucleomodulin discovered in Lm (189): it targets the chromatin repressor BAHD1 and fine-tunes transcription of interferon (IFN)-stimulated genes (ISGs), required for efficient in vivo infection (190,191). More recently, the nucleomodulin OrfX has been shown to interact and decrease the levels of the regulatory protein RybP, dampening the oxidative response in macrophages probably through modulation of host transcriptional responses (192). The secreted internalin InlC interferes with innate immune responses by targeting the IκB kinase subunit IKK⍺, inhibiting NF-κB translocation to the nucleus [START_REF] Gouin | The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the I{kappa}B kinase subunit IKK{alpha}[END_REF]. LLO modulates gene transcription with opposite effects for infection: LLO induces a dramatic dephosphorylation of histone H3 and deacetylation of histone H4 that leads to reduced transcriptional activity of key immunity host genes (185); LLO has been also shown to modulate the functionality of the promyelocytic leukemia protein (PML) nuclear bodies, activating a signaling response that decreases Lm infection (193). Finally, an InlB/PI 3-kinase pathway is required for the SIRT2dependent deacetylation of histone H3 on lysine 18, involved in efficient bacterial infection in vitro and in vivo (194).

CONCLUSIONS

The study of the interactions of Lm with eukaryotic host cells during bacterial invasion, intracellular growth and cell-to-cell spread has proven to be fundamental to better understand the exquisite adaptation of this bacterial pathogen to mammalian hosts, as Lm is able to hijack multiple cellular functions including receptor signaling, membrane trafficking, cytoskeletal rearrangements, organellar dynamics, DNA stability and gene transcription. The work reviewed in this chapter also highlights that Lm is an extraordinary tool to manipulate and to unravel host cell signaling cascades, in particular innate immune responses that allow to expand our understanding on the control of bacterial intracellular infections.

  Clostridium perfringens and streptolysin O (SLO) from Streptococcus pyogenes (101). PFO studies indicate that a conserved undecapeptide provides a structural conformation for a threonineleucine pair at the C-terminal region of CDCs responsible for cholesterol binding (102, 103).Theoretically predicted to form large pores(20-80 monomers) based on initial PFO studies (104), electron microscopy (EM) and atomic force microscopy (AFM) analyses indicates that LLO oligomers actually form arc-like structures that assemble into lineactants, and these heterogenous structures are responsible for membrane disruption and vacuolar rupture (105, 106). Membrane perforation by LLO not only facilitates Lm translocation to the cytoplasm, it also controls the vacuolar pH and calcium concentration, delaying the maturation of the bacterial-containing compartment and inhibiting lysosomal fusion (107, 108).Several physical parameters and host molecules modulate the activity of LLO. Removal of LLO pores from the host cell plasma membrane is mediated by a LLO Pest-like sequence recognized by the clathrin adaptor Ap2a2, favoring pore endocytosis and protection of the plasma membrane integrity (109). At 37°C, at neutral pH LLO undergoes denaturation, but it is in a stable conformation at acidic conditions (105). Consequently, within mammalian hosts the LLO poreforming activity is compartmentalized to slightly acidified bacterial-containing compartments (110, 111) and cytoplasmic LLO activity, which is cytotoxic to host cells and detrimental to intracellular Lm (112) is limited by translational regulation of LLO synthesis (113) and by cytoplasmic LLO degradation by the ubiquitin system (114). LLO is activated by reducing agents (115) and within the vacuole of macrophages, the ɣ-interferon-inducible lysosomal thiol reductase (GILT) is responsible for reducing and activating LLO (116). The increase in vacuolar chloride concentration mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to enhance LLO oligomerization and Lm cytoplasmic escape (117). LLO-disrupted vacuoles trigger the recruitment of the protein kinase C (PKC) ε, suggesting that this enzyme is involved in the recognition of damaged membrane organelles (118). Phospholipases PlcA / PlcB and vacuolar disruption Lm secretes two phospholipases, a phosphatidylinositol-specific phospholipase C named PlcA (119), and a broad-range phospholipase C/sphingomyelinase named PlcB (120, 121) activated by the metalloprotease Mpl (122-124). Both enzymes have been shown to contribute to Lm escape from primary vacuoles and from secondary vacuoles during bacterial cell-to-cell spread (4, 125-128). PlcA from Lm is the only bacterial phosphatidylinositol-specific phospholipase C that lacks a Vb β-strand that increases activity towards GPI-anchored proteins (129); interestingly, expression of this β-strand in PlcA impairs bacterial escape from vacuoles and cell-to-cell spread (130), suggesting that a Lm adaptation to the intracellular environment requires reduced activity against GPI-anchored proteins. It has been proposed that PlcA via LLO pores reaches the host cell cytoplasm, and in this compartment PlcA cleaves intracellular phosphatidylinositol into inositol phosphate and diacylglycerol (DAG) (131); production of DAG, which might also take place through activation of host phospholipases C and D in an LLO-dependent signaling pathway (132), leads to activation of PKC βI and βII, which are required for vacuolar disruption (133). As both PKC βI and βII are recruited to the Lm internalization vacuole (134), it is speculated that the phosphorylation of PKC βI and βII targets at the surface of the bacterial-containing compartment is critical for a still non-identified signaling cascade leading to vacuolar rupture (133). PlcB maturation and activation by the metalloprotease Mpl requires acidification of the vacuole (135, 136) and as it has been observed for LLO, compartmentalization of this phospholipase C activity is critical for intracellular survival of Lm (137). Both PlcA and PlcB have been found to activate the NOX2 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase during Lm infection, which might be harmful to internalized bacteria via the production of reactive oxygen species; however, modulation of vacuolar maturation by LLO restricts NADPH oxidase localization to the Lm-containing compartments (138).

  abnormal or superfluous subcellular components. The pioneering work of Rich et al. (150) demonstrated that cytoplasmic and metabolically arrested Lm can be targeted by the autophagic machinery for destruction. Different mechanisms have been proposed to participate in the active escape of cytoplasmic Lm from autophagy: polymerization of actin by the surface protein ActA favors cytoplasmic motility and avoidance of autophagosomes (151); polymerized actin or Arp2/3

FIGURE 1 .

 1 FIGURE 1. Cellular receptor for Lm in host cells. The receptor for InlA in non-phagocytic polarized

FIGURE 2 .

 2 FIGURE 2. Lm intracellular stages. Lm is able to induce its entry into non-phagocytic cells mainly

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Acknowledgements

We would like to thank members of the Cossart lab for helpful discussions. We apologize upon those people who have contributed to advancements in the field of listeriosis but whose work was not cited in our chapter due to size limitations. J.P.C. received support from the Institut Pasteur 'Transversal Research Program' PTR521 and from the National Research Agency (ANR) grant ANR-15-CE15-0017 'StopBugEntry'. Work in P.C. laboratory received financial support from Institut Pasteur, INSERM, INRA, ANR-13-IFEC-0004-02 'ERANET Infect-ERA PROANTILIS', the French Government's 'Investissement d'Avenir' program, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (ANR-10-LABX-62-IBEID), the European Research Council (ERC) (Advanced Grant H2020-ERC-2014-ADG 670823-BacCellEpi), Human Frontier Science Program Organization (RGP011/2013), the Fondation le Roch les Mousquetaires and the Fondation Balzan. P.C. is a Senior International Research Scholar of the Howard Hughes Medical Institute.


domain: sdv shs

text:
S Bellon

De Abreu

The relationships between organic farming and agroecology

Keywords:

While acknowledging an extension of agroecology in the organic sector and a growing influence of agroecology in the academic world, we explore their relationships. These relationships cannot be reduced to an opposition between a scientific field and a practical domain. A Brazilian case study based on the analysis of researchers and social actors trajectories exemplifies the diversity of existing relations, whether inclusive or exclusive. With a literature review, this allows characterising the specific attributes of both organic agriculture and agroecology. We discuss them in the light of current challenges for organic farming research and development.

Introduction

Both organic farming (OF) and agroecology (AE) claim they can contribute to many challenges faced by agriculture today. Among these are the interrelated challenges of providing food security and preserving the environment. Apart from common objectives, both OF and AE also refer to ecology and question the prevalent technological model designed during the XX th century. However, albeit AE can be considered as scientifically rooted and equipped for a holistic study of agroecosystems, at least three main interpretations of AE are possible: as a scientific field, as a social movement or as an agricultural practice [START_REF] Wezel | Agroecology as a science, a movement and a practice. A review[END_REF]. Likewise, diversity also exists within organic farming, which cannot be summarised into a set of certified practices [START_REF] Sylvander | Facing the organic reality : the diversity of development models and their consequences on research policies[END_REF].

Since there is a continuous confusion about both terms [START_REF] Francis | Education in Organic Farming and Food Systems[END_REF], we intend to contribute to clarify the relationships between OF and AE, while opening a debate and suggesting guidelines for research agendas. Our analysis starts with the extension of AE in various arenas, based on literature reviews. The following section addresses the relationships between OF and AE, based on interviews with stakeholders and case studies. The third section intends to generalise the previous analyses. The discussion opens avenues for further research work.

Materials and methods

Our approach combined 3 methods: (i) scientometric analysis based on Web of Science (WoS); (ii) case studies and comparisons among regions and countries [START_REF] Wezel | Agroecology as a science, a movement and a practice. A review[END_REF]; (iii) direct interviews with Brazilian actors involved in research and training in OF and AE [START_REF] Abreu | Trajetórias da Agroecologia no Brasil: entre Movimentos Sociais, Redes Científicas e Políticas Públicas. VI Congresso Brasileiro de Agroecologia. II Congresso Latino-americano de Agroecologia[END_REF]. The scientometric analysis, developed in [START_REF] Ollivier | Thematic and citation structure dynamics of Organic Food & Farming research[END_REF], was done to analyse the dynamics of AE and OF domains as well as the publications co-using both terms.

Comment citer ce document : Bellon, S., Lamine, C., Ollivier, G., de Abreu, L. (2011). The relationships between organic farming and agroecology. In: Organic is life. Knowledge for tomorrow. Proceedings (p. 235-238). Presented at 3. ISOFAR Scientific Conference at the 17. IFOAM Organic World Congress, Gyeonggi Paldang, KOR (2011-09-28 -2011-10-01). Bonn, DEU : ISOFAR -International Society of

Results

Extension and influence of AE in agricultural sciences

Fig 1 . shows a growing number of publications related to AE since 2004, after a rather stable period. In spite of this recent extension, the corpus is still limited in AE (around 370 references), as compared with OF [START_REF] Ollivier | Thematic and citation structure dynamics of Organic Food & Farming research[END_REF]. WoS notices mentioning both OF and AE represent 15% of the AE litterature and 1% of the OF one. Document co-citation analysis point out the variety of addressed topics. They can be aggregated in 4 main categories: (i) definitions of AE, (ii) enhancing biodiversity, (iii) transition studies, (iv) agroecosystems studies, Many reference books are cited documents in AE, reflecting a process of knowledge building.

Main authors in AE are also influential in the field of alternative agricultures, including OF. For instance, Altieri, one of the leading authors in AE, is the 14 th most cited author in OF after 2003 [START_REF] Ollivier | Thematic and citation structure dynamics of Organic Food & Farming research[END_REF]. Other leading authors in AE consider it as an «umbrella» for alternative agricultures, or view OF as an «area of practice» for AE. [START_REF] Guthman | Raising organic: An agro-ecological assessment of grower practices in California[END_REF] assessed OF practices in the light of AE principles, arguing that in many cases organic practices fall quite short of agro-ecological ideals. Likewise, [START_REF] Altieri | Agroecology rescuing organic agriculture from a specialized industrial model of production and distribution[END_REF] suggested that AE would rescue OF from an industrialisation model. Such positions reflect the conventionalisation debate in OF, and show the importance of organic values, rules and regulations in shaping organic production [START_REF] Luttikholt | Principles of organic agriculture as formulated by the IFOAM[END_REF]. However OF is not only questioned but also utilised by AE, e.g. to support conversion issues where OF remains a key-reference [START_REF] Gliessman | Agroecology: the ecology of sustainable food systems[END_REF].

Development of agroecology in Brazil

The Brazilian trajectory of AE can be described through three main phases: (i) emergence of social movements against the industrialisation of agriculture, (ii) structuration of social groups and economic organisations, (iii) institutionalisation of AE [START_REF] Brandenburg | Movimento agroecologico: trajetoria, contradiçoes e perspetivas[END_REF]. The author mentions the same phenomena and phases regarding OF. Indeed, the last phase of institutionalisation links AE and OF with a law (n° 10.831/2003) acknowledging AE under the umbrella of organic production. In this law, participatory guarantee systems and the political dimension of AE were considered as important to support small farmers and foster rural communities.

On the scientific side, AE is gaining in importance. AE was officially recognised in 2006 as a scientific framework by the Brazilian Agricultural Research Corporation (EMBRAPA). In 2009 the VIth congress of AE gathered close to 4000 participants; it was organised by the Brazilian Association of Agroecology (ABA), created in 2004. On the civil society side, the National Articulation of Agroecology (ANA) appeared in 2002 as a space for convergence of social movements, networks and organisations.

Comment citer ce document : Bellon, S., Lamine, C., Ollivier, G., de Abreu, L. (2011). The relationships between organic farming and agroecology. In: Organic is life. Knowledge for tomorrow. Proceedings (p. 235-238). Presented at 3. ISOFAR Scientific Conference at the 17. IFOAM Organic World Congress, Gyeonggi Paldang, KOR (2011-09-28 -2011-10-01). Bonn, DEU : ISOFAR -International Society of

The case of Brazil shows the complexity of relationships between AE and OF and the diversity of visions they encompass.

Interviews with Brazilian scientists and actors of the civil society enabled to distinguish three trajectories corresponding to specific visions: (i) actors coming from NGOs and social movements who got involved in policy making and maintained or developed strong links with scientific communities, (ii) research or extension workers involved in social movements and policy making through participatory research projects mostly anchored in AE, (iii) scientists interested in OF and AE as research fields and much less involved in social movements and policy making. The two first types of actors claim a political vision of AE which is considered far beyond OF ("OF is at best a stage along the way to AE in the process of transition", as said an interviewee). The third category of actors claim a scientific and "objective" version of AE which is seen as close to OF. The types of farms (family farms vs. all farms), the relationships to markets and consumers (short food chains vs. all types of food chains), the key scholars and the research methods (participatory approach vs more classical research projects) can also be distinguished in these competing visions of AE.

General relationships between OF and AE

Some commonalities appear between OF and AE. Both promote a "closed system" approach, use multiple and diverse crops or animals, rely on biological processes for building soil fertility and controlling pests and diseases, support transition pathways towards ecologically-based agricultural systems [START_REF] Abreu | Trajetórias da Agroecologia no Brasil: entre Movimentos Sociais, Redes Científicas e Políticas Públicas. VI Congresso Brasileiro de Agroecologia. II Congresso Latino-americano de Agroecologia[END_REF]. They are also both a suited way to introduce practical systems research into academia. Some differences can however be identified (Table 1).

Discussion

The case of Brazil showed a combined movement of institutionalisation of both OF and AE, which also occurs in other latin-american countries [START_REF] Nelson | Institutionalizing agroecology: successes and challenges in Cuba[END_REF]. In most European countries, AE is not yet as institutionalised as OF, despite a growing influence of AE on agricultural sciences. However, in many countries, various scholars extended their research from OF to AE, both in research or in education. In the case of France, the Institute for Agricultural Research (INRA) recently mentioned AE as one

Figure 1 :

 1 Figure 1: Dynamics in AE and their crossing over with OF (Source: Web of Science)

Table 1 : Comparative analysis of central attributes in OF and AE

 1

		Organic Farming	Agroecology
	Definition	System of farm management	Various e.g. Interdisciplinary study
		and food production	and design of agricultural and food
			systems (Gliessman, 2007)
	Initial paradigms	Soil fertility (and soil sciences)	Ecology (and entomology)
	Key concepts	Farming system ; Value chain	Agroecosystem; Food sovereignty
	Reference models	Mixed livestock-cropping	Traditional multistratified systems
	Agricultural forms	Biological, Biodynamic, Organic	Alternative, Sustainable agriculture,
	associated		Integrated Pest Management
	Key actors	Farmers, processors, consumers	Diversified small farmers
	Technologies	Use of natural substances and	Nutrient cycling; biological crop
		processes; no GMOs	protection; possibly chemical inputs
	Food	Quality, content, health	Agri-food systems, sovereignty
	Biodiversity	Impact oriented (effect of	Resource oriented, enhancing
		practices on biodiversity)	agrobiodiversity
	Regulations	Historical recognition, IFOAM	No international standards
		principles, and national rules	acknowledged
	Certification	Mostly third-party	Participatory guarantee systems

INRA SAD, Ecodevelopment Research Unit (UR 767), Site Agroparc, 84143 Avignon Cedex 9 France E-Mail bellon@avignon.inra.fr

EMBRAPA Environment, Jaguariuna, Sao Paulo, Brazil

Comment citer ce document : Bellon, S., Lamine, C., Ollivier, G., de Abreu, L. (2011). The relationships between organic farming and agroecology. In: Organic is life. Knowledge for tomorrow. Proceedings (p. 235-238). Presented at 3. ISOFAR Scientific Conference at the 17. IFOAM Organic World Congress, Gyeonggi Paldang, KOR (2011-09-28 -2011-10-01). Bonn, DEU : ISOFAR -International Society of of its main priorities. However, it is mainly in some professional or in civil society organisations that AE is mobilized as a way to express an opposition to mainstream agriculture, and sometimes to organic movements and definitions. This phenomenon is rather recent, while in Brazil AE has been for long an available frame for alternative agricultures' networks. Further comparative analyses would probably confirm the fluidity which exists between OF and AE, since their fontiers and contents are evolving, despite the specificity of conceptual attributes which we assessed.

More generally and beyond the academic arenas (see Fig. 1), AE becomes a catchword in many public and private organisations. In some civil society organisations, the reference to AE allows enhancing the importance of key values (such as quality of life and work; social justice) which are de facto also present in the organic movements (but not in the organic legal rules) as well as the notion of food system (producers/consumers links and relocalization of food production and consumption) which is considered by many as negelected in OF.

Conclusions

OF is still a reference, due to its history (almost a century), its principles for action (set of rules) and codified practices (regulations), its controls and certifification, its growing economic importance and its identification by consumers. AE which is gaining importance in the academic world and in many social movements has to strengthen its identity as an action-oriented interdisciplinary project. This entails (i) deepening relationships between AE and OF as built in literature, in social movements and in individual curricula and (ii) developing beneficial convergences through crossfertilisation. In this respect, research efforts should contribute to the design of dynamic agricultural models embedded in social-ecological systems.


Marek Spinka

Céline Tallet

Pavel Linhart

Emotional valence : Is it reflected in call types and in acoustic properties of piglet vocalizations?

starlings conformed to economic rationality on both tests, showing strong stochastic transitivity and no violation of the independence principle. The results endorse both axiomatic microeconomics and optimality assumptions in behavioural ecology. How emotional valence is encoded in mammalian vocalizations is a prominent question in animalwelfare-oriented bioacoustics. We assessed this question using 1513 calls produced by 84 piglets in 11 situations. The negativity of the situations was ranked based on judgments of 28 pig behaviour experts. The acoustic quality of calls was described with 8 acoustic parameters. K-means clustering method was used to classify the calls into 5 call types. Statistics were calculated based on situation means (N=11, P adjusted for multiple testing). More negative situations were associated with longer calls (r s =-0.80, P=0.025) but no other acoustic parameter was associated with the experts' ranking of situations. The proportion of high-pitched, stable frequency "scream" calls was higher in more negative situations (b=-0.55, P=0.012) while medium-pitched tonal "croaking" calls were increasing in more positive situations (b=0.34, P=0.012). Thus the acoustic quality and the call types were related to the expert-ranked negativity of the situation but not as much as expected. The experts might have overrated negativity of some human-induced situations (eg, brief isolation) and underrated negativity pig-induced situations such a fighting for teats. Emotional valence is encoded in piglet vocalizations but animal-based measures of the situations' negativity are needed to assess the relationship precisely.

C5: Applied Ethology

C5.2 Chronic noise affects predator avoidance behaviour via trade-offs in resource use

Sophie Holles, University Of Bristol Stephen Simpson, Erica Morley, Andrew Radford Trade-offs in resource allocation are vital to survival through developmental stages. Many recent studies have revealed impacts of anthropogenic noise on a wide variety of taxa including birds, mammals and fish varying from stress responses, to disruption of vital cue detection via distraction and masking. In this study we investigated the impact of chronic noise through early stages of development in an economically important vulnerable species where recruitment is vital for population dynamics; Atlantic cod (Gadus morhua).

Three experiments revealed that 1) acute exposure to ship noise playback caused startle responses; 2) chronic exposure to ship noise playback lead to reduced body condition and 3) reduced body condition was linked with reduced survival time in a predator avoidance experiment. We propose two mechanisms where trade-offs may affect body condition and as a result, predator avoidance behaviour: a cognitive trade-off in the allocation of attention to noise versus foraging, affecting resource acquisition; and a physiological trade-off in resource allocation to growth and development versus the metabolic load incurred by chronic adrenal activation.

These experiments reveal that anthropogenic noise has the potential to impact behaviour, survival, population dynamics and selection through tradeoffs in resource allocation during developmental stages.

C5.3 Simple environmental enrichments improve

reproductive success in a model captive carnivore, the American mink (Neovison vison) Rebecca Meagher, University Of Guelph / University Of British Columbia Jamie Dallaire, Dana Campbell, Maria Diez-Leon, Misha Buob, Georgia Mason Unstimulating enclosures typically reduce welfare, and some hypothesise that they impair reproduction (e.g. in breeding centres/zoos). We tested this hypothesis via a large-scale experiment on three mink farms. We selected two 3-month-old male-female pairs from each of 260 families, providing one pair/family with balls and a hose or chain. Temperament (in "stick tests") and play were assessed two-three months later; and fur-chewing scored in the c. 60% subjects "pelted" at 7 months. Enrichments increased play, and reduced fearfulness (plus aggression on one farm) and furchewing. As adults (9-10 months), faecal cortisol metabolites (FCM) and locomotor stereotypies (e.g. pacing) were assessed; mating observed in a male sub-sample; and 380 new age-matched individuals recruited (half enriched). Enrichments did not reduce stereotypies, and only reduced FCM on one farm. However, enriched males copulated for longer than controls, and life-long enriched females had reduced infertility. Once litters were born, c. 20% of females were given elevated shelves for resting.

C5. 1

 1 Emotional valence: Is it reflected in call types and in acoustic properties of piglet vocalizations? Marek Spinka, Institute Of Animal Science Prague Celine Tallet, Pavel Linhart