Neural Correlates of Threat Perception: Neural Equivalence of Conspecific and Heterospecific Mobbing Calls Is Learned

Songbird auditory areas (i.e., CMM and NCM) are preferentially activated to playback of conspecific vocalizations relative to heterospecific and arbitrary noise [1]–[2]. Here, we asked if the neural response to auditory stimulation is not simply preferential for conspecific vocalizations but also for the information conveyed by the vocalization. Black-capped chickadees use their chick-a-dee mobbing call to recruit conspecifics and other avian species to mob perched predators [3]. Mobbing calls produced in response to smaller, higher-threat predators contain more “D” notes compared to those produced in response to larger, lower-threat predators and thus convey the degree of threat of predators [4]. We specifically asked whether the neural response varies with the degree of threat conveyed by the mobbing calls of chickadees and whether the neural response is the same for actual predator calls that correspond to the degree of threat of the chickadee mobbing calls. Our results demonstrate that, as degree of threat increases in conspecific chickadee mobbing calls, there is a corresponding increase in immediate early gene (IEG) expression in telencephalic auditory areas. We also demonstrate that as the degree of threat increases for the heterospecific predator, there is a corresponding increase in IEG expression in the auditory areas. Furthermore, there was no significant difference in the amount IEG expression between conspecific mobbing calls or heterospecific predator calls that were the same degree of threat. In a second experiment, using hand-reared chickadees without predator experience, we found more IEG expression in response to mobbing calls than corresponding predator calls, indicating that degree of threat is learned. Our results demonstrate that degree of threat corresponds to neural activity in the auditory areas and that threat can be conveyed by different species signals and that these signals must be learned

Au-delà de l’invasion cellulaire, la stratégie du touché-coulé : un nouveau paradigme pour la pathogénicité de Shigella

L’invasion de la muqueuse du côlon humain par les entérobactéries à Gram négatif du genre Shigella aboutit à une rectocolite aigue appelée dysenterie bacillaire qui reste un problème de santé publique majeur. Shigella possède un Système de Sécrétion de Type Trois (SST3) codé par un plasmide de virulence qui permet la translocation d’effecteurs bactériens dans le cytoplasme des cellules eucaryotes pour manipuler leurs fonctions. Ces effecteurs détournent les cellules épithéliales pour constituer une niche de réplication intracellulaire et interagissent avec les cellules immunes pour affecter l’initiation de la réponse immune adaptative. En conséquence, plusieurs épisodes infectieux sont nécessaires afin d’établir une protection immunitaire humorale qui est toutefois de courte durée. Ces travaux de thèse avaient pour but (i) d’approfondir les connaissances sur l’interaction de Shigella avec l’hôte en se concentrant sur les mécanismes dépendant du SST3 interférant avec les lymphocytes et (ii) de déterminer si des effecteurs du SST3 non encore identifiés sont codés par le plasmide de virulence. Nos résultats démontrent que la translocation d’effecteur du SST3 peut être découplée de l’invasion cellulaire, conduisant à des cellules « injectées-seulement ». Nous rapportons que Shigella induit l’apoptose des lymphocytes B par un mécanisme dépendant de la protéine située à l’extrémité du SST3 mais indépendant de la translocation d’effecteurs. Ces résultats établissent un nouveau paradigme concernant la pathogénicité de Shigella au-delà de l’invasion cellulaire, basé sur des mécanismes de type « touché-coulé » qui sont au cœur des interactions entre ce pathogène et les cellules immunes. Par ailleurs, nous décrivons la capacité de Shigella jusque-là inconnue d’interférer avec la sécrétion d’anticorps par les lymphocytes B, ce qui pourrait contribuer à moduler la réponse spécifique humorale de l’hôte. Enfin, nous avons identifié cinq nouveaux effecteurs potentiels de Shigella codés par le plasmide de virulence et injectés par le SST3 dans les cellules eucaryotes. Ces travaux de thèse apportent donc de nouveaux éléments concernant la pathogénicité de Shigella par la découverte de nouveaux mécanismes ciblant les cellules immunes et l’identification de nouvelles protéines bactériennes injectées dans le cytoplasme des cellules de l’hôte.Invasion of the human colonic mucosa by the Gram-negative enterobacteria Shigella spp. results in an acute recto-colitis named bacillary dysentery that still remains a major public health concern. Shigella expresses a Type Three Secretion System (T3SS) encoded on a virulence plasmid and mediating translocation of bacterial effectors into eukaryotic cell cytoplasm to manipulate their functions. These effectors hijack epithelial cells to create a bacterial intracellular replicative niche and also interact with immune cells to affect the priming of the adaptive immune response. As a result, several episodes of infection are required to mount a protective humoral immunity that is nevertheless of short-duration. This thesis work aimed at (i) further documenting Shigella cross-talks with its host, with a particular focus on T3SS-mediated mechanisms towards lymphocytes and (ii) investigating if the Shigella virulence plasmid encodes for yet unidentified T3SS-effectors. We report that translocation of Shigella T3SS-effectors into lymphocytes can be uncoupled from cellular invasion, resulting in “injected-only” cells. We demonstrate that Shigella mediates B lymphocyte apoptosis through a mechanism depending on the secretion apparatus needle tip protein but independent from effectors translocation. These findings set up a new paradigm for Shigella pathogenicity beyond cellular invasion, with “kiss-and-run” mechanisms proposed to be at the core of the interactions between this pathogen and immune cells. In addition, we describe a so far not known capacity of Shigella to interfere with B lymphocyte antibody secretion that could contribute to divert Shigella- specific humoral immunity. We also identify five new putative Shigella effectors encoded by the virulence plasmid and translocated by the T3SS into eukaryotic cells. Thus, this thesis work brings new insights into Shigella pathogenicity by unraveling novel mechanisms towards host immune cells and identifying new bacterial proteins that might constitute additional molecular weapons for this pathogen

Host Cell Targeting by Enteropathogenic Bacteria T3SS Effectors

International audienceMicrobial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities

Foraging zones of the two sibling species of giant petrels in the Indian Ocean throughout the annual cycle: implication for their conservation

We studied the year-round distribution and at-sea activity patterns of the sibling species, northern giant petrel Macronectes halli and southern giant petrel M. giganteus. Loggers combining light-based geolocators and immersion sensors were used to provide year-long data on large-scale distribution and activity of both species from the Crozet Islands (46°25’S, 51°51’E) and northern giant petrels from the Kerguelen Islands (49°19’S, 69°15’E) in the southern Indian Ocean. Argos platform transmitter terminals (PTTs) were used to track fine-scale movements of breeding adults and juveniles. Overall, adults remained within the Indian Ocean during and outside the breeding season, whereas juveniles dispersed throughout the Southern Ocean. In accordance with previous studies, differences in adult distribution and behaviour were greater between sexes than species: females dispersed more widely than males and also spent more time sitting on the water, particularly during the winter. Observed differences in distribution have important conservation implications: adults, especially males, overlap to a large extent with longline fisheries for Patagonian toothfish Dissostichus eleginoides in shelf areas within national Exclusive Economic Zones (EEZs), whereas adult females and juveniles are more likely to encounter high-sea longline fleets targeting tuna in subtropical waters. The circumpolar wide ranging behavior of naïve juvenile birds makes them particularly susceptible to interaction with a wide range of longline fisheries

Controlling Nanoparticle Interconnectivity in Thin-Film Platinum Catalyst Layers

The optimization of conventional hydrogen fuel cell catalyst layers suffers from a poor understanding of their composite nanostructure during both initial preparation and its evolution during use. We demonstrate how highly active, ultralow loading platinum (Pt) catalyst layers can be fabricated in a single, solution-processable step using electroless deposition. Growing Pt nanoparticles directly in the surface of a polyelectrolyte Nafion membrane yields a mechanically robust film with tunable optical reflectance and electronic conductivity. Small changes in the polymer hydration and Pt film thickness critically modulate nanoparticle interconnectivity near the percolation threshold. Conductive atomic force microscopy (AFM) and electron microscopy reveal how the film’s dynamic nanoscale morphology allows control over bulk electrochemical and optical properties. Well-defined composition and structure make these layers an experimentally accessible model system for studying thin-film electrocatalyst architectures

B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection mediated by the virulence factor IpaD

International audienceShigella is a gram-negative enteroinvasive bacterium and the causative agent of bacillary dysentery, an acute recto-colitis. Antibody-mediated natural immunity to Shigella requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. Here we show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and invades B lymphocytes. We observe the induction of a type three secretion apparatus (T3SA)-dependent B cell death in vitro, both in lamina propria B lymphocytes and the human CL-01 B cell line. This cell death and the parallel reduction of the B cell pool can also be observed in an in vivo mouse infection model. Intriguingly, Shigella-induced B cell death does not require bacterial invasion or injection of virulence effectors via the T3SA in vitro. Instead, the virulence factor IpaD triggers mitochondrial B cell apoptosis in the presence of bacterial co-signals that render B lymphocytes prone to die. We provide evidence that IpaD binds to and induces apoptosis via TLR2, a signaling pathway that has thus far only been considered as a mitogenic stimulus for B lymphocytes. Apoptotic B lymphocytes in close contact with Shigella displaying IpaD are also detected in isolated lymphoid follicles of rectal biopsies of naturally-infected individuals. These findings reveal a novel mechanism of T3SA action to induce B cell death by the binding of a virulence factor and reveal an efficient strategy by which entero-invasive pathogens could impair the priming of a protective immune response

Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes

International audienceThe enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection

Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed

Glycan-Glycan Interaction Determines Shigella Tropism toward Human T Lymphocytes

International audienceDirect interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis.IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells