<p><br></p> <p>Thrips, Thysanoptera, represent an ancient (~407 m.y.a.) order of ~6000 tiny insects from 9 families. Despite the small size of the order, thrips have a diversity of life histories, diets, and survival strategies. Thrips represent a challenge to fieldworkers and axonomists alike due to the morphological similarity between species and the lack of homologies between families. Recent </p> <p>molecular evidence has reopened debate over the phylogenetic relationships of the families of Thysanoptera.</p> <p>In this thesis we use genomic approaches to elucidate and clarify the early nodes in order to answer evolutionary questions about the Thysanoptera, their mitochondrion symbiotes, and their </p> <p>coevolutionary interactions with a group of economically important viruses; tospoviruses. Our results support previous ordinal hypotheses and show families in both sub-orders radiating </p> <p>around the emergence of the angiosperms ~120 m.y.a. We show that all thrips lineages likely have highly rearranged mitochondrial genomes, even on an intraspecies level, and that this rearrangement phenomena occurs very quickly in evolutionary time. We provide comment on the caution that must be taken with mitochondrial loci in any phylogenetic analysis with this new </p> <p>evidence and argue for the impact of among-site-rate-heterogeneity to be further investigated within thrips hylogenetics. We show that much more data is needed before thrips and tospovirus relationships can be fully elucidated but that two dueling hypotheses are emergent from our studies: either 5 very new separate vector/virus relationships, or one very old relationship that has been lost by the vast amount of thrips. We call for targeted taxa selection and show how new genomic methods can target certain taxa based upon the identification of </p> <p>assembled proteins from illumine shotgun read data.</p>

Artenübergreifende Interaktionen helfen uns, Krankheitsmechanismen zu verstehen und Targets für Therapien zu finden. Die Koexpression von Genen, gemessen an der mRNA-Häufigkeit, kann Interaktionen zwischen Wirt und Pathogen aufzeigen. Die RNA-Sequenzierung von Wirt und Pathogen wird als "duale RNA-Sequenzierung" bezeichnet. Malaria ist eine der am besten untersuchten parasitären Krankheiten, so dass eine Fülle von RNA-seq-Datensätzen öffentlich zugänglich ist. Die Autoren führen entweder duale RNA-seq durch, um den Wirt und den Parasiten gleichzeitig zu untersuchen, oder sie erhalten kontaminierende Sequenzierungs-Reads aus dem Nicht-Zielorganismus. Ich habe eine Meta-Analyse durchgeführt, bei diese beiden Arten von RNA-seq-Studien verwendet wurden, um über korrelierte Genexpression auf Wirt-Parasit-Interaktionen zu schließen. Ich habe Studien mit Homo sapiens, Mus musculus und Macaca mulatta als Wirte und ihre Plasmodium-Parasiten einbezogen. Ich benutzte orthologe Einzelkopien von Genen, um ein Repertoire von Interaktionen bei Malaria und in diesen Modellsystemen zu erstellen. Ich verknüpfte die Daten von 63 Plasmodium-Phasen-spezifischen Studien und reduzierte die Zahl der Interaktionen von potenziell 56 Millionen auf eine kleinere, relevantere Menge. Die Zentralität in den Netzwerken der Blutphasen konnte die Essentialität der Plasmodium-Gene erklären. Das aus den verketteten Daten sagte die Genessenzialität besser vor als die einzelnen Studien - ein Vorteil der Meta-Analyse. Neutrophile und Monozyten Immunmarkergene waren überrepräsentiert, was auf eine Fülle von phagozytären und respiratorischen Reaktionen hindeutet. Die Analyse der Leberphase ergab Wirts- und Parasitenprozesse in frühen und späten Entwicklungsphasen. Ich fand bekannte Wirt-Parasit-Interaktionen, die für beide Phasen gleich sind, sowie bisher unbekannte Interaktionen. Dieses Prinzip lässt sich auch auf andere Krankheiten anwenden, um Mechanismen und therapeutische Ziele zu verstehen. / Cross-species interactions help us understand disease mechanisms and find targets for therapy. Gene co-expression, measured by mRNA abundance, can identify host-pathogen interactions. The RNA-sequencing of host and pathogen is termed “dual RNA-sequencing”. Malaria is one of the most studied eukayotic parasitic diseases, making an abundance of RNA-seq data sets publicly available. Authors either perform dual RNA-seq to study the host and parasite simultaneously or acquire contaminant sequencing reads from the non-target organism. I performed a meta-analysis using these two kinds of RNA-seq studies to infer host-parasite interactions using correlated gene expression. I included studies of Homo sapiens, Mus musculus and Macaca mulatta as hosts and their corresponding Plasmodium parasites. I used single-copy orthologous genes to generate a repertoire of interactions in human malaria and in these model systems. I found 63 malaria RNA-seq studies. I concatenated sequencing runs from Plasmodium stage-specific studies and reduced the number of interactions from a potential 56 million to a smaller, more relevant set. Centrality in the blood stage networks was able to explain Plasmodium gene essentiality. The network from the concatenated data predicted gene essentiality better than the individual studies, indicating a benefit of the meta-analysis. Immune marker genes for neutrophils and monocytes were over-represented, suggesting an abundance of phagocytic and respiratory burst-related responses. The liver stage analysis revealed linked host and parasite processes at early stages until late developmental stages. I found linked host and parasite processes that are common to the two stages, e.g. parasite cell gliding and invasion and host response to hypoxia and immune response. I showed that existing data can be explored for new information. This principle can be applied to other diseases to understand mechanisms and therapeutic targets.

Made available in DSpace on 2015-07-22T20:50:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-30. Added 1 bitstream(s) on 2015-08-11T03:26:36Z : No. of bitstreams: 1 Publico-Tese%20Paloma%20Korehiza%20Maza%20versao%20final.pdf: 1568579 bytes, checksum: 8a94eab5c79046acd8162d9a949d2c36 (MD5) / Muitos patógenos são capazes de manipular a sinalização de células do hospedeiro para facilitar sua infecção. Nesta tese, demonstramos que o fungo Paracoccidioides brasiliensis promove um aumento na ativação de ERK1/2 e SFKs em células epiteliais A549 (de pulmão humano), de aproximadamente 6 e 7 vezes, respectivamente, em relação aos níveis basais. Utilizando PP2, inibidor da atividade de SFKs, e PD98059, inibidor da ativação da via ERK1/2, verificamos que a ativação de ERK1/2 é parcialmente dependente de SFKs ativadas e que provavelmente outras quinases também estão envolvidas neste evento. Além da modulação da sinalização de células do hospedeiro, diversos estudos têm demonstrado que patógenos seqüestram domínios presentes nas membranas da célula hospedeira denominados “membrane rafts”, os quais são enriquecidos em esfingolipídeos e colesterol e estão envolvidos em diversos eventos celulares, incluindo a sinalização celular. Por diferentes metodologias, como a desorganização de “membrane rafts” por drogas que extraem (metil-β- ciclodextrina, MβCD) ou que se ligam (nistatina) ao colesterol, e a localização do gangliosídeo GM1, um marcador de “membrane rafts”, utilizando a subunidade B da toxina da cólera (CTB), mostramos o envolvimento destes domínios de membranas de células epiteliais na adesão de P. brasiliensis. A partir do isolamento de membranas resistentes a detergente (DRMs) de células epiteliais após incubação com o fungo, mostramos a ativação e o deslocamento de SFKs para as frações que contêm os “membrane rafts”. Além disso, verificamos que a depleção do colesterol com MβCD inibe completamente a ativação de SFKs, corroborando a importância dos domínios de membranas para a ativação destas quinases. Os resultados apresentados nesta tese demonstram, pela primeira vez, que fungos patogênicos modulam a organização e a atividade de “membrane rafts” de células hospedeiras para o estabelecimento da infecção. / Many pathogens are able to manipulate host cell signaling in order to facilitate infection. In this thesis, we demonstrated that the fungus Paracoccidioides brasiliensis promotes an increase of ERK12 and SFK activation in A549 epithelial cells (human lung cells), by approximately 6- and 7-fold over basal levels, respectively. By using PP2, inhibitor for SFK activity, and PD98059, inhibitor for ERK1/2 activation, we verified that ERK1/2 activation is partially dependent of activated SFKs and probably other kinases are involved in this event. Besides modulation of host cell signaling, several studies have been demonstrated that pathogens hijack domains that are present in host cell membranes called membrane rafts, which are cholesterol- and sphingolipidenriched domains, that are involved in several cell events, including cell signaling. By using several approaches, such as membrane rafts disruption with cholesterolextractor (methyl-β-cyclodextrin, MβCD) or –binding (nystatin) drugs, and the localization of ganglioside GM1, a membrane raft marker, by using cholera toxin subunit B (CTB), we showed the involvement of these epithelial cell membrane domains in P. brasiliensis adhesion. By isolating detergent-resistant membrane (DRM) from epithelial cells after incubation with this fungus, we showed the activation and the dislodgment of SFKs to fractions which contain membrane rafts. Moreover, we verified that cholesteroldepletion with MβCD completely inhibits SFK activation, corroborating the importance of membrane domains in activation of these kinases.The results presented in this thesis demonstrate for the first time that pathogenic fungi may modulate the organization and activity of host cell membrane rafts for infection establishment. / TEDE / BV UNIFESP: Teses e dissertações

Les interactions intimes et répétées entre hôtes et parasites peuvent engendrer la spécialisation d’un parasite à son hôte, grâce à des adaptations comportementales, morphologiques et/ou génétiques, combinées avec un flux de gènes limité. C’est un processus clef car il participe à l’évolution de la biodiversité parasitaire et peut ainsi permettre de mieux comprendre l’émergence d’organismes pathogènes. Encore peu étudié, une spécialisation d’hôtes a néanmoins été démontré lors de précédentes études chez deux espèces de tiques nidicoles : chez Ixodes uriae une tique dure, parasite des oiseaux marins coloniaux en zone arctique, et dans un complexe de tiques molles Ornithodoros capensis sensu lato, parasitant aussi de nombreuses espèces d’oiseaux marins, mais cette fois-ci en zones tempérées et tropicales. Ces deux espèces sont vectrices d’une grande diversité d’agents pathogènes incluant des virus, des bactéries et des protozoaires. Cependant, les facteurs impliqués dans le phénomène de spécialisation d’hôte restent inconnus. Dans ce cadre, le but de ma thèse était donc de déterminer 1) si l’évolution des divergences en fonction des hôtes est toujours accompagnée par les mêmes changements phénotypiques et 2) si ces changements pourraient permettre d’identifier les facteurs de sélection sous-jacents. Dans ce contexte, des campagnes d’échantillonnage de tiques ont été menées durant la période de reproduction des hôtes oiseaux dans les différentes zones de leur répartition et nous avons réalisé des analyses morphométriques, basées sur l’utilisation de landmarks et de contours sur chaque individu tique et des analyses phylogénétiques et génétiques des populations sur les mêmes individus. L’ensemble de ces résultats suggère la présence de convergences morphologiques au sein de ces systèmes et souligne un rôle de la sélection dans ce processus de divergence. En effet, les caractéristiques écologiques des hôtes mais aussi le micro-habitat exercent des pressions sélectives importantes dans ces deux systèmes pouvant être à l’origine de la divergence observée entre les populations. De plus, les caractéristiques biologiques de chaque espèce de tiques, telle que la capacité de dispersion, entrent également en jeu et peuvent fortement modifier l’épidémiologie des agents infectieux dont elles sont vectrices.Mots clés : Argasidae, écologie de la transmission, évolution convergente, interactions hôte-parasite, Ixodidae, oiseaux marins. / Intimate and repeated interactions between hosts and parasites can lead to parasite specialization to a given host via behavioral, morphological and/or genetic adaptations that act in combination with restricted gene flow. Specialization is a key process leading to the generation of parasite biodiversity and can help us understand the emergence of pathogenic organisms. Although little studied, host specialization has already been demonstrated to occur in previous studies of two nidicolous tick species: Ixodes uriae a hard tick parasitizing colonial seabirds in polar regions, and soft ticks of the complex Ornithodoros capensis sensu lato, that also exploit colonial seabirds, but this time in temperate and tropical zones. Both of these species act as vector to a wide variety of pathogenic organisms, including viruses, bacteria and protozoa. However, the factors involved in host specialization remain unknown. In this context, the aim of my thesis was to determine 1) whether the evolution of host specialization is always accompanied by the same phenotypic changes and 2) whether these changes could help to identify the selective factors that influence this phenomenon. In this context, tick collections were conducted during the breeding period of the host birds in different areas of their distribution and morphometric analyses, based on landmark and contour methods, were performed on each individual tick. Phylogenetic and population genetic analyses were also carried out using the same individuals. Overall, the results demonstrate that morphological convergence occurs within these systems, highlighting the role of selection in the divergence process. Indeed, the ecological characteristics of the hosts, but also their micro-habitat, may exert significant selective pressures on ticks and may cause the observed divergence among populations. Likewise, the biological characteristics of each tick species, particularly in relation to dispersal capacity, may also come into play and will greatly modify the epidemiology of associated infectious agents.Keywords: Argasidae, convergent evolution, host-parasite interactions, Ixodidae, transmission ecology, seabirds.

<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

Pathogens and parasites are increasingly recognized as important components within host populations, communities, and ecosystems. Parasite contributions to ecosystem function most likely manifest as density-mediated impacts of parasites on their hosts, the direct contributions of parasite biomass to a system, and via parasite-induced changes in host behavior and physiology (trait-mediated impacts). Here, a framework was constructed that can be used to conceptualize parasite contributions to ecosystem function (Chapter 1). Then the influence of parasite attack on host movement was explored to further evince the mechanistic underpinnings of trait-mediated parasite impacts (Chapter 2). Additionally, mesocosms were created across a gradient of parasitism to examine how these mechanisms are likely to unfold at larger biological scales (Chapter 3). Lastly, a series of differential equations was created to model host-parasite-ecosystem interactions and generate theoretical predictions about how and when parasites are likely to influence ecosystem processes (Chapter 4). Parasites have many characteristics of ecosystem engineers, but their role has historically been ignored. These studies begin to explore the role that parasitism may have as one of the drivers of ecosystem processes.

Giardia intestinalis is a unicellular protozoan parasite responsible for 280 million gastrointestinal infections every year. When colonizing its host, Giardia interacts closely with the small intestine epithelium by attaching to enterocytes and releasing multiple proteins to the extracellular environment. Some of the released proteins have been shown to aid the parasite’s survival in the intestine by disrupting various host defense mechanisms. Here, we attempt to characterize the specific localization of five proteins after their secretion by Giardia. In parallel we aim to produce and identify parasite’s molecules potentially working as triggers of the immune response built during infection. To study the localization of specific secreted proteins during in vitro interactions with differentiated Caco-2 cells, we started by creating transgenic parasites expressing the ADI, EF1α and G3PD proteins with a downstream detectable tag. To identify candidate proteins from Giardia, thought by our lab to be involved in immune system activation, we established a mammalian expression system for the production of recombinant versions of the selected candidate giardial PAMPs. We achieved the expression of the VSP1267 protein, natively present on the parasite’s surface. However, we found that this protein was not secreted after expression, thus complicating its purification and later use in TLR-activation experiments. In the future, we aim to localize the tagged proteins, expressed by the produced transgenic trophozoites, and optimize the mammalian expression system in order to identify candidate immune triggers during giardiasis.

Le parasitisme est omniprésent dans l’environnement et une attention croissante est récemment apportée sur son impact sur les communautés écologiques. En effet, les parasites peuvent affecter la valeur adaptative des animaux sauvages, en altérant leur physiologie et/ou leur comportement. Ainsi, le rôle des parasites dans le maintien ou l’érosion des différences persistantes comportementales et cognitives entre individus est le sujet de nombreux débats et recherches. La relation entre l’infection parasitaire et le comportement de l’hôte est souvent complexe. Le comportement des individus agit sur leur susceptibilité au parasitisme, mais l’infection parasitaire peut aussi modifier le comportement de l’hôte, favorisant parfois la transmission du parasite. En outre, l’inclusion d’un proxy de santé, tel que la condition corporelle est importante à considérer dans des populations naturellement infectées où la santé des individus peut varier. Dans cette étude, nous avons examiné la relation entre un gradient de densité parasitaire, la personnalité (exploration et témérité), la cognition (apprentissage par stimuli aversifs) et la condition corporelle (Indice K de Fulton) chez les crapets-soleil (Lepomis gibbosus) sauvages, naturellement infectés par des endoparasites, comme le trématode responsable de la maladie du point noir (Trematoda : Apophallus sp. et Uvulifer sp.) et le ver solitaire de l’achigan (Cestoda : Proteocephallus ambloplites). Nous avons trouvé que l’exploration, mais pas la témérité, était répétable ce qui suggère que ce trait reflète la personnalité. De plus, l’exploration a diminué avec l’augmentation de la densité de parasites et la diminution de la condition corporelle de l’hôte. Ainsi, étant donné que la relation entre le comportement explorateur et la densité de parasites variait avec la condition corporelle, il est possible que les parasites aient un effet indirect sur le comportement de l’hôte en impactant sa physiologie. L’exploration variait également selon la densité de points noirs et la densité de cestodes, suggérant un potentiel conflit entre ces deux parasites, leurs hôtes finaux étant différents. Les individus avec plus de cestodes ont moins bien exécuté la tâche d’apprentissage, ce qui laisse à penser que ces parasites imposeraient un coût énergétique qui réduit les performances cognitives de l’hôte. Nos résultats contribuent à démontrer que les parasites et la condition corporelle de l’hôte doivent être pris en considération dans les études écologiques, comportementales ou physiologiques afin de mieux comprendre le maintien des variations inter-individuelles au sein des populations sauvages. / Parasites are ubiquitous in nature and increasing attention is given to their impact on ecological communities. Indeed, parasites can affect host fitness through changes in physiology and/or behaviour. Thus, their role in maintaining or eroding persistent inter-individual differences in behaviour (i.e. personality) and cognitive abilities in hosts is the subject of increasing study and debate. The relationship between parasite infection and host behaviour can sometimes be complex. For instance, personality traits may affect an individuals’ susceptibility to parasites. Conversely, parasite infection can itself modify host behaviour, sometimes favouring the parasite’s own transmission. In addition, including a general fitness proxy, such as body condition, is important when studying naturally infected populations, where individual health can vary greatly among individuals. Here, we examine the relationships among host body condition (Fulton’s K index), personality (i.e. exploration, boldness), cognition (aversive learning) and parasite density in wild pumpkinseed sunfish (Lepomis gibbosus), naturally infected with endoparasites, including trematodes causing blackspot disease (Trematoda: Apophallus sp. and Uvulifer sp.) and bass tapeworms (Cestoda: Proteocephallus ambloplites). We found that exploration but not boldness was repeatable, which suggests that this trait reflects personality. Host exploration decreased with both increasing parasite density and decreasing host body condition. Because the relationship between exploration and parasite density varied with body condition, this suggests a possible indirect effect of parasites on host behaviour through effects on host physiology. Exploration varied depending on blackspot and bass tapeworm density suggesting a possible conflict between these two parasites, as their final hosts are different. Inhibitory avoidance learning decreased with increasing cestode density, suggesting that these parasites could impose an energetic cost which decreases host cognitive performances. Our results provide more evidence that including host body condition and parasite density in ecological, behavioural or physiological studies can help better understand the persistence of inter-individual differences in wild populations.

Die Einschleppung gebietsfremder Parasiten durch den Menschen ist einer der Hauptgründe für das Auftreten neuer Krankheiten in the letzten Jahrzehnten. Neue Wirte sind oftmals anfälliger für diese invasiven Parasiten als die ursprünglichen Wirte. In schwerwiegenden Fällen können invasive Parasiten zu Massensterben und zum Aussterben ihrer neuen Wirte führen. Der ursprüngliche Wirt des Aalparasiten Anguillicola crassus ist der Japanische Aal. In den frühen 1980er Jahren wurde der Parasit in die Population des Europäischen Aals eingeschleppt. Er ist einer der Faktoren, die für den Populationsrückgang des Europäischen Aals verantwortlich sind. Die molekularen Prozesse, die zur stärkeren Anfälligkeit des Europäischen Aals im Vergleich zum Japanischen Aal führen, sind noch nicht zureichend bekannt. Die Analyse transkriptomweiter differenzieller Genexpression von Immungewebe ergab, dass im Europäischen Aal sowohl Immun- als auch Nichtimmungene differenziell exprimiert waren. Dies war im Japanischen Aal nicht der Fall und deutet darauf hin, dass der Europäische Aal eine ineffiziente und kostspielige Immunantwort auslöst. Die Resultate ensprechen jenen die schon in anderen Wirbeltierwirt-invasiven Parasiten-Systemen beobachtet wurden. Ausserdem stützen diese Resultate die Hypothese, dass neuen Wirten eine wirksame Immunantwort fehlt und sie deuten darauf hin, dass Nichtimmunprozesse wesentlich zur höheren Anfälligkeit von neuen Wirten beitragen. Als Reaktion of die negativen Fitnesseffekte können neue Wirte Abwehrmechanismen entwickeln. Im Europäischen Aal entspricht das der Einkapselung von A. crassus. Einkapselung führte zu eine niedrigere Abundanz adulter A. crassus. Dies deutet darauf hin, dass das Einkapseln sich positiv auf die Gesundheit infizierter Aale auswirkt. Jedoch war die Abundanz zweier nativer Parasiten höher in Aalen, die A. crassus einkapselten. Eine verbesserte Abwehr des eingeschleppten Parasiten könnte daher die Abwehr nativer Parasiten beeinträchtigen. / The human-mediated translocation of non-native parasites into foreign regions is one of the primary factors for the emergence of new diseases in recent decades. Novel hosts are often more susceptible to these invasive parasites than the native host. In severe cases, invasive parasites can lead to population declines and extinctions of their novel hosts. The eel parasite Anguillicola crassus is native to the Japanese eel. In the early 1980s it was introduced into the European eel population and is now considered to be one factor contributing to the population decline of its novel host. The underlying molecular processes determining higher susceptibility in the European eel compared to the Japanese eel are not well understood. Using whole-transcriptome differential gene expression analysis of immune organs, I found that genes involved in both immune and non-immune processes were differentially expressed in the European eel but not the Japanese eel, suggestive of an ineffective and costly immune response in the former. These results are in line with those observed between susceptible and resistant hosts in other vertebrate host-invasive parasite systems. Furthermore, the results support the hypothesis that novel hosts lack an effective immune response. The results also suggest that alteration of non-immune processes contributes substantially higher susceptibilities of novel hosts. In response to the negative fitness effects of invasive parasites, novel hosts can evolve coping mechanisms. The European eel has the capacity to encapsulate and kill A. crassus. Using natural infections, I found a lower abundance of adult A. crassus, the most costly parasitic stage in those eels encapsulating the parasite, suggesting that encapsulation can potentially improve health of infected eels. At the same time, the abundance of two native parasites was higher in those eels encapsulating A. crassus. Thus, coping with A. crassus may come at the expense of coping with native parasites.

<p>The crossing of host barriers (intestinal, blood-brain, and placental) is a critical step for systemic infections caused by entero-invasive pathogens. In the intestine, the epithelial cells are the first line of defense against enteric pathogens. <i>Listeria monocytogenes</i> is a facultative-intracellular foodborne pathogen that first crosses the intestinal barrier to cause a systemic infection. However, the underlying mechanism is not well understood.</p><p><br></p> <p>We demonstrate that <i>Listeria</i> adhesion protein (LAP) promotes the translocation of <i>L. monocytogenes </i>across the intestinal barrier in mouse models (A/J and C57BL/6). Relative to the wild-type (WT; serotype 4b) or the isogenic bacterial invasion protein Internalin A mutant (Δ<i>inlA</i>) strain, the <i>lap^─</i> strain showed significant defect in translocation across the intestinal barrier and colonization of the mesenteric-lymph nodes, liver and spleen in the early phase of infection (24 h and 48 h). LAP induces intestinal epithelial barrier dysfunction for increased translocation as evidenced by increased permeability to 4-kDa FITC-dextran (FD4), a marker of paracellular permeability, in the serum and urine of WT and Δ<i>inlA</i>- infected mice and across Caco-2 cell barrier, but not the <i>lap^─</i> mutant strain. Microscopic examination confirmed localization of the WT and Δ<i>inlA</i> strains in the tight junction, a crucial barrier of intestinal paracellular permeability, in the mouse ileal tissue but the <i>lap^─</i> strain remained confined in the lumen. LAP also upregulates TNF-α and IL-6 in intestinal epithelia of mice and in Caco-2 cells for increased permeability. </p><p><br></p> <p>Investigation of the underlying molecular mechanisms of LAP-mediated increase in intestinal permeability by using <i>lap^─</i> mutant strain, purified LAP and shRNA-mediated Hsp60 suppression, we demonstrate that LAP interacts with its host receptor, Hsp60, and activates the canonical NF-κB signaling, which in turn facilitates myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of major epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of NF-κB or MLCK in cells or genetic ablation of MLCK in mice (C57BL/6) prevents mislocalization of epithelial junctional proteins, intestinal permeability and <i>L. monocytogenes</i> translocation across the intestinal barrier.</p> <p><br></p><p>Furthermore, LAP also promotes <i>L. monocytogenes </i>translocation across the intestinal barrier and systemic dissemination in a Mongolian gerbil that are permissive to the bacterial invasion proteins; InlA-and InlB-mediated pathways; similar to that in humans. We show a direct LAP-dependent and InlA-independent pathway<i> </i>for <i>L. monocytogenes</i> paracellular translocation across the intestinal epithelial cells that do not express luminally accessible E-cadherin. Additionally, we show a functional InlA/E-cadherin interaction pathway that aids <i>L. monocytogenes</i> translocation by targeting cells with luminally accessible E-cadherin such as cells at the site of epithelial cell extrusion, epithelial folds and mucus-expelling goblet cells. Thus, <i>L. monocytogenes</i> uses LAP to exploit epithelial innate defense in the early phase of infection to cross the intestinal epithelial barrier, independent of other invasion proteins.</p><p><br></p> <p>This work fills a critical gap in our understanding of <i>L. monocytogenes </i>pathogenesis and sheds light to the complex interplay between host-pathogen interactions for bacterial crossing of the crucial intestinal barrier.</p> <br>