Dissertationen zum Thema „Malaria virulence“

From an evolutionary perspective, natural selection is expected to maximize transmission to new hosts. If a live, mobile host often benefits parasite transmission, the question arises as to why malaria parasites are virulent? The favoured trade-off view of virulence evolution assumes that virulence arises as an unavoidable consequence of parasite resource exploitation within the host that is necessary to maximise parasite transmission. However, virulence is not always a simple function of parasite density and can arise as a result of immune-mediated virulence (immunopathology). This thesis explores how immunopathology contributes to virulence on the one hand, and parasite transmission on the other, in order to improve our understanding of parasite virulence evolution. In tackling this question, the role parasite genetic diversity plays in determining immunopathology induced during malaria infection was also addressed. Using the rodent malaria Plasmodium chabaudi chabaudi (P.c.c.) in C57BL/6 mice, I explored whether immune factors – in terms of specific host cytokines central to the protection-pathology balancing act of the immune response elicited against malaria parasites – help to determine the virulence induced during infection with genetically distinct parasites, and if so, what effect this may have on transmission-stage parasites. I showed that the cytokine milieu induced by P.c.c. parasites during primary infection varies with parasite genotype and that virulence can arise independent of parasite density, via immunopathology. Specifically, I showed propensity to induce the pro-inflammatory cytokine tumour necrosis factor [TNF]-a contributes to the virulence induced, regardless of P.c.c. clone. Importantly, I also showed that across P.c.c. genotype, TNF-a reduces the density of transmission-stage parasites. Thus, virulence is not always a simple function of parasite replication, having an immune-mediated component which acts to reduce transmission potential. The importance of parasite genotype in determining the degree of immunopathological virulence induced during malaria infection was revealed by studying the anti-inflammatory arm of the immune response. The extent to which the anti-inflammatory cytokines interleukin [IL]-10 or transforming growth factor [TGF]-b limited the immunopathology induced during P.c.c. infection depended on parasite clone. In addition, parasite genotype played a key role in determining how such anti-inflammatory manipulations affected the density of transmission-stage parasites; being detrimental, beneficial or incidental to parasite fitness, depending on P.c.c. clone. Although the general mechanisms of immune regulation are qualitatively unchanged across distinct P.c.c. clones, these data emphasize the importance of parasite genotype: distinct clones differ quantitatively in immune regulation, which contributes towards their distinct virulence and fitness schedules. Overall, I found that even within a parasite species – in this case P. chabaudi – the effect of immunopathology on the virulence-transmissibility relationship may be genetically variable and may not conform to that predicted by the trade-off hypothesis, having the potential to alter the costs and benefits of virulence, depending on parasite genotype. Thus, the host immune response may play a role shaping virulence evolution and defining the limit to malaria virulence in nature.

This thesis focuses on the determinants of virulence in single and mixed-clone malaria infections, and the consequent impact of these infections for host- and parasite-fitness. Controlled experiments were conducted using a rodent model of malarial disease, Plasmodium chabaudi. Mice were infected with precise numbers of virulent and avirulent parasites. In the mixtures, known ratios of two clones that differed in virulence were used. Virulence was quantified in terms of host morbidity and mortality. Experiments investigating how virulence is determined in mixtures revealed that both the proportion of virulent clone in the innocula, and the genetic diversity of the infection determine virulence. Replacing virulent parasites with avirulent ones in a mixture was shown to confer protection for the host. These results challenge the various assumptions made in the models of the evolution of virulence about how virulence is determined in mixtures. They also suggest that selection against virulence can be reduced if virulent clones coinfect with avirulent ones, because host mortality is reduced in mixtures when the avirulent clone dominates. In single infections, the inoculating dose of virulent and avirulent parasites affected the virulence of the infection. Larger doses caused greater anaemia. They also caused additional weight loss, and death, but only for the virulent clone. Clone differences in virulence were maintained over the range of doses. Dose effects were manifested through the timing and/or magnitude of peak parasite densities, broadly supporting the idea that disease severity is due to the time the host has to control parasite densities and ameliorate the effects of parasites. To investigate the correlates of mortality, multivariate analyses were conducted. These generally showed that both the initial weight and red blood cell density of mice, and the rate at which they lost red blood cells and weight affected their probability of survival.

A laboratory study with the rodent malaria parasite P. chabaudi and A. stephensi vector indicated that mosquito morality varied with parasite genotype, infection diversity and nutrient availability. In standard conditions, mixed clone infections were the most lethal, but when glucose water was limited, mortality was highest in mosquitoes infected with CR. A second experiment showed that under standard conditions, mixed infections also had the greatest impact on vector fecundity. The virulence of mixed infections could not be explained by parasite load, nor their rate of resource uptake by parasites within the mosquitoes. During the parasite development period, infected mosquitoes had the same amount of three key physiological resources (lipids, glycogen, proteins), as those that were uninfected. Furthermore, mosquitoes infected with the most virulent parasite genotypes had an increased abundance of glucose relative to the controls. This is consistent with Plasmodium manipulating mosquito sugar-feeding behaviour in order to increase its own transmission. Several laboratory studies of malaria parasites and some field observations suggest that Plasmodium virulence in vertebrates is positively correlated with transmission to mosquitoes. A final experiment was undertaken to test whether the transmission advantage of infections that are virulent to vertebrates could be offset by an increased probability of causing death to their vectors. Mice were infected with one of seven distinct genetic clones of P. chabaudi that are known to vary in virulence. Infection virulence in mice (weight loss and anaemia) was positively correlated with mosquito infection rate but not with mosquito survival. Vector survival was influenced only by parasite clone and oocyst burden (negative association). These results suggest that vector fitness should not place an upper limit on malaria virulence. Overall, this research demonstrates that Plasmodium can be virulent to its vector, and that the magnitude of virulence is dependent on parasite genotype, infection diversity and environmental conditions. Although P. chabaudi virulence in vectors was not correlated with virulence in vertebrates, parasite genetic differences do impact vector fitness. Thus differential vector mortality could play an important role in determining the genetic composition of Plasmodium populations.

Drug resistance is one of the most medically relevant forms of pathogen evolution. To date, vaccines have not failed with the same depressing regularity as drugs. Does that then make vaccines evolution-proof? In the face of vaccination, pathogens are thought to evolve in two ways: by evolving epitope changes at the antigenic target of vaccination (epitope evolution); or by evolving changes at other antigenic loci, some of which may involve virulence (virulence evolution). The fundamental difference between these two forms of evolution is that virulence evolution could lead to disease outcomes in unvaccinated people that are more severe than would have been seen prior to evolution. One of the theoretical assumptions of virulence evolution is that more virulent parasites will have a selective advantage over less virulent parasites in an immunized host, and are thus more likely to be transmitted. The assumption is that more virulent parasites may be competitively more superior in mixed infections, or may be better able to evade/modulate the host immune response. Thus, the aim of this thesis was to experimentally test whether more virulent parasites have a within-host selective advantage in an immunized host or whether vaccine efficacy is more likely to depend on genetic differences at the targeted sites of vaccination. I used clones (genotypes) of the rodent malaria Plasmodium chabaudi originally derived from wild-caught Thicket (Thamnomys rutilans) rats to infect laboratory mice and a rodent analogue of the candidate blood-stage malaria vaccine apical membrane antigen 1 (AMA-1). I found that within-host selection did not depend on parasite virulence, and that protective efficacy depended on genotype-specific differences at the vaccine target. Vaccine-induced protection was not enhanced by including a number of allelic variants. However, such genotype-specific responses were only observed when the vaccine was tested against genetically distinct P. chabaudi parasites. When one P. chabaudi genotype was serially passaged through naïve mice the derived line was more virulent and was subsequently less well controlled by vaccine-induced immunity. In other experiments I found within host competition not to be immune-mediated. Thus my results suggest that vaccination has the potential to select for more virulent parasites but that the selective advantage is likely to be independent of competition. The selective advantage may be attributable to the enhanced immune evasion of more virulent parasites. However, without genetic markers of virulence, the mechanisms that mediate this selection remain unknown. My thesis contributes towards a growing body of evidence that vaccines have the potential to differently alter the within-host parasite dynamics of particular pathogen genotypes and that the selection imposed is likely to be system specific, depending on the fine specificity of the vaccine-induced responses and the identity of infecting parasites. Although vaccine potency may not be enhanced by including more than one allelic variant of an antigen, multi-valent vaccines may be one of the best ways to avoid the inadvertent selection for more virulent malaria parasites.

The biology of malaria parasites, Plasmodium spp., may be influenced by the presence of genetically distinct conspecific clones within a single infection, resulting in competition for host resources and transmission, and increased virulence for the vertebrate host. The extent of within host diversity, however, may be limited because overall clonal diversity could be reduced by the transmission biology of Plasmodium and variation in local prevalence. I examined clonal diversity of a natural malaria parasitehost association, P. mexicanum in its hosts, the western fence lizard, Sceloporus occidentalis, and sandflies, Lutzomyia vexator and L. stewarti, at a site in California ("Hopland"). Using microsatellite markers I characterized for the parasite, I examined (i) diversity within and among infections over time and space, (ii) transmission success of clones into both vector and lizard, (iii) the effects of clonal diversity on the parasite's infection dynamics and virulence for the lizard. From 1996 to 2006, clonal diversity varied both temporally and spatially, with slightly more multiclonal infections detected during years of high vs. low parasite prevalence (88% vs. 78% for sites with the highest prevalence at Hopland). Spatially, low prevalence sites (< 1% of lizards infected) had fewer multiclone infections (50%). Thus, even when prevalence drops over time, or at sites with chronically low prevalence, clonal diversity of the parasite remains high. Using natural and induced infections in the lizard, I found that multiclonal infections are no more infectious to vectors than single-clone infections, and almost all clones transfer successfully when the insect takes a blood meal. A competition experiment demonstrated that infections block new genotypes from entering a lizard host. Thus, multiclone infections are likely to be established when vectors feed on a complex infection and transmit those parasite clones to an uninfected lizard. The transmission biology of Plasmodium thus allows for the maintenance of genetic diversity in the parasite population. Finally, I examined the effects of multiclonality on the parasite's infection dynamics and virulence to the lizard host. Induced infections harboring a single or multiple clones had similar overall growth rates and maximal parasitemia, but multiclonal infections had significantly higher investment in gametocytes, suggesting competition for transmission. In addition, variation in parasite growth and density was greater for multiclonal infections, with approximately 1/3 displaying high replication rates and final parasitemia. Virulence measures indicated that weight change and proportion of immature erythrocytes was consistent for infections with 1, 2, 3 or > 3 clones, but the highly diverse infections had greater blood hemoglobin and glucose and more rapid clotting rates. Compared with the noninfected control lizards, highly diverse infections (3+) had higher blood glucose levels but similar hemoglobin levels. I have found that genetic diversity of the malaria parasite Plasmodium mexicanum varies both temporally and spatially, although overall diversity remains high. The transmission dynamics of the parasite maintains high genetic diversity within infections. Additionally, diversity within hosts plays a significant role in variation of infection dynamics and virulence.

L'étude des interactions hôtes/parasites est actuellement un thème de recherche incontournable des sciences de l'évolution. Une des questions majeures soulevée par ces études est de comprendre pourquoi certains parasites provoquent des maladies mortelles, alors que d'autres restent relativement bénins pour leurs hôtes. Dans ce défi que tentent de relever les biologistes de l'évolution mais également les sciences médicales, nous avons choisi de nous intéresser à la malaria aviaire, et plus précisément à son représentant le plus commun dans les populations naturelles d'oiseaux : Plasmodium relictum. En procédant à des infections expérimentales de canaris domestiques (Serinus canaria), nous avons voulu comprendre quels facteurs liés aux caractéristiques du parasite et de l'oiseau (au niveau de l'individu ou de la population) pouvaient influencer la virulence de Plasmodium relictum. Nous avons ainsi pu mettre en évidence que des facteurs, tels que le passé infectieux de l'hôte, la dose de parasites reçue, la prévalence de ces derniers dans la population d'oiseaux ou bien encore les interactions sociales entre individus, pouvaient moduler les coûts d'une telle infection. La virulence est un trait composite qui dépend, certes, de l'exploitation de l'hôte par les parasites mais également de la réponse immunitaire de ce dernier contre l'infection. Nous avons pu le vérifier dans notre système expérimental en utilisant une approche immuno-écologique. Nous nous sommes enfin intéressée aux conditions favorisant l'évolution de la virulence : ce qui est essentiel pour comprendre l'émergence ou la réémergence de maladies infectieuses et pour développer des stratégies de contrôle de ces maladies.

Le parasite protozoaire Plasmodium falciparum est l'agent causal de la forme la plus mortelle de paludisme humain. Ce pathogène utilise l'expression monoallélique de molécules d'adhésion de surface variantes, codées par la famille de gènes var, pour échapper au système immunitaire de l'hôte et provoquer une pathogenèse. On ne sait toujours pas comment l'activation du gène var fonctionne au niveau moléculaire et si des facteurs environnementaux peuvent moduler l'expression du gène var. Notre laboratoire a montré qu'une famille de gènes d'ARN non codants transcrits par Pol III, appelée RUF6, agit comme un trans-activateur des gènes var. Une association physique entre l'ARNnc RUF6 transcrit et le locus du gène var actif a été observée par FISH. La répression transcriptionnelle de tous les RUF6 par une stratégie d'interférence CRISPR spécifique a entraîné une régulation négative de la transcription de toute la famille des gènes var, suggérant une fonction potentielle de type amplificateur pour l'expression des gènes var. Une compréhension de la façon dont l'ARNnc RUF6 médie l'activation du gène var fait défaut. Ici, nous avons développé un protocole robuste de découverte protéomique dirigée par l'ARN (ChIRP-MS) pour identifier les interactions in vivo des protéines ARNnc RUF6. Des oligonucléotides antisens biotinylés ont été utilisés pour purifier l'interactome d'ARNnc RUF6. La spectrométrie de masse a identifié plusieurs protéines enrichies de manière unique qui sont liées à la transcription génique, telles que les sous-unités d'ARN Pol II, les protéines d'assemblage des nucléosomes et un homologue de la Dead-Box Helicase 5 (DDX5). La purification par affinité de PfDDX5 a identifié plusieurs protéines trouvées à l'origine par notre protocole RUF6-ChIRP, validant la robustesse de la technique pour l'identification des interactomes d'ARNnc chez P. falciparum. Le déplacement inductible de PfDDX5 nucléaire a entraîné une importante régulation à la baisse du gène var actif. Notre travail identifie un complexe protéique RUF6 ARNnc qui interagit avec l'ARN Pol II pour soutenir l'expression du gène var. Nous postulons que l'hélicase DDX5 peut résoudre les structures secondaires G-quadruplex hautement enrichies en gènes var pour faciliter l'activation et la progression de la transcription. De plus, nous découvrons des facteurs environnementaux qui déclenchent une régulation négative de la transcription du gène var. Nous observons que la privation d'isoleucine et les concentrations élevées de MgCl2 dans le milieu inhibent les gènes transcrits par l'ARN polymérase III. Il est important de noter que cela inclut une famille de gènes ARNnc régulateurs spécifiques de P. falciparum (codée par la famille de gènes RUF6) qui est un régulateur clé de l'activation du gène var. Nous avons identifié un gène homologue à l'eucaryote Maf1 hautement conservé, en tant qu'effecteur négatif de la transcription de l'ARNnc RUF6. Des concentrations élevées de MgCl2 ont entraîné un déplacement de PfMaf1 cytoplasmique vers le compartiment nucléaire. Nous avons utilisé un système de dégradation inductible des protéines pour montrer que les stimuli externes dépendent de PfMaf1 pour déclencher une expression plus faible des gènes RUF6. Nos résultats indiquent une voie indépendante de TOR qui répond aux changements de l'environnement et réprime la transcription Pol III. Ce travail fournit des informations conceptuelles nouvelles et importantes sur la répression de la virulence du parasite dépendante de PfMaf1 qui peuvent être très pertinentes pour établir la persistance subclinique du parasite pendant la saison sèche. Pris ensemble, ces résultats aident à mieux comprendre la fonction et la régulation d'un ARNnc impliqué dans la régulation de la variation antigénique et de la pathogenèse chez P. falciparum. Notre validation de la technique ChIRP-MS permet de futures études dans l'identification des protéines de liaison à l'ARN pour les ARNnc dont la fonction [...]
The protozoan parasite Plasmodium falciparum is the causative agent of the deadliest form of human malaria. This pathogen uses monoallelic expression of variant surface adhesion molecules, encoded by the var gene family, to evade the host immune system and cause pathogenesis. It remains unclear how monoallelic expression of var gene activation works at the molecular level and if environmental factors can modulate var gene expression. Our laboratory showed a Pol III transcribed GC-rich non-coding RNA gene family, termed RUF6, acts as a trans-activator of var genes. A physical association between the transcribed RUF6 ncRNA and the active var gene locus was observed through FISH. Transcriptional repression of all RUF6 by a specific CRISPR interference strategy resulted in transcriptional down regulation of the entire var gene family, suggesting a potential enhancer-like function to var gene expression. An understanding of how RUF6 ncRNA mediates var gene activation is lacking. Here we developed a robust RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. Biotinylated antisense oligonucleotides were used to purify the RUF6 ncRNA interactome. Mass spectrometry identified several uniquely enriched proteins that are linked to gene transcription such as RNA Pol II subunits, nucleosome assembly proteins, and a homologue of the Dead-Box Helicase 5 (DDX5). Affinity purification of PfDDX5 identified several proteins originally found by our RUF6-ChIRP protocol, validating the robustness of the technique for the identification of ncRNA interactomes in P. falciparum. Inducible displacement of nuclear Pf-DDX5 resulted in the significant down-regulation of the active var gene. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression. We postulate that DDX5 helicase may resolve G-quadruplex secondary structures highly enriched in var genes to facilitate transcriptional activation and progression. Furthermore, we discovered environmental factors that trigger downregulation of var gene transcription. We observe that isoleucine starvation and high MgCl2 concentrations in the medium inhibit RNA Polymerase III transcribed genes. Importantly, this includes a P. falciparum-specific regulatory ncRNA gene family (encoded by the RUF6 gene family) that is a key regulator in var gene activation. We identified a homologous gene to the highly conserved eukaryotic Maf1, as a negative effector of RUF6 ncRNA transcription. Elevated MgCl2 concentrations led to a shift of cytoplasmic PfMaf1 to the nuclear compartment. We used an inducible protein degradation system to show that external stimuli depend on PfMaf1 to trigger lower expression of RUF6 genes. Our results point to a TOR independent pathway that responds to changes in the environment and represses Pol III transcription. This work provides new and important conceptual insights into PfMaf1-dependent repression of parasite virulence that may be highly relevant for establishing subclinical parasite persistence in the dry season. Taken together, these results help to better understand the function and regulation of a ncRNA involved in regulating the antigenic variation and pathogenesis in P. falciparum. Our validation of the ChIRP-MS technique allows for future studies in identifying RNA-binding proteins for ncRNAs whose function remains to be fully characterized

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.

La Peste Porcine Classique est une maladie infectieuse virale ayant un grand impact économique sur l’industrie porcine. Les isolats récents sont suspectés être des souches de virulence modérée rendant le diagnostic difficile augmentant ainsi le risque de propagation de la maladie. L’étude de l’influence de la virulence sur la propagation de la maladie en calculant le taux de reproduction de base (R0) ont confirmé : l’influence de la virulence sur le R0 et le haut niveau de contagion en intra-parc. Les porcs infectés présentent une leucopénie et une atrophie des organes lymphoi͏̈des qui semble être causé par l’apoptose indirecte des lymphocytes. L’apoptose induite par une souche moyennement virulente dans le sang et la mise en place d’un modèle in vitro de co-culture ont révélé: 1) une leucopénie dès les premières 48 heures accompagnée d’une apoptose des cellules de la périphérie sanguine, 2) la nécessité d’un contact cellulaire pour induire l’apoptose.

Les changements globaux provoquent une augmentation des infections associées aux Vibrio. Les vibrioses affectent les humains, mais aussi des animaux marins. Un problème majeur est le manque de connaissances sur les vibrios présents dans l’environnement. Pour mieux comprendre et gérer ces maladies, nous nous sommes intéressés à l’écologie, l’évolution et la virulence de ces agents infectieux. Tout d’abord, nous avons montré que dans la nature, certains génotypes de Vibrio sont virulents pour les huîtres et que leurs mécanismes de virulence sont impliqués dans la différentiation écologique de ces souches. En France, les parcs ostréicoles font face à des évènements de mortalités massives associés à la présence d’un virus et de bactéries du genre Vibrio. Nous avons montré que le virus n’est ni essentiel, ni suffisant pour induire de fortes mortalités, contrairement aux vibrios qui jouent un rôle majeur. Les huîtres juvéniles malades sont toujours infectées par plusieurs populations, mais une seule, Vibrio crassostreae, est détectée en abondance. La virulence de cette population dépend d’un système de sécrétion de type IV codé par un plasmide conjugatif. Nos résultats suggèrent que V. crassostreae était un colonisateur peu virulent et que l’invasion du plasmide l’a rendu hautement pathogène. La distribution restreinte du plasmide de virulence suggère qu’il a été sélectionné par les hautes densités d’élevages. Enfin, nous avons montré que le transfert ou la sélection du plasmide était favorisé dans l’huître, suggérant que l’huître représente une niche propice aux transfert latéraux de gènes. Ce travail pourra permettre le développement d’outils de diagnostic et le suivi épidémiologique des vibrios pathogènes
Global change, including anthropogenic activities, have resulted in an increase in the incidence of Vibrio-associated illnesses. These diseases not only affect humans but also marine animals. Despite strong ecological and economic consequences, little is known about population structure and virulence mechanisms of vibrios in the environment. To better understand and manage those diseases, we explored the ecology, the evolution and the virulence of these infectious agents. First, we found that some environmental strains were virulent towards oyster and we identified original virulence mechanisms related to their ecology and evolution. In France, oyster-farms are facing massive mortality events associated with the presence of a virus and bacteria of the genus Vibrio. We showed that the virus appears neither essential nor sufficient to cause high mortality rates contrary to the vibrios that play a major role. Juvenile diseased oysters were always co-infected by several populations, but only one, Vibrio crassostreae, was detected systematically and in abundance. Its virulence is dependent on a type VI secretion system that is carried by a conjugative plasmid. Our results suggest that V. crassostreae first differentiated into a low-virulent oyster colonizer and turned into a pathogen after acquisition of the virulence plasmid. The narrow distribution of the plasmid suggests that it has been selected by high density farming areas. Finally, we showed that the plasmid transfer or selection was enhanced in oysters, which suggests that oysters represent a niche for horizontal gene transfer. Overall, this work can lead to the development of diagnostic tools and epidemiological monitoring of pathogenic vibrios

Sur la façade Atlantique, de la Norvège à l'Espagne, de nombreuses souches de Vibrio tapetis ou apparentées ont été isolées depuis 1988. Elles sont issues soient de palourdes présentant les symptômes de la maladie de l'anneau brun, soit d'autres mollusques. De plus, V. Tapetis a récemment été impliquée dans des vibrioses de poissons. Au cours de ce travail, une caractérisation systématique des isolats disponibles (21) a été menée afin de réaliser une étude complète de leur variabilité phénotypique et génétique ainsi que de leur virulence in vivo. Il a ainsi été montré qu'à l'exception de trois d'entre eux, tous les isolats appartiennent à l'espèce V. Tapetis sont virulentes avec une intensité variable. Par ailleurs, l'étude des mécanismes de pathogénie de V. Tapetis a été réalisée à partir de deux approches complémentaires : l'une cellulaire et l'autre moléculaire. L'approche cellulaire a permis de montrer d'importantes capacités cytotoxiques de cette bactérie vis à vis des hémocytes : altérations morphologiques (arrondissement et gonflement cellulaire), inhibition des capacités d'adhésion et du métabolisme oxydatif, mortalité. L'approche moléculaire a permis la mise en évidence d'un gène d'h émolysine, de type lécithinase, identifié chez toutes les souches de V. Tapetis. Il s'agit du premier gène de virulence potentiel identifié chez V. Tapetis. Cependant, son implication dans la virulence et le développement de la maladie de l'anneau brun n'est pas encore élucidée. L'ensemble du travail sur la caractérisation des souches de V. Tapetis constitue une base indispensable pour les recherches futures sur la biogéographie et la phylogénie de ce pathogène. De plus, pour le développement conjoint des approches in vivo, cellulaire et moléculaire a permis de faire progresser la connaissance sur la pathogénie de V. Tapetis et sur ces mécansimes d'interactions avec les cellules immunitaires de la palourde
Along the Atlantic coast, from Norway to Spain, many strains of Vibrio tapetis or related species have been isolated from diseased clams or other molluscs since 1988. Also, more recently, V. Tapetis have been implicated in fish vibriosis. As these isolates had previously been partially characterised, a comprehensive study was undertaken to assess and compare their phenotypic, genetic and in vivo virulence characteristics. With the exception of three isolates, all were grouped in a unique and homogeneous phylogenetic clade. In addition, all V. Tapotis strains were pathogenic for the Manila clam Ruditapes philippinarum although the virulence of these strains varied widely. Two approaches were used to study V. Tapetis pathogenicity mechanisms and their regulation : one cellular and one molecular. Vibrio tapetis showed strong cytotoxic activity towards clam hemocytes causing morphological alterations (cell rounding and swelling ), inducing inhibition of their adhesion capacities and of their oxidative metabolism, ultimately leading to cell death. Molecular approaches led to the discovery of a lecithinase hemolysin gene in all V. Tapetis strains. This is the first putative virulence gene identified in V. Tapetis. However, its role in virulence and in the developement of brown ring disease remains to be elucidated. The work carried out on characterisation of these V. Tapetis strains has yielded essential knowledge for future research on the biogeography and the phylogeny of this pathogen. Moreover, by combining experimental infection with cellular and molecular approaches the present study has contributed to a better understanding of V. Tapetis pathogenicity and of its interaction mechanisms with clam immune cells

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie est multifactorielle. Les lésions iléales des patients atteints de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC pour " Adherent-Invasive E. coli ". Ces souches sont capables d'adhérer et d'envahir les cellules épithéliales intestinales, et ont la capacité de survivre et de se multiplier fortement en macrophages en induisant une synthèse intense de TNF-α. L'objectif de ce travail s'inscrit dans la compréhension des mécanismes permettant aux bactéries AIEC de coloniser la muqueuse intestinale et d'induire les stades précoces de la pathologie. Une précédente étude menée au laboratoire avait permis de mettre en évidence l'importance de l'activation de la voie de régulation dépendante du facteur bactérien sigma alternatif RpoE (ou σE) dans le processus d'adhésion et d'invasion des cellules épithéliales intestinales par la souche AIEC de référence LF82 via l'expression des pili de type 1 et des flagelles. En continuité de ces travaux, nous montrons que l'activation de la voie de signalisation dépendante du facteur σE est également primordiale pour la capacité des souches AIEC à former des biofilms, et une analyse bioinformatique ayant pour but d'identifier les gènes régulés par σE a montré que l'opéron waaWVL, impliqué dans la biosynthèse du lipopolysaccharide, est primordial pour la formation de biofilm par les souches AIEC. De plus, nous avons mis en évidence que les long polar fimbriae (LPF) sont impliqués dans le ciblage de l'épithélium associé aux plaques de Peyer par les bactéries AIEC, et ceci en leur permettant de cibler spécifiquement les cellules M. L'inactivation du gène Nod2, gène de susceptibilité à la MC, conduit à une augmentation du nombre de plaques de Peyer ainsi que des cellules M à leur surface, indiquant que les bactéries AIEC pourraient tirer avantage d'une susceptibilité génétique pour cibler les plaques de Peyer.

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie reste mal connue. Les lésions iléales de patients atteints de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC pour Adherent-Invasive E. coli. Ces souches adhérent et envahissent fortement les cellules épithéliales intestinales. Elles sont également capables de se multiplier fortement en macrophages et d'induire une sécrétion élevée de TNF-α. L'objectif de ce travail est d'analyser les systèmes de régulation de la souche AIEC de référence LF82 impliqués dans l'expression maximale de virulence au sein la lumière intestinale et/ou en conditions intracellulaires. Nous avons étudié la co-régulation de l'expression des pili de type 1 et des flagelles en partant de l'observation qu'un mutant n'exprimant plus de flagelles perd de façon concomitante sa capacité à synthétiser des pili de type 1. Nous avons pu montrer que cette co-régulation complexe met en jeu la concentration intracellulaire en di-GMP cyclique et en protéine de type-histone Fis. Ces systèmes de régulation transcriptionnelle permettent une expression maximale des pili de type 1 qui, en conditions intestinales, confèrent aux AIEC un avantage par rapport à la flore commensale. L'annotation et l'analyse du génome de la souche AIEC LF82 a permis de mettre en évidence l'existence d'îlots de pathogénicité, des gènes spécifiques codants de potentiels facteurs de virulence mais également des mutations ponctuelles, de type pathoadaptatives, dans des gènes de virulence connus permettant vraisemblablement aux souches AIEC de coloniser la muqueuse intestinale de patients génétiquement prédisposés à développer une MC.

La colonisation de l’intestin humain par le parasite Entamoeba histolytica, l’agent étiologique de l’amibiase, demeure le plus fréquemment asymptomatique. Suite à des stimuli inconnus, le parasite devient invasif et détruit la barrière muco-intestinale ; ceci conduit à la dysenterie amibienne et aux formes cliniques graves : l’amibome intestinal et l’abcès hépatique amibien, qui sont les causes d’une centaine de milliers de morts par an. Par la comparaison des transcriptomes d’amibes virulentes et avirulentes de même fond génétique, nous avons identifié une réponse au stress et à l’oxydation chez les parasites virulents, ainsi qu’une famille de protéines riches en lysine. L’inhibition de la production de KERP1, un de ces facteurs, bloque la formation des foyers inflammatoires dans un modèle animal. Par ailleurs, l’expression de kerp1 est modulée au cours de l’infection hépatique, ainsi que par le monoxyde d’azote, un effecteur de la réponse inflammatoire, et par un choc thermique. La mise en évidence de l’implication de KERP1 dans la digestion des globules rouges phagocytés par l’amibe et dans l’adhérence aux cellules périsinusoïdales hépatiques nous a conduits à proposer un modèle expliquant le rôle de cette protéine au cours de l’infection : KERP1, par son association à la membrane plasmique des cellules de l’hôte, permettrait de les lyser et servirait de ligand pour des facteurs amibiens. Autrement, KERP1 permettrait le trafic vers la cellule cible de facteurs accomplissant ces activités. KERP1 ne partage d’homologie avec aucune protéine connue et les anticorps générés pour notre étude permettent d’identifier exclusivement le parasite E. Histolytica ; ainsi, nous les avons utilisés pour développer un test de diagnostic de l’amibiase.

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie reste mal connue. Les lésions iléales de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC pour Adherent-Invasive E. Coli. Ces souches sont capables d'adhérer et d'envahir les cellules épithéliales intestinales et ont la capacité de survivre et de se multiplier fortement en macrophages, en induisant une synthèse intense de TNF-α. L'objectif de ce travail s'inscrit dans la compréhension des mécanismes permettant aux bactéries AIEC d'adhérer et d'envahir les cellules épithéliales intestinales. Le criblage de la banque de mutants d'insertion du transposon Tn5phoA de la souche AIEC de référence LF82 a permis de mettre en évidence le rôle de la lipoprotéine YfgL dans le pouvoir invasif de la souche LF82. En effet, cette lipoprotéine participe à la formation de vésicules membranaires qui pourraient constituer un système de sécrétion et de transport de protéines bactériennes effectrices délivrées dans la cellule hôte et nécessaires à l'invasion des cellules par les bactéries. De plus, l'oxydoréductase DsbA est impliquée dans l'adhésion de la souche AIEC LF82 en régulant l'expression des flagelles et des pili de type 1, mais est également essentielle à la survie des bactéries AIEC LF82 en macrophages. Enfin, la protéine de membrane externe OmpC est essentielle chez la souche AIEC LF82 à l'adhésion et l'invasion des cellules épithéliales intestinales, mais son rôle dans la virulence est indirect. L'expression de la protéine OmpC est fortement induite à forte osmolarité, condition rencontrée par les bactéries AIEC au sein du tube digestif. Une surexpression de OmpC permet, chez la souche AIEC LF82, l'activation de la voie de régulation dépendante du facteur sigma alternatif RpoE (ou σE) qui régule l'expression des pili de type 1, des flagelles et d'autres facteurs nécessaires au processus d'adhésion et d'invasion des cellules épithéliales intestinales. En conclusion, sur la base des facteurs de virulence identifiés chez la souche AIEC LF82 et de leur régulation, les souches aIEC semblent avoir évolué de façon à pouvoir coloniser et envahir la muqueuse intestinale.

Trypanosoma cruzi, agent de la maladie de chagas, est un parasite tres variable pour beaucoup de parametres biologiques. De plus, les populations naturelles du parasite presentent une structure des populations clonale. L'etude presentee ici a pour hypothese de travail que les divergences phylogeniques, s'etant accumulees entre clones naturels de t. Cruzi, ont un impact important sur la biodiversite de ce parasite. Il existe une forte correlation entre les distances genetiques (divergences phylogeniques) existant entre les clones, et les differentes caracteristiques biologiques. Ce travail souligne l'interet du cadre de genetique des populations dans l'etude de la variabilite genetique des microorganismes, et confirme l'utilite du concept de clone majeur en microbiologie

L'objectif de ma thèse était de caractériser l'interaction de Pseudomonas fluorescens avec les cellules épithéliales de l'intestin. En effet, des anticorps anti I2, un antigène appartenant à cette bactérie, ont été retrouvés dans le sérum de patients atteints de maladie de Crohn, suggérant l'implication potentielle de P. Fluorescens dans cette maladie. Ainsi, nous avons souhaité étudier la virulence de cette bactérie envers les cellules épithéliales de l'intestin, sur les modèles cellulaires Caco-2/TC7 et HT-29. Dans un premier temps, nous avons étudié et comparé les effets cytotoxiques et proinflammatoires de la souche clinique P. Fluorescens MFN1032 avec ceux d'une souche psychrotrophe de référence P. Fluorescens MF37 et un pathogène opportuniste reconnu, Pseudomonas aeruginosa PAO1. Nous avons pu montrer que bien qu'inférieur à la cytotoxicité induite par P. Aeruginosa PAO1, la souche clinique P. Fluorescens MFN1032 est capable d'exercer un effet cytotoxique élevé sur les cellules Caco-2/TC7 et HT-29. La cytotoxicité de P. Fluorescens MF37 reste au contraire relativement faible. De plus, les deux souches de P. Fluorescens, tout comme P. Aeruginosa PAO1, induisent la sécrétion de la cytokine proinflammatoire IL-8. Cette induction est dépendante de la voie AP-1 pour les deux souches P. Fluorescens tandis que pour P. Aeruginosa PAO1, c'est la voie NF-kB qui est impliquée dans la sécrétion d'IL-8. Par la suite, nous nous sommes intérssés à l'étude de l'effet de ces bactéries sur la perméabilité intestinale en utilisant des cellules Caco-2/TC7 sous forme différenciée. Les résultats montrent que les deux souches de P. Fluorescens provoquent une augmentation de la perméabilité paracellulaire, associée à des réarrangements du cytosquelette d'actine F et une modification de l'expression des jonctions serrées. Nous avons pu montrer également dans cette étude, que lors de l'infection, les bactéries testées sont capables de transloquer du coté apical de la monocouche de cellules Caco-2/TC7 vers le milieu basolatéral en utilisant plus probablement la voie transcellulaire que paracellulaire. Enfin, nous avons étudié l'effet de la b-défensine-2, un peptide antimicrobien présent au niveau intestinal, sur la virulence de P. Fluorescens. Nos résultats montrent qu'une bactérie considérée généralement comme inoffensive telle que P. Fluorescens MF37, originairement isolée du lait, voit sa virulence envers les Caco-2/TC7 fortement augmentée losqu'elle est pré-traitée avec ce peptide. L'ensemble de ce travail démontre pour la première fois que P. Fluorescens est capable de se comporter comme un véritable pathogène opportuniste et pourrait ainsi participer à l'étiologie des phénomènes inflammatoires tels que ceux rencontrés chez les patients atteints de maladie de Crohn. Une plus grande importance devrait donc lui être accordée dans le futur afin de pouvoir identifier plus précisément son mode de virulence et son rôle éventuel dans les pathologies humaines
Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. Fluorescens (biovar 1) are able to adapt at a growth temperature of 37°C or above. Moreover, a highly specific antigen of P. Fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In this study, we aimed at determining the virulence of P. Fluorescens on human intestinal epithelial cells (IECs) Caco-2/TC7 and HT-29. First, the behaviour of the clinical strain of P. Fluorescens MFN1032 was compared to that of the psychrotrophic strain P. Fluorescens MF37 and to the opportunistic pathogen Pseudomonas aeruginosa PAO1. Both strains of P. Fluorescens were found cytotoxic on IECs and the cytotoxicity of the clinical strain MFN1032 was higher than that of MF37 but lower than P. Aeruginosa PAO1. The two strains of P. Fluorescens also induced IL-8 secretion in Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. Aeruginosa PAO1 potentially used the NF-kB pathway. Secondly, we examined the ability of a psychrotrophic and a clinical strain of P. Fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial cells model, reorganize the actin cytoskeleton, translocate across the epithelium and invade the target cells. Both strains of P. Fluorescens were found to be able to decrease the transepithelial resistance (TER) of Caco-2/TC7 differentiated monolayers. This was associated to a modification of the expression of tight junction proteins. Moreover, the translocation and invasion tests demonstrated that the two strains used in this study can invade and translocate across differentiated Caco-2/TC7 cell monolayers, and more probably via the transcellular route. Finally, we investigated the effect of b-defensin-2 (hBD-2), an antimicrobial peptide present in the intestine, on the virulence of P. Fluorescens. The results show that a pre-treatment of the bacteria with hBD-2 highly increases the virulence of P. Fluorescens MF37, a psychrotrophic bacterium originated from milk and generally consider as an inoffensive strain. The present work shows for the first time, that P. Fluorescens is able to behave very similarly to an opportunistic pathogen and thus may be able to participate in the etiology of the inflammatory phenomenon like those observed in Crohn’s disease patients. Additional studies may focus on better understanding the virulence capacity of P. Fluorescens and its possible role in human diseases

Les bactéries du genre Nocardia sont des Actinobactéries filamenteuses. Ces microorganismes sont saprophytes du sol. Les infections à Nocardia ou nocardioses se manifestent dans la majorité des cas (60%) par des infections pulmonaires et plus rarement par des infections cérébrales. La souche Nocardia cyriacigeorgica GUH-2 à causé la mort d'un patient dans les années 70. Des études sur cette souche ont montré qu'elle avait un haut pouvoir pathogène et qu'elle était capable dans certaines conditions de déclencher chez la souris et les primates le développement de troubles moteurs similaires à ceux observés dans la maladie de Parkinson. Afin d'appréhender l'implication de l'espèce pathogène opportuniste N. cyriacigeorgica dans l'une des plus importantes maladies neurodégénératives du 21ème siècle, le séquençage du génome de la souche N. cyriacigeorgica GUH-2 à été entrepris. Il a permis l'identification d'un grand nombre de gènes liés à la virulence et à la résistance aux antibiotiques ainsi que l'identification d'ilots génomiques et de séquences d'insertions reflétant une plasticité plus grande que celle qui était décrite pour ce genre bactérien. L'étude de groupements de gènes impliqués dans la production de métabolites secondaire a montré que ces molécules pourraient être responsables des propriétés neurodégénératives de la souche. L'espèce de N. cyriacigeorgica étant retrouvée fréquemment en clinique sans que son réservoir naturel n'ai été mis en évidence à ce jour, la mise au point de marqueurs de détection génétique a été réalisé afin de permettre la recherche de la niche écologique de cette espèce mais également de faciliter le diagnostic des nocardioses.

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l'intestin, dont l'étiologie est multifactorielle. Elle résulte de l'interaction complexe entre des prédispositions génétiques, des facteurs environnementaux et des altérations de la composition du microbiote intestinal, induisant une dérégulation du système immunitaire intestinal. À ce jour, la MC est incurable, seuls des traitements visant à soulager les symptômes et à prévenir les récidives et complications sont disponibles. Chez les patients atteints de la MC, une augmentation de la prévalence de souches particulières d'Escherichia coli, appelées les AIEC (adherent-invasive E. coli), a été rapportée. Les AIEC sont des pathobiontes capables d'adhérer et d'envahir les cellules épithéliales intestinales ainsi que de se répliquer en macrophages sans induire la mort cellulaire, entraînant une réponse immunitaire dérégulée. Par ailleurs, des études ont montré que plusieurs polymorphismes dans les gènes de l'autophagie (ATG16L1, IRGM, ULK1, etc.) sont associés à un risque augmenté de développer la MC. L'autophagie est un processus essentiel au maintien de l'homéostasie cellulaire permettant la dégradation et le recyclage de composants cytoplasmiques et de pathogènes via le lysosome. Toutefois, certains pathogènes intracellulaires développent diverses stratégies pour échapper à la dégradation par l'autophagie. Dans ce contexte, l'objectif de mes travaux de thèse était d'identifier les récepteurs autophagiques responsables de la reconnaissance des AIEC, ainsi que les gènes nécessaires aux AIEC pour échapper à l'autophagie.Le 1er axe de mes travaux de thèse a montré que la déficience de p62 ou NDP52 dans les cellules HeLa entraîne une augmentation de la réplication intracellulaire de la souche de référence AIEC LF82 et de la production de cytokines pro-inflammatoires. L'analyse par microscopie confocale a révélé la colocalisation de p62 ou NDP52 avec la bactérie AIEC LF82 et la protéine LC3, un marqueur de l'autophagie. Ainsi, nos résultats suggèrent que p62 et NDP52 agiraient comme des récepteurs autophagiques pour contrôler la réplication intracellulaire des AIEC. De plus, nous avons étudié l'impact d'un polymorphisme du gène NDP52 associé à une susceptibilité augmentée de développer la MC, appelé NDP52Val248Ala, sur le contrôle des AIEC. Aucune différence n'a été observée dans le nombre de bactéries AIEC LF82 intracellulaires entre les cellules HeLa exprimant le variant à risque NDP52Val248Ala et celles exprimant l'allèle sauvage, suggérant un autre rôle de ce variant, probablement dans le contrôle de l'inflammation.Le 2ème axe de mes travaux de thèse s'est concentré sur l'identification de gènes nécessaires aux AIEC pour échapper au contrôle par l'autophagie en utilisant la technique Transposon Sequencing (Tn-Seq). Brièvement, une banque de mutants de la souche AIEC LF82 a été créée de manière « saturée », c'est-à-dire que chacun des gènes du génome bactérien a été interrompu par au moins un transposon, conduisant à son invalidation. Cette banque de mutants a été utilisée pour infecter des cellules HeLa contrôles et déficientes pour l'autophagie. À 24h post-infection, l'ADN des mutants a été extrait et les sites d'insertion du transposon déterminés par séquençage ont permis d'identifier 68 gènes différentiellement représentés entre nos deux conditions. Les gènes sur-représentés dans les cellules HeLa déficientes pour l'autophagie par rapport aux cellules contrôles, sont les gènes potentiellement nécessaires aux AIEC pour échapper à l'autophagie. Ainsi, cette étude permettrait d'identifier de nouvelles cibles pour limiter la virulence des AIEC.En conclusion, ces travaux contribuent à la compréhension des divers aspects de l'interaction entre les cellules hôtes et les bactéries AIEC associées à la MC et permettront, à l'avenir, de mieux caractériser la pathogenèse de cette maladie
Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology is multifactorial. It results from the complex interaction between genetic predispositions, environmental factors and alterations in the intestinal microbiota composition, inducing a deregulation of the intestinal immune system. To date, CD is incurable, only treatments aimed at alleviating symptoms and preventing recurrences and complications are available. In CD patients, an increase in the prevalence of particular strains of Escherichia coli, called AIEC (adherent-invasive E. coli) strains, has been reported. AIEC are the pathobionts able to adhere to and to invade intestinal epithelial cells as well as replicate inside macrophages without inducing cell death, leading to a dysregulated immune response. Furthermore, it has been shown that several polymorphisms in autophagy-related genes (ATG16L1, IRGM, ULK1, etc.) are associated with an increased risk to develop CD. Autophagy is an essential process for maintaining cellular homeostasis, which allows the degradation and recycling of cytoplasmic components and pathogens via the lysosome. However, some intracellular pathogens develop various strategies to escape autophagy degradation. In this context, the aim of my thesis was to identify the autophagic receptors responsible for AIEC recognition, as well as the genes necessary for AIEC to escape autophagy.The first part of my thesis showed that the depletion of p62 or NDP52 in HeLa cells leads to an increase in the intracellular replication of the AIEC LF82 reference strain and the production of pro-inflammatory cytokines. Confocal microscopy analysis revealed the colocalization of p62 or NDP52 with AIEC LF82 bacteria and LC3 protein, a marker of autophagy. Thus, our results suggest that p62 and NDP52 could act as autophagic receptors to control AIEC intracellular replication. Additionally, we investigated the impact of a polymorphism in the NDP52 gene associated with increased susceptibility to develop CD, called NDP52Val248Ala, on the control of AIEC. No difference was observed in the AIEC LF82 intracellular number between HeLa cells expressing the NDP52Val248Ala risk variant and those expressing the wild-type allele, suggesting another role for this variant, probably in the control of inflammation.The second part of my thesis focused on the identification of the genes necessary for AIEC to escape from autophagy control by the Transposon Sequencing (Tn-Seq) technique. Briefly, a mutant library of the AIEC LF82 strain was created in a “saturated” manner, meaning that each gene of the bacterial genome was inserted by at least one transposon, leading to its invalidation. This mutant library was used to infect control and autophagy-deficient HeLa cells. At 24 hours post-infection, the mutants DNA was extracted and transposon insertion sites determined by sequencing allowed the identification of 68 genes differentially represented between our two conditions. The genes over-represented in autophagy-deficient HeLa cells compared to control cells, are potential genes necessary for AIEC to escape from autophagy control. Thus, this study could allow the identification of new targets to limit the virulence of AIEC.In conclusion, this work contributes to the understanding of various aspects of the interaction between host cells and CD-associated AIEC bacteria and, in the future, will aide to better characterize the etiopathogenesis of this disease

Les maladies à transmission vectorielle, qui représentent dix-sept pour cent de l’ensemble des maladies infectieuses, sont un réel problème de santé publique. Parmi elles, la borréliose de Lyme est la maladie vectorielle la plus répandue de l’hémisphère nord. Elle est causée par la transmission d’une bactérie, Borrelia burgdorferi sensu lato, par une tique dure du genre Ixodes. Le premier contact entre l’hôte vertébré et la tique, et donc a fortiori entre l’hôte vertébré et la bactérie, se fait au niveau cutané. La peau est donc une interface essentielle dans le développement précoce de l’immunité envers Borrelia. La tique effectue un repas de plusieurs jours qui induit une lésion cutanée par ses pièces piqueuses et sa salive, créant une cavité lui permettant de se nourrir. Ce processus facilite le repas sanguin, mais aussi la transmission de Borrelia. Nous avons caractérisé une protéine présente dans la salive de tique responsable de la formation de la cavité : l’histone H4. Celle-ci lyse les fibroblastes et possède une activité bactéricide envers les bactéries commensales de la peau. Ces deux activités pourraient être exploitées par Borrelia afin de favoriser sa transmission et son développement dans la peau. Une fois injectées, Borrelia et la salive interagissent avec les cellules de la peau telles que les kératinocytes de l’épiderme, les fibroblastes et les cellules immunitaires du derme. Nous avons montré que l’inflammation induite par la lésion augmente la réponse inflammatoire des kératinocytes contre Borrelia. Cependant, la salive de tique inhibe efficacement cette coopération inflammatoire dont la mise en place dépend des voies inflammatoires TLR3/TRIFF et TLR2/MyD88. Une fois la tique détachée de l’hôte vertébré et la salive de tique disparue, la coopération inflammatoire ne serait plus inhibée et pourrait expliquer en partie l’apparition de l’érythème migrans. L’inflammation cutanée précoce implique d’autres cellules que les kératinocytes et les fibroblastes telles que les macrophages, les cellules dendritiques ou encore les neutrophiles. Nous avons étudié l’implication d’un autre type cellulaire peu exploré dans le contexte de la borréliose de Lyme : les mastocytes. Ces cellules sont capables de répondre efficacement contre Borrelia par la sécrétion d’IL-6 et la dégranulation. Les antigènes bactériens nécessaires à l’activation des mastocytes semblent liés à la bactérie intacte et vivante. La salive de tique réduit la sécrétion d’IL-6 induite par Borrelia, mais ne l’inhibe pas complètement. A ce stade de nos études, les mastocytes ne semblent pas jouer un rôle majeur. Lors de la transmission, Borrelia exprime différents gènes qui lui permettront notamment de disséminer vers différents organes cibles de l’hôte vertébré : le système nerveux, l’articulation et la peau à distance. Nous avons identifié chez un clone de B. burgdorferi sensu stricto aux propriétés disséminatrices importantes, l'expression de deux gènes, bb0347 et bb0213, qui semblent associés à cette virulence. bb0347 code une protéine d’adhésion à la matrice extracellulaire, ce qui pourrait favoriser la migration de la bactérie à travers les tissus cutanés de l’hôte vertébré et donc sa dissémination. Le rôle de bb0213 n’est pas connu à ce jour
Vector-borne diseases account for seventeen percent of world-wide infectious diseases. They are amajor threat to public health. Lyme borreliosis is the first vector-borne disease of the northernhemisphere. It is caused by a bacteria, Borrelia burgdorferi sensu lato, inoculated by a hard tickbelonging to the Ixodes genus. The first contact between the vertebrate host and the tick, and sobetween the vertebrate host and the bacteria, occurs at the skin interface. The skin is then of majorimportance for the early development of the immune response against Borrelia.The tick bite induces a skin injury owing to its biting pieces, the hypostome and two chelicerae. The ticksaliva also creates a feeding pool allowing the tick to feed efficiently. This process also facilitates Borreliatransmission. We have characterized a tick saliva protein which might participate to the formation ofthe feeding pool: histone H4. This protein lyses fibroblasts and harbors bactericidal properties againstcommensal bacteria. These two activities might help Borrelia to infect the vertebrate host by sustainingits development in the skin. Once bacteria have been injected into the skin, they interact with residentskin cells, keratinocytes and fibroblasts, and immune cells. We have shown that the inflammationinduced by the tick bite increases the keratinocyte inflammatory response against Borrelia. However,the saliva inhibits this cross-talk which depends on TLR3/TRIFF and TLR2/MyD88 pathways. Once thetick has detached and the saliva has disappeared, the cross-talk might explained the inflammationobserved during the erythema migrans.Other skin cells than keratinocytes and fibroblasts are involved in the early inflammatory responseagainst Borrelia such as dendritic cells, macrophages and neutrophils. We have explored theinvolvement of another poorly-studied cell-type: mast cells. We have shown that these cells can secreteIL-6 and degranulate in response to Borrelia. Bacteria antigens responsible of the activation mightdepends on the living state of Borrelia. The tick saliva is able to negatively control the secretion of IL-6,but not to completely inhibit it. At this point, we cannot conclude in a WSH mouse model deficient inmast cells, to a major role of these cells in the inflammatory response against Borrelia.While in the skin, Borrelia expresses many genes which will facilitate the dissemination across thevertebrate host, to reach different target organs (brain, joint, distant skin). We have characterized twogenes potentially involved in the dissemination of a virulent clone of B. burgdorferi sensu stricto: bb0347and bb0213. bb0347 encodes for an adhesion which can specifically interact with the extracellularmatrix of the skin while the role of bb0213 is unknown. bb0347 might help the bacteria to migratethrough skin tissues and then increases the infection rate

Cette étude, in vitro et sur modèle murin, de souches cliniques de Borrelia burgdorferi sensu lato (Bb sl) comporte une étape de mise au point d’un milieu de culture BSK solide permettant le clonage sur boîte de 29 souches de Bb sl, pré-requis pour l’étude de la pathogénicité bactérienne ; une étape de mise au point d’une technique d’identification moléculaire pour 11 espèces de Bb sl ; une étape d’étude sur modèle murin de 20 souches natives de Bb sl en fonction de leur espèce et de leur pathotype humain. Cette étude a révélé que le tropisme tissulaire et l’arthritogénicité murins ne sont pas liés à l’espèce de Bb sl infectante mais plutôt au pathotype humain initial de ces souches. L’étude des clones de 14 de ces souches a permis de réaliser une collection de clones dont le profil infectieux expérimental est variable par rapport à celui de la souche native homologue, base précieuse pour l’étude des facteurs de virulence de Bb sl
This study of clinical strains of Borrelia burgdorferi sensu lato (Bb sl) have been performed in vitro and in mouse model. A solid BSK culture medium have been set to clone 29 Bb sl strains, prerequisite for bacterial virulence factors study. A molecular typing technique was performed to identify 11 species of Bb sl. An experimental study of 20 Bb sl clinical strains revealed that mouse organotropism and mouse arthritis was not linked to the species of Bb sl infecting strains, but to their human pathotype. Based on the study of clones from 14 of these strains in mouse model, a valuable clonal strains collection have been set, suitable for Bb sl virulence factors studies

Le Gallid herpesvirus 2 (GaHV-2), responsable de lymphomes T du poulet, est contrôlé par le vaccin CVI988/Rispens. Mes travaux de thèse ont montré que le vaccin était composé, au contraire des souches virulentes, d’une population dynamique de variants viraux majoritairement délétés de la région promotrice et d’une partie variable de l’extrémité 5’ du gène LAT codant des microARN et associé à la latence virale. Dans une approche vaccinale, un virus recombinant correspondant à l’un des variants majoritaires du vaccin CVI988/Rispens a été généré à partir d’une souche GaHV-2 hypervirulente, clonée en bacmide. Nous avons montré que ce recombinant, présentant une perte de pathogénicité presque totale, était capable de protéger significativement les poulets lors d’une épreuve avec des souches GaHV-2 hypervirulentes. Ces travaux posent les bases du développement de nouveaux vaccins à partir de souches hypervirulentes émergentes
Gallid herpesvirus 2 (GaHV-2), responsible for T-cell lymphomas chicken, is controlled by the vaccine CVI988/Rispens. My work has shown that the vaccine contains, unlike virulent strain, a viral variants population mostly deleted from the promoter region and a variable portion of the 5' end of the gene LAT encoding microRNA and associated with viral latency. In a vaccine approach, a recombinant virus corresponding to a majority variant of the CVI988/Rispens vaccine was generated from a hypervirulent strain GaHV-2, cloned as bacmid. We showed that recombinant, with an almost total loss of pathogenicity, was able to significantly protect chickens against challenge with virulent strains GaHV-2. This work lays the basis for the development of new vaccines from emerging virulent strains

Dans la forêt de pin maritime (Pinus pinaster) des Landes de Gascogne (sud-ouest de France), la mortalité des pins causée par le champignon pourridié Armillaria ostoyae (Basidiomycète) a augmenté au cours des 30 dernières années. Les premiers cas de cette maladie ont été signalés quelques années après un changement majeur dans l'utilisation des terres, qui a eu lieu dans cette région suite au remplacement des landes et marais d'origine par une forêt plantée et gérée da façon intensive. Notre objectif était de comprendre les facteurs à l'origine de cette maladie émergente. Pour cela, nous avons étudié la distribution spatiale des dommages causés par le pathogène en relation avec des facteurs historiques, estimé la variabilité des traits fongiques liés au parasitisme et saprophytisme, et étudié l'histoire démographique d'A. ostoyae. La répartition actuelle de la mortalité induite par A. ostoyae est apparue dépendre de la présence des forêts préexistantes, ce qui suggère qu'A. ostoyae était fréquent dans ces zones forestières anciennes, qui ont agi comme un réservoir pour la colonisation des forêts plantées récentes. La production de rhizomorphes était significativement corrélée avec la virulence, suggérant que ce trait joue un rôle important dans le stade parasitaire d'A. ostoyae. Aucune relation significative entre le parasitisme et saprophytisme n'a été détectée, suggérant une absence de compromis évolutif entre ces traits. Enfin, le meilleur scénario démographique pour expliquer la structure de la population d'A. ostoyae dans la forêt des Landes est un scénario en deux étapes : il y aurait eu d'abord une diminution puis une expansion de la population fongique, qui semblait suivre la dynamique de la population d'hôtes. Le temps de génération d’A. ostoyae a été estimé entre 10 et 20 ans
In the maritime pine (Pinus pinaster) forest of the Landes de Gascogne (south-western France), pine mortality due to the root rot fungus Armillaria ostoyae (Basidiomycete) has been increasing over the last 30 years. The first cases of this disease were reported a few years after a major change in land use which occurred in this region following the replacement of original moors by an intensively managed planted forest. Our aim was to understand the factors driving this disease emergence. For this, we investigated the spatial distribution of pathogen damage related to historical factors, estimated the variation in fungal traits related to parasitism and saprophytism and investigated the demographic history of A. ostoyae. The current distribution of A. ostoyae mortality appeared depending on the pre-existing forests, suggesting that A. ostoyae was commonly distributed in pre-existing forest areas which acted as a reservoir for the colonization of recent planted forests. The rhizomorphs production was significantly correlated with virulence, suggesting that this trait plays an important role in the parasitic stage of A. ostoyae, but no significant relationship between parasitism and saprophytism components was detected, which may suggest that there is no trade-off between these traits. Finally, the best demographic scenario to explain A. ostoyae population structure in the Landes forest is a two step scenario: there was first a decrease and then an expansion in the fungal population, which appeared to follow the dynamics of the host population. The generation time of A. ostoyae was estimated between 10 and 20 years

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.