During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.

Leptosphaeria maculans ‘brassicae’ (Lmb) est un ascomycète de la classe des Dothideomycètes faisant partie d’un complexe d’espèces présentant différents niveaux d’adaptation au colza. Lmb est responsable d’une des maladies les plus dommageables sur colza : la nécrose du collet. Lmb présente un cycle de vie complexe au cours duquel il alterne différents modes de vie, traduisant l’existence de mécanismes de régulation fine de l’expression des gènes lui permettant de s’adapter rapidement à de nouvelles conditions. Le séquençage de son génome a révélé une structure originale, avec l’alternance de deux types de régions : les isochores GC et les isochores AT. Alors que les isochores GC sont riches en gènes, les isochores AT sont pauvres en gènes et présentent des caractéristiques de l’hétérochromatine (régions génomiques riches en éléments transposables et présentant un faible taux de recombinaison). Bien que pauvres en gènes, les isochores AT représentent une « niche écologique » pour les gènes codant des effecteurs puisque 20 % des gènes des isochores AT codent des effecteurs putatifs contre seulement 4 % des gènes localisés en isochores GC. Les gènes codant des effecteurs situés en isochores AT présentent un comportement transcriptionnel différent de ceux localisés en isochores GC : une faible expression pendant la croissance mycélienne et une forte induction d’expression pendant l’infection primaire du colza. Sur la base de ces observations, l’objectif de ma thèse était de caractériser le déterminisme de la co-expression des effecteurs situés dans les isochores AT et en particulier d’évaluer si la régulation de l’expression de ces gènes se fait par un contrôle épigénétique lié à leur localisation particulière et/ou par l’intervention de régulateurs communs. Afin de déterminer le rôle de la structure des isochores AT, l’analyse fonctionnelle de protéines impliquées dans le remodelage de la chromatine (i.e. HP1, DIM-5 et DMM-1) a été réalisée et leur implication dans la régulation de l’ensemble des gènes prédits dans le génome de L. maculans a été évaluée. Cette étude a permis de démontrer l’implication de la structure hétérochromatinienne des isochores AT dans la répression de l’expression pendant la croissance mycélienne des gènes situés dans cet environnement génomique, en particulier les gènes codant des effecteurs. Parmi les gènes sous contrôle épigénétique, nous avons pu observer qu’en plus des gènes localisés en isochores AT, des zones en isochores GC étaient aussi affectées et pouvaient constituer des « hot-spots » de contrôle épigénétique. Afin d’identifier des régulateurs candidats pouvant être impliqués dans le contrôle de l’expression des effecteurs pendant l’infection, le répertoire des gènes codant des facteurs de transcription (FTs) chez Lmb a été établi et l’analyse de la conservation de ce répertoire parmi les autres espèces du complexe d’espèces Leptosphaeria a permis d’identifier les FTs spécifiques, ou spécifiquement sur-exprimés pendant l’infection du colza, chez Lmb. Des candidats ont été sélectionnés pour réaliser leur analyse fonctionnelle : des gènes codant des FTs sur-exprimés pendant l’infection (9 FTs) ainsi que les orthologues de FTs qui avaient été décrits chez d’autres espèces comme régulateurs majeurs de la pathogénie (StuA, Sge1 et Fox1). L’analyse fonctionnelle de FTs candidats a permis d’établir que StuA, comme chez d’autres champignons phytopathogènes, joue un rôle important dans la mise en place de l’infection et l’expression des effecteurs chez L. maculans. Le « silencing » d’un FT de type AT-Hook, famille de FTs se fixant préférentiellement au niveau de séquences riches en AT, a un fort effet sur la pathogénie du champignon et entraîne une diminution d’expression de 2 effecteurs. Cette thèse a permis d’apporter de nouveaux éléments concernant la régulation des gènes codant des effecteurs chez un champignon phytopathogène impliquant, pour la première fois, un mécanisme épigénétique. / Leptosphaeria maculans is an ascomycete belonging to the Dothideomycete class and is part of a species complex showing different level of adaptation toward oilseed rape. Within this species complex, Lmb is responsible for the most damaging disease of this crop: “stem canker”. Lmb presents a complex life cycle during which it alternates between different life styles and nutritional strategies underlying the involvement of precise regulatory networks for gene expression to rapidly adapt to new conditions. The sequencing of the Lmb genome has revealed an unusual structure, alternating two types of regions, GC- and AT-isochores. While GC-isochores are gene-rich, AT-isochores are gene-poor and have several characteristics of heterochromatin (they are rich in transposable elements and present a lower rate of recombination compared to GC-isochores). Although gene-poor, AT-isochores are “ecological niches” for effector genes as 20% of the genes in these regions encode for putative effectors against only 4% of the genes in GC-isochores. Effector-encoding genes located in AT-isochores present a different transcriptional behavior compared to those located in GC-isochores: a very low expression in axenic culture and a drastic increase in expression during primary leaf infection. On these bases, the aim of my thesis was to characterize the determinism of the concerted effector gene expression. Are AT-isochores targets of reversible epigenetic modifications that affect the regulation of effector genes? and/or are one or several common regulators involved in the control of the concerted expression of effector genes? To assess the role of the structure of AT-isochores, functional analysis of three key players involved in chromatin remodeling (i.e. HP1, DIM-5 and DMM-1) was performed and their role in global gene expression was assessed. This study validated that heterochromatic structure of AT-isochores represses expression of genes located in such a genomic environment, notably effector genes. Among genes under an epigenetic control, we also identified genes located in GC-isochores that were similarly influenced and may represent “hot spots” for epigenetic control. To identify putative regulators of effector gene expression, we established the complete repertoire of transcription factors (TFs) of Lmb and by analyzing the conservation of this repertoire among species of the Leptosphaeria species complex, we identified TFs specific of Lmb, or specifically induced during infection. Functional analysis of 12 TFs was set up: nine TF-encoding genes induced during infection and three orthologs of TFs described as required for pathogenesis in other phytopathogenic fungi (StuA, Sge1, Fox1). This functional analysis showed that StuA, as in other phytopathogenic fungi, plays a major role in infection and expression of effector genes in Lmb. The silencing of an AT-Hook type TF, family of TFs that specifically interact with AT-rich sequences, was associated with a reduction of the expression of two effector genes during infection and with pathogenicity defects. This study brought new insights into the regulation of effector genes in a phytopathogenic fungus involving, for the first time, an epigenetic mechanism.

<i>Legionella pneumophila,</i> the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates over 330 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK (Lpg0969) as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H₉₅E_XXH₉₉ (x, any amino acid) motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage occurs during <i>L. pneumophila </i>infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, <i>L. pneumophila</i> exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

L. pneumophila est l'agent responsable de la légionellose, une pneumonie sévère associée à 10% de mortalité. Cette bactérie intracellulaire a acquis la capacité de survivre et de se répliquer dans des cellules humaines. Notamment, L. pneumophila sécrète un grand nombre d'effecteurs par son système de sécrétion de type IV, qui interagissent avec différentes voies cellulaires, dont l'autophagie. L'autophagie est une voie de dégradation conservée qui permet aux cellules eucaryotes de réguler l'homéostasie cellulaire et d'éliminer les agents pathogènes intracellulaires. Néanmoins, nombre d'entre eux ont évolué pour manipuler cette voie à leur propre avantage. Même si l'interaction entre L. pneumophila et l'autophagie a été rapportée, aucun modèle clair n'est déterminé. Dans cette étude, nous montrons qu'une infection à L. pneumophila induit une stimulation globale de l'autophagie, mais que ce phénotype dépend des souches utilisées, et notamment de la présence de certains effecteurs. De plus, l'inhibition de l'autophagie est liée à un défaut de réplication intracellulaire suggérant que cette voie est bénéfique à la bactérie. Afin de rechercher les déterminants génétiques impliqués dans cette interaction, nous avons identifié des effecteurs communs sécrétés par le système de sécrétion de type IV entre L. pneumophila et Coxiella burnetii, une bactérie de l'ordre des Legionellales connue pour stimuler et détourner l'autophagie. La capacité des mutants de ces effecteurs à stimuler l'autophagie chez L. pneumophila a été analysée. Si aucun d'entre eux ne semble impliqué dans la modulation de l'autophagie, cette étude suggère d'autres fonctions pour ces effecteurs conservés / Legionella pneumophila is responsible for the legionellosis disease, a severe pneumonia associated with 10% mortality rate. This intracellular bacterium has evolved the ability to survive and replicate within human cells. Notably, L. pneumophila secretes a high number of type IV secretion system effectors that interfere with many cellular pathways including autophagy. Autophagy, a highly conserved degradative pathway, allows eukaryotic cells to regulate cell homeostasis and fight intracellular pathogens. Nevertheless numerous microorganisms have evolved strategies to subvert this mechanism to their own advantage. The interaction between L. pneumophila and autophagy has been reported but remains unclear. In this study, we show that L. pneumophila infection induces a global stimulation of autophagy, but importantly this autophagy stimulation depends on the bacterial strain. Moreover, we also observed that inhibition of autophagy results in decreased intracellular bacterial proliferation suggesting that host cell autophagy is benificial for L. pneumophila. In order to decipher the molecular determinants involved in the interaction with autophagy, we identified common effectors secreted by the type IV secretion system between L. pneumophila and Coxiella burnetii, a bacterium from the order Legionellale responsible for Q fever and known to stimulate and hijack host cell autophagy. Mutant of these common effectors in L. pneumophila were analysed. While, none of them seems to be implicated in autophagy modulation, this study suggests other functions for these conserved effectors

Since their first discovery in the 19th century, microsporidian species have been found to be successful obligate intracellular parasites capable of infecting a wide variety of hosts including economically and ecologically important organisms as well as model organisms for biomedical research. Recently, cases of infection of the widely used animal model Danio rerio, commonly known as the zebrafish, by the newly described microsporidium Pseudoloma neurophilia have been reported in an increasing number of research facilities. Current knowledge of the biology of this parasite found in 75% of the Zebrafish Resource Center facilities is limited to microscopic analyses on its lifecycle as well as its physical, behavioral and psychic impact on its hosts. Despite the growing relevance of this parasite in biomedical research no current data is available on its genome. In this dissertation, I provide additional knowledge on the basic biology of P. neurophilia by acquiring and exploring the content and structure of the first genome draft of the zebrafish parasite. My findings reveal that the 5.25 Mb genome of P. neurophilia harbors an unusually high amount of transposable elements as well as numerous inserts found in coding regions typically conserved in microsporidia and other organisms. This peculiar obligate parasite demonstrates strong phylogenetic and genetic relationships with other fish-mosquito microsporidia. Similar to what is observed in closely related species, intra-genomic analyses of P. neurophilia’s genome suggest that it is diploid and possesses a large repertoire of over a thousand putative genes unique to this specie. Overall, my findings provide new insights into the basic biology of this parasite and represent a milestone in the understanding of P. neurophilia and D. rerio host-parasite interaction and ultimately in the development of treatments against this parasite that has been infecting the zebrafish research industry for the past decades.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21311号 / 農博第2296号 / 新制||農||1064(附属図書館) / 学位論文||H30||N5145(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Maize (Zea mays) is globally considered as an important cereal crop, and a major staple food in developing countries such as Africa (WARD et al. 1999). In South Africa, maize is considered the most important grain crop as it is the main feed grain used for animals and a staple food for the population (FAO 2012). Maize can also be used for the production of maize-based ethanol, which can be used as a bio-fuel. In the USA, approximately 40% (11 million tonnes) of maize produced in 2012 was used for the production of bio-fuel (FAO 2012). Maize production in Africa was estimated to be less than two and on average 1.4 tons per hectare and remains below world average (FAO 2012). It was expected that South African crop production would decrease by approximately six percent during the 2012/2013 growing season as droughts during February and March 2013 in the North West and Free State provinces led to below-average maize yields in these production areas (FAO 2012; USDA 2013). Over the last few years maize production output has not been increasing together with the increasing population growth rate and thus puts pressure on commercial farmers to produce more maize for food security purposes and economical growth. The FAO states that agricultural production still needs to increase by up to 60% (80% in developing countries) within the next four years to be able to cope with an estimated global population growth of 39% by 2050. / Dissertation (MSc)--University of Pretoria, 2014. / National Research Foundation (NRF) / Plant Science / MSc / Unrestricted

Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine transcriptomic data of the infection between Fo47 and A. thaliana for putative effectors from the non-pathogenic isolate using a pipeline that is validated by identifying known effectors in the interaction between Fo5176 and A. thaliana. Phenotypic characterization of A. thaliana plants inoculated with Fo47 or Fo5176 has revealed a significant increase in rosette biomass of Fo47 inoculated plants when compared to mock (sterile water) inoculated plants. As is observed in other systems, treatment of plants with Fo47 prior to challenging with pathogenic Fo significantly reduces the disease severity over time. The results of this study suggest that Fo47 is a possible biocontrol agent against Fo5176, and that inoculation with non-pathogenic Fo alters the physiology of A. thaliana such that it has a higher rosette biomass without alterations to the water status of the plant. Our pipeline for extracting putative effectors using transcriptomic data as a critical filter generated 13 candidate genes for further experimentation to determine their role in the Fo47-A. thaliana interaction. This research reports the first known observation that Fo47 increases the shoot biomass of the host plant it is interacting with, and that the model plant A. thaliana can be used as a host to examine the spectrum of interactions capable within the Fusarium oxysporum species complex.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast caused by the ascomycetous fungus Magnaporthe oryzae remains a threat to global sustainable agriculture and food security. This pathogen infects staple cereal crops such as rice, wheat, barley and millets, as well as turf grasses, in a distinct way among fungal plant pathogens, which we described in the first chapter. In addition to economical importance, rice blast is a model pathosystem for difficult-to-study biotrophic fungi and fungal-plant interactions. We are studying proteins that fungi secrete inside living cells to block plant defenses and control host cell processes; these proteins are called effectors. To date mechanisms for secretion and delivery of effectors inside host cells during disease establishment remain unknown. This step is critical to ensure the successful infection. So far, the only commonality found among all unique small-secreted blast effector proteins is their accumulation in a novel in planta structure called the biotrophic-interfacial complex (BIC). Identifying effectors and understanding how they function inside rice cells are important for attaining durable disease control. In the second chapter, we presented one approach to address this challenge. We characterized four candidate effector genes that were highly expressed specifically during the rice cell invasion. Using transgenic fungi that secrete fluorescently-labeled versions of each protein allowed me to follow them during invasion in vivo by live cell imaging. These candidates show distinct secretion patterns suggesting a spatially-segregated secretion mechanism for effectors. Results revealed a BIC-located strong candidate cytoplasmic blast effector, two putative cell-to-cell movement proteins and a putative extrainvasive hyphal membrane (EIHM)-matrix protein, which has become a valuable tool for assessing successful infection sites. In the third chapter, we test if normal secretion components of filamentous fungi are involved in accumulation of effectors into BICs. We report localization studies with M. oryzae orthologs of conserved secretion machinery components to investigate secretion mechanisms for effectors showing preferential BIC accumulation and for non-BIC proteins such as BAS4. Especially bright fluorescence adjacent to BICs from Mlc1p (Myosin Light Chain, a Spitzenkörper marker), from Snc1p (a secretory vesicle marker), and from Yup1p (a putative t-SNARE endosomal protein) suggest secretion actively occurs in the BIC-associated cells. Localization of Spa2p (a polarisome marker), as a distinct spot at the tips of the bulbous invasive hyphae (IH) in planta, suggests the existence of two secretion complexes after the fungus switches growth from the polarized filamentous primary hyphae to bulbous IH. In the final chapter on future perspectives, we present some strategies towards the molecular understanding of the M. oryzae secretion mechanism during biotrophic invasion, which will lead to novel strategies for disease control.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.