京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17813号 / 医博第3811号 / 新制||医||999(附属図書館) / 30628 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 武藤 学, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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<p>Coronaviruses are the largest known RNA viruses and infect a wide range of hosts. Human coronaviruses traditionally have been known to be the cause of the common cold and have been vastly understudied due to low morbidity and mortality. The emergence of SARS-CoV and MERS-CoV has altered the landscape of coronavirus research and proven the deadly capabilities of human coronaviruses. With two recent zoonotic events, it is increasingly important to understand the molecular biology of human coronaviruses. The coronavirus nucleocapsid protein is an essential structural protein that complexes with the viral genome. Though nucleocapsid formation is the protein’s major role, it has also been found to have other functions and effects during infection. The following research aimed to examine how the human coronavirus nucleocapsid protein affects the innate immune response <em>in vitro</em>. Modulation of the type I interferon response by the nucleocapsid was first investigated and the nucleocapsids were shown to have the ability to block interferon signalling. Additionally, the nucleocapsid protein was found to cause a dysregulation of transcription factor NFKB1. We propose a novel mechanism of this NFKB1 negative regulation interference. Taken together, we have further characterized the significant role of the coronavirus nucleocapsid protein in innate immune evasion.</p> / Doctor of Philosophy (Medical Science)

Herpes simplex virus (HSV) infections are common in all human populations and for most people they represent relatively mild lifelong infections. To facilitate the persistent infection of hosts, HSV has evolved immune evasion strategies which suppress various aspects of the immune response including the actions of complement and antibodies. Previously in our laboratory, an HSV immediate early protein called ICP47 was shown to inhibit the MHC class I antigen presentation pathway and thereby block recognition of virus infected cells by CD8+ cytotoxic T lymphocytes (CTL). This thesis explores the potential cellular targets of ICP47. Using immunoprecipitation I found ICP47 associates with the transporter associated with antigen presentation (TAP). By blocking the transport of peptide antigens into the endoplasmic reticulum, MHC class I molecules become unstable and are subsequently degraded before displaying HSV antigens on the cell surface. Thus, CTL destruction of cells infected with HSV is blocked. In addition, an interaction between an ICP47 bacterial fusion protein, called GSTICP47-1 and a cellular protein, calcyclin, was examined. The functions of calcyclin are largely unknown. However, based on its association with ICP47, it was possible that calcyclin might play a role in the class I pathway -perhaps as the peptide shuttle. Nevertheless, the results of several experiments were consistent with the notion that calcyclin and ICP47 may not interact in vivo and that calcyclin may not play a role in the MHC class I antigen presentation pathway. / Thesis / Master of Science (MS)

The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.

Vibrio splendidus LGP32 est une bactérie pathogène associée aux épisodes de mortalités estivales qui affectent la production d'huître Crassostrea gigas depuis des décennies. Nous avons montré ici que la porine OmpU était un effecteur majeur de l'interaction V. splendidus / C. gigas. Nous avons pour cela construit un mutant ΔompU de V. splendidus. Celui-ci nous a permis de mo ntrer l'implication de OmpU (i) dans la résistance de V. splendidus aux antimicrobiens, incluant ceux de l'huître, (ii) dans la « fitness » chez l'huître, et (iii) dans la virulence en infections expérimentales (mortalités de 56 % pour le sauvage versus pour le 11% mutant). En accord avec ces résultats, nous avons montré que la délétion de ompU modifiait la sécrétion de protéines dont l'expression est contrôlée par les voies de régulation de la virulence (ToxR) et de l'intégrité membranaire (SigmaE). Par ailleurs, nous avons montré que OmpU jouait un rôle essentiel dans la reconnaissance par les hémocytes. En effet, (i) in vivo, les gènes hémocytaires répondent différemment à l'infection par le Vibrio sauvage ou ΔompU, et (ii) in vitro, OmpU est nécessaire à l'invasion hémocytaire par V. splendidus. Cette invasion utilise la phagocytose dépendante de l'intégrine b et la SOD extracellulaire du plasma d'huître comme opsonine qui lie OmpU. Ainsi, OmpU est un facteur de virulence majeur qui permet l'infection des hémocytes dans lesquels il est capable de survivre en inhibant la formation de radicaux oxygénés et de vacuoles acides. La résistance du Vibrio aux antimicrobiens hémocytaires de l'huître, elle-même dépendante de OmpU, est probablement un élément supplémentaire favorable à la survie intra-cellulaire. / Vibrio splendidus LGP32 is a bacterial pathogen associated to the summer mortality outbreaks that have affected the production of Crassostrea gigas oysters over the past decades. We showed here that the OmpU porin is a major effector of the V. splendidus / C. gigas interaction. For that, we have constructed a ΔompU mutant of V. splendidus, and shown that the OmpU porin is implicated (i) in the resistance of V. splendidus to antimicrobials, including those of oyster, (ii) in its in vivo fitness, and (iii) in its virulence in oyster experimental infections (mortalities have been reduced from 56 % to 11 % upon mutation). In agreement, we have shown that the ompU deletion modified the expression of secreted proteins controlled by the virulence (ToxR) and the membrane integrity (SigmaE) regulation pathways. Furthermore, we have shown that OmpU has a major role in the recognition of V. splendidus by oyster hemocytes. Indeed, (i) in vivo, hemocyt e genes displayed differential responses to an infection with the wild-type or the ΔompU mutant, and (ii) in vitro, OmpU was necessary for hemocyte invasion by V. splendidus. This invasion process required the hemocyte b-integrin and the oyster plasma extracellular SOD, which was found to act as an opsonin recognizing OmpU. Thus, OmpU is a major virulence factor that allows infection of hemocytes in which V. splendidus is able to survive by inhibiting the production of reactive oxygen species and the formation of acidic vacuoles. Resistance of V. splendidus to hemocyte antimicrobials, which is also OmpU-dependant, is probably an additional determinant of V. splendidus intracellular survival.

Le parasitisme est un des principaux problèmes dans les élevages des ruminants. Les nématodes parasites du tractus digestif des ovins et caprins sont responsables d’importantes baisses de rendement. La maîtrise de ces parasitoses a été longtemps basée sur l’utilisation de molécules anthelminthiques. Cependant, l’efficacité des traitements est fréquemment remise en cause par l’émergence d’isolats résistants à une ou plusieurs de ces molécules. Dans ce contexte, une meilleure connaissance des mécanismes impliqués dans l’installation et la survie des parasites dans leur hôte est essentielle pour le développement de méthodes de lutte efficaces. Les P-glycoprotéines sont des pompes membranaires de la superfamille des transporteurs ABC. Ces pompes transportent des molécules très variées qui ont en commun leur caractère hydrophobe. Nous avons émis l’hypothèse de l’implication de ces transporteurs dans l’interaction hôte-parasite. Dans le contexte de ce travail nous avons identifié des séquences partielles ou complètes d’ADNc de 9 Pgps du nématode parasite Haemonchus contortus. Une forte activation des Pgps des nématodes en présence des produits de dégranulation des éosinophiles de l’hôte a été observée, démontrant ainsi l’interaction entre les Pgps des nématodes et les produits issus de l’hôte. De plus, l’exposition in vitro des nématodes parasites aux produits de l’hôte montrent après analyse par PCR quantitative une induction significative de l’expression de deux Pgps (Hco-pgp-3, et Hco-pgp-16). Chez le nématode murin Heligmosomoides polygyrus bakeri 5 Pgps ont été identifiés. D’autre part, l’analyse du niveau d’expression des Pgps d’H. bakeri a permis de montrer que le gène Hba-pgp-2 est exprimé uniquement chez les stades en contact avec les produits de l’hôte (oeufs, L4 et adultes). De plus, une induction spécifique d’Hba-pgp-2 par le cholestérol a été observée suggérant ainsi l’implication d’Hba-pgp-2 dans la capture et/ou la distribution des stérols des cellules de l’hôte indispensable aux nématodes. Ce travail constitue la première mise en évidence de l’interaction entre les Pgps des nématodes parasites et des produits issus de leur hôte. Ces résultats constituent une base solide pour le développement d’une méthode efficace permettant de bloquer ces transporteurs et d’éliminer les nématodes parasites. / Gastrointestinal nematodes cause significant economic loses in goat and sheep livestocks. Control of these parasites is mainly based on anthelmintic treatments. However, the efficacy of these molecules is questioned by the emergence of isolates resistant to one or several antiparasitic drugs. In this context, a better understanding of the mechanisms involved in the nematode parasites establishment and survival in the host is essential for the development of an effective control methods. P-glycoproteins are membrane pumps belonging to the ABC transporter family. These pumps transport a wide range of hydrophobic molecules. In the present study, we hypothesized that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as P-glycoproteins (Pgps) may also be involved in the transport of host products. Using the sheep parasitic nematode Haemonchus contortus, we investigated the modulation and expression of parasite Pgps activity in response to host eosinophil granule products. These works allowed to identify nine partial or complete H. contortus Pgps. Using a rhodamine efflux assay, we provided functional evidence that host eosinophil granule products can activate Pgps from the parasite suggesting that granule products could act as potential modulators of the ABC transporters activity. We showed by quantitative RT-PCR that among nine different H. contortus Pgp genes; Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitical life stages suggesting a potential involvement of these Pgps during the host-parasite interaction. Using exsheated L3 larvae, we demonstrated that eosinophil granules induced in a dose response manner an overexpression of Hco-pgp-3 and the closely related Hco-pgp-16 gene highlighting the possible involvement of these Pgps in host product transport. . The mice parasitic nematode Heligmosomoides polygyrus bakeri was used for studying the involvement of Pgps in the cholesterol transport. These works allowed identifying five Pgps in H. bakeri. The analysis of the mRNA expression level of H. bakeri Pgps has shown that Hba-pgp-2 gene is expressed only in stages in contact with host products. In addition, a specific induction of Hba-pgp-2 by cholesterol was observed suggesting the involvement of Hba-pgp-2 in the capture and / or distribution of cholesterol from host cells. Taken together, our results provide the first evidence that a subset of helminth Pgps could be involved in the transport of host products. This opens the way for further studies aiming to explore the function of helminth Pgps in host-parasite interactions including host immune response evasion.

Mais de 25% das mortes em humanos são causadas por doenças infecciosas. Muitas dessas doenças são emergentes e de importância zoonótica. A leptospirose é considerada uma das mais importantes doenças zoonóticas emergentes. Sua distribuição global e seu potencial epidêmico constituem um problema de Saúde Pública. Estima-se que ocorram anualmente 1.03 milhão de casos e 58.900 mortes por leptospirose em todo o mundo, mas, em se tratando de uma doença negligenciada, a real prevalência da doença é subestimada. O Rattus norvegicus é o principal reservatório associado a epidemias urbanas. Leptospiras possuem a capacidade de aderir às células dos túbulos renais e interagem com muitos componentes da matriz extracelular do hospedeiro, o que facilita a invasão e colonização. Possuem também mecanismos de evasão ao sistema complemento do hospedeiro. A identificação destes mecanismos tem sido alvo de pesquisas desenvolvidas por vários grupos. Enolase e Gliceraldeído-3-fosfato desidrogenase (GAPDH) pertencem à categoria de proteínas conhecidas como proteínas moonlighting. Estas englobam um grupo de proteínas multifuncionais. Enolases são metaloenzimas citossólicas que catalisam a conversão de 2-D-fosfo-glicerato em fosfoenolpiruvato. Apesar de não possuírem sequência clássica de ancoragem à membrana, são encontradas na superfície de uma variedade de células eucarióticas e procarióticas, tendo a capacidade de interagir com plasminogênio. GAPDH é uma enzima da via glicolítica responsável pela conversão de gliceraldeído 3-fosfato em D glicerato 1,3-bifosfato. Estudos recentes mostram que a GAPDH tem múltiplas funções independentes do seu papel no metabolismo de energia. Neste trabalho demonstrou-se que GAPDH de Leptospira está localizada na superfície da bactéria, e que tanto GAPDH como enolase interagem com plasminogênio, o qual é convertido em sua forma ativa, a plasmina, na presença do ativador exógeno uPA. A capacidade da plasmina gerada sobre a enolase de clivar substratos fisiológicos foi testada. A cadeia &#946; do fibrinogênio foi totalmente degradada, e a molécula vitronectina parcialmente clivada em fragmentos de 61- 64 kDa. Ainda, mostrou-se que a enolase interage com os reguladores do complemento C4BP e FH. Ambos os reguladores permanecem funcionais, agindo como co-fatores de Fator I na degradação de C3b (FH) e C4b (C4BP). No que diz respeito à GAPDH, os dados claramente mostram que a plasmina ligada à GAPDH degrada as cadeias &#945; e &#946; do fibrinogênio, assim como a isoforma de 75 kDa da vitronectina, de forma tempodependente. Ainda, na presença de GAPDH, a plasmina degradou totalmente a cadeia &#945; de C5, mas não degradou C3b. Por fim, resultados obtidos por Far Western Blot revelam que GAPDH interage com C1q, molécula-chave da via clássica do sistema complemento, e também com fibronectina plasmática, podendo, portanto, contribuir para a adesão da bactéria durante a colonização do hospedeiro. Em suma, no presente estudo caracterizamos duas novas proteínas moonlighting de Leptospira: enolase e GAPDH. A caracterização funcional destas proteínas, assim como a identificação das moléculas-alvo do hospedeiro com as quais essas proteínas são capazes de interagir, podem contribuir para a compreensão dos mecanismos de invasão, disseminação e evasão imune utilizados por leptospiras patogênicas. / More than 25% of human deaths are caused by infectious diseases, among which a large number are emerging and of zoonotic importance. Leptospirosis is considered one of the most important emerging zoonotic diseases. Its global distribution and its epidemic potential constitute a Public Health problem. It is estimated that approximately 1,03 million cases and 58,900 deaths from leptospirosis occur annually worldwide, but as a neglected disease, its actual prevalence is underestimated. Rattus norvegicus is the main reservoir associated with urban epidemics. Leptospires have the capacity to adhere to renal tubule cells which facilitates invasion and colonization. They also have mechanisms to evade the host\'s complement system. The identification of these mechanisms has been the object of research developed by several groups. Enolase and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) belong to the category of proteins known as moonlighting proteins. These encompass a group of multifunctional proteins. Enolases are cytosolic metalloenzymes that catalyze the conversion of 2-D-phosphoglycerate to phosphoenolpyruvate. Although they do not have a classic membrane anchor sequence, they are found on the surface of a variety of eukaryotic and prokaryotic cells, and have the capacity to interact with plasminogen. GAPDH is an enzyme of the glycolytic pathway responsible for the conversion of glyceraldehyde 3-phosphate to D-glyceryl 1,3-bisphosphate. Recent studies show that GAPDH has multiple functions independent of its role in energy metabolism. In this work we demonstrated that Leptospira GAPDH is located on the surface of the bacterium, and that both GAPDH and enolase interact with plasminogen, which is converted into its active form, plasmin, in the presence of the exogenous activator uPA. The capacity of plasmin-bound enolase to cleave physiological substrates was tested. The &#946;-chain of fibrinogen was totally degraded, and vitronectin was partially cleaved into fragments of 61-64 kDa. Further, enolase interacts with the complement regulators C4BP and FH. Both regulators remain functional, acting as cofactors for Factor I on the degradation of C3b (FH) and C4b (C4BP). With regard to GAPDH, the date clearly show that plasmin bound to GAPDH degrades the &#945; and &#946; chains of fibrinogen as well as the 75-kDa isoform of vitronectin, in a time-dependent manner. Furthermore, in the presence of GAPDH, plasmin totally degraded C5 &#945;-chain, but did not degrade C3b. Finally, our Far Western Blot data reveal that GAPDH interacts with C1q, a key molecule of the classical pathway of the complement system, and also interacts with plasma fibronectin, and may therefore contribute to bacterial adhesion during host colonization. Briefly, in the present study we characterized two novel moonlighting proteins of Leptospira: enolase and GAPDH. The functional characterization of these proteins, as well as the identification of the host target molecules with which these proteins are capable of interacting, may contribute to the understanding of the mechanisms of invasion, dissemination and immune evasion used by pathogenic leptospires.

A leptospirose é causada por bactérias que pertencem ao gênero Leptospira. Em um estudo realizado por nosso grupo, observou-se que as proteases secretadas por leptospiras patogênicas foram capazes de clivar a molécula C3 do Complemento e seus fragmentos C3b e iC3b, além de Fator B, C4b e C2. Neste trabalho expandimos a análise da atividade proteolítica sobre os componentes do Complexo de Ataque à Membrana (MAC): C6, C7, C8 e C9. Nós observamos que essas proteases clivam todos os componentes do MAC inclusive o complexo solúvel formado e que essas clivagens ocorrem de modo tempo-dependente. Além disso, as clivagens dessas moléculas ocorrem de modo seletivo, pois mesmo utilizando quantidades reduzidas de sobrenadantes ainda foi possível observar produtos de clivagem. A atividade proteolítica foi inibida pela 1,10fenantrolina, indicando a participação de metaloproteases. O reconhecimento de quais moléculas do MAC são clivadas por proteases de leptospiras patogênicas pode contribuir para o desenvolvimento de estratégias terapêuticas na infecção por estes patógenos. / Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. In a previous study, our group observed that the proteases secreted by Pathogenic Leptospira were capable of cleaving C3 of Complement, as well as the fragments C3b and iC3b, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Membrane Attack Complex (MAC). We observed that the protease cleaves all MAC components including soluble complex formed and that these cleavages occur in a time-dependent manner and in a selective way, since even when reduced quantities of supernatants were used, the cleavage products were still observed. The proteolytic activity was inhibited by 1,10phenanthroline, indicating the participation of metalloproteinases. The recognition that MAC molecules are cleaved by proteases of pathogenic leptospires can contribute to the development of therapeutic strategies for the infection by these pathogens.

A leptospirose é uma zoonose causada por leptospiras patogênicas que possuem estratégias para driblar a ação do sistema complemento ao interagir com diversos reguladores negativos, como a vitronectina. Neste projeto, avaliou-se a interação de vitronectina com três proteínas de membrana de Leptospira: LigA, LigB e LcpA. Verificou-se que essas interações ocorrem nas porções C-terminais das proteínas e nos domínios de ligação com heparina da vitronectina. Essas proteínas também se ligam a C9 e são capazes de impedir a formação do poro in vitro e a formação do MAC em um ensaio clássico de hemólise. Essas proteínas de Leptospira interagem com diversos reguladores negativos do sistema complemento. Ensaios de competição demonstram que estes reguladores interagem simultaneamente com as proteínas através de sítios distintos. A caracterização funcional de proteínas e a descoberta de novos mecanismos utilizados por esse patógeno para sobreviver no hospedeiro podem auxiliar nossa compreensão no que diz respeito a aspectos relacionados à clínica e à prevenção da leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira that have strategies to escape the action of the complement system interacting with several negative regulators, such as vitronectin. In this project, we evaluated the interaction of vitronectin with three Leptospira membrane proteins: LigA, LigB and LcpA. It has been found that such interactions occur at the C-terminal portions of these proteins and the heparin binding domain of vitronectin. These proteins also bind to C9 and are capable of preventing the formation of the pore in vitro and the formation of MAC in a classical test of hemolysis. These proteins interact with various Leptospira negative regulators of the complement system. Competition assays demonstrated that both interact with these regulatory proteins through distinct sites. The functional characterization of these proteins and the discovery of new mechanisms used by this pathogen to survive in the host may help our understanding with regard to clinical aspects and prevention of leptospirosis.