京都大学 / 新制・課程博士 / 博士(医学) / 甲第24475号 / 医博第4917号 / 新制||医||1062(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ziel der Arbeit war es, die regulatorischen Mechanismen von Virulenz-assoziierten Genen in den Pathogenen Francisella novicida und Streptococcus pyogenes zu untersuchen. Kapitel eins befasst sich mit der Regulation des Virulenzfaktors Streptolysin S (SLS) von S. pyogenes. Wir untersuchten die Rolle der Ribonuklease (RNase) Y in der transkriptionellen und posttranstrikptionellen Regulation des Gens sagA. RNase Y begünstigte die Produktion einer kleinen RNA (sRNA) vom sagA Transkript, war jedoch nicht an der posttranskriptionellen Regulierung der sagA RNA beteiligt. Dennoch förderte RNase Y die Transkription von sagA indirekt. Wir konnten weiterhin zeigen, dass die 5′- untranslatierte Region (UTR) der sgaA RNA eine Sekundärstruktur besitzt, die möglicherweise einen Liganden bindet und damit die Zugänglichkeit der ribosomalen Bindungsstelle beeinflusst. Die Deletion einzelner Abschnitte der 5′ UTR hat einen negativen Effekt auf die sagA Expression. Wir haben eine Methode entwickelt um die Aktivität von Riboswitches, (u.a. die sagA 5‘ UTR) zu analysieren und konnten damit drei putative Riboswitches in S. pyogenes validieren. In Kapitel zwei charakterisierten wir den Mechanismus mit dem CRISPR-Cas9 aus F. novicida (FnoCas9) die Expression bakterieller Lipoproteine (BLPs) unterdrückt, um dem Immunsystem des Wirtes zu entgehen. Wir zeigen, dass FnoCas9 eine duale Funktion besitzt, die es dem Protein ermöglicht nicht nur DNA zu schneiden, sondern auch Transkription zu regulieren. In dieser erstmals beschriebenen Aktivität bindet FnoCas9 an den tracrRNA:scaRNA Duplex, wodurch der Protein-RNA Komplex an einen DNA Abschnitt hinter dem Promoter der blp Gene bindet und somit deren Transkription verhindert. Diese Bindungsstelle besitzt ein protospacer-adjacent motif (PAM) und eine scaRNA-komplementäre Sequenz, an die der FnoCas9-RNA Komplex bindet, allerdings nicht schneidet. Dieses System könnte in Zukunft das Repertoire an CRISPR-basierten Anwendungsmöglichkeiten erweitern. / The aim of this thesis was to study regulatory mechanisms of virulence-related genes in the bacterial pathogens Francicella novicida and Streptococcus pyogenes. Chapter one focuses on the regulation of the virulence factor streptolysin S (SLS) in S. pyogenes. First, we investigated the role of the ribonuclease (RNase) Y in the transcriptional and post-transcriptional regulation of SLS-coding gene, sagA. We found that RNase Y promotes the production of a small RNA (sRNA) from the sagA transcript but we observed no regulation at the post-transcriptional level. Yet, RNase Y promotes sagA transcription indirectly and affects hemolysis levels. We next showed that the sagA 5′ untranslated region (UTR) contains a secondary structure that is is possibly modulated by direct binding to a ligand and may affect the accessibility to the ribosomal binding site (RBS). Our results indicate that removing fragments of the 5′ UTR has a negative effect on sagA expression. We developed a method for testing the activity of putative riboswitches, including sagA 5′ UTR. Using this method, we validated three predicted riboswitches in S. pyogenes. In chapter two, we characterized the mechanism by which F. novicida CRISPR-Cas9 (FnoCas9) represses the expression of bacterial lipoproteins (BLPs), allowing evasion of the host immune system. We show that FnoCas9 is a dual-function protein that, in addition to its canonical DNA nuclease activity, evolved the ability to regulate transcription. In this newly-described mechanism, the non-canonical RNA duplex tracrRNA:scaRNA guides FnoCas9 to the DNA target located downstream of the promoter of the BLP-coding genes, causing transcriptional interference. The endogenous targets contain a protospacer-adjacent motif (PAM) and a sequence that is complementary to scaRNA, promoting FnoCas9 binding but not DNA cleavage. Engineering this system expands the toolbox of CRISPR applications by allowing repressing other genes of interest.

Bacillus amyloliquefaciens FZB42 ist ein grampositives Bodenbakterium. Es kann in der Rhizosphäre das Wachstum von Pflanzen fördern und durch die Produktion von Sekundärmetaboliten phytopathogene Organismen hemmen. Aus der Genomanalyse und den dazugehörigen Arbeiten war bekannt, dass Bacillus amyloliquefaciens FZB42 nicht-ribosomal je drei antimikrobielle Polyketide und Lipopeptide herstellt, sowie zwei Siderophore und das Dipeptid Bacilysin. Für Bacillus typische Lantibiotika oder große Bacteriocine wurden nicht gefunden. In dieser Arbeit wird erstmalig gezeigt, dass Bacillus amyloliquefaciens FZB42 auf ribosomale Weise antibakterielle Peptide herstellt. Zwei bisher unbekannte Bacteriocine, Amylocyclicin A und Plantazolicin, und deren dazugehörigen Gencluster konnten identifiziert und charakterisiert werden. Amylocyclicin A ist ein unmodifiziertes Peptid, dessen N- und C-Terminus kovalent verbunden sind. Es wurde der Gruppe I der zirkulären Bacteriocine zugeordnet, dessen Mitglieder sich durch schwache Homologie untereinander, aber durch wahrscheinlich ähnliche 3D-Strukturen auszeichnen. Die Masse beträgt 6381 Da und die Substanz ist stark aktiv gegen grampositive Bakterien. Das Biosynthesecluster umfasst sechs Gene für die Synthese, den Export, die Zyklisierung und die Immunität. Plantazolicin ist ein hydrophobes, stark modifiziertes Peptid aus der TOMM-Gruppe, einer Gruppe aus Microcin B17-ähnlichen Peptiden, die nach neueren Erkenntnissen verbreiteter ist, als bisher bekannt. Plantazolicin ist schwach aktiv gegen grampositive Bakterien und besitzt die Masse 1335 Da. Das Biosynthesecluster umfasst zwölf Gene, mit allen nötigen Genen für Synthese, Modifikation, Regulation, Immunität und Export. / Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Five gene clusters direct the non-ribosomal synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide and the iron-siderophore bacillibactin. Three gene clusters direct the non-ribosomal synthesis of the antibacterial acting polyketides macrolactin, bacillaene and difficidin; in addition to the non-ribosomal synthesis of the antibacterial dipeptide bacilysin. Genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Only two incomplete gene clusters directing immunity against mersacidin and subtilin were found. In this work two ribosomally synthesized antibacterial peptides, amylocyclicin A and plantazolicin, and their corresponding gene clusters were identified. Amylocyclicin A is a circular peptide with a mass of 6381 Da and strong activity against Gram-positive bacteria. Six genes are responsible for the synthesis, maturation, export and immunity of this peptide belonging to group I of circular bacteriocins. Plantazolicin is a strongly modified hydrophobic peptide bearing a molecular mass of 1,335 Da and displaying antibacterial activity toward closely related Gram-positive bacteria. Essential modification contains the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide and addition of two methyl groups. Twelve genes are responsible for synthesis, modification, export and immunity of this peptide belonging to the TOMM group of thiazol/oxazol modified microcins.

Streptokinase is clinically used as a thrombolytic agent for the treatment of patients with acute myocardial infarction and venous and arterial thrombosis. In the present work, a substrate chromogenic assay was validated for the potency evaluation of biopharmaceutical formulations. Method validation investigated parameters such as the linearity (r²=0.9988) intra- and inter- day precision, accuracy and robustness; the method yielded good results with a quantitation limit of 2.50 IU/mL and a detection limit of 1.10 IU/mL. The biological assay was carried out by chromogenic end point giving potencies between 98.42% and 108.97%. The results demonstrated the validity of the chromogenic assay for the potency assessment of streptokinase in biopharmaceutical formulations. Besides, the activities of streptodornase and streptolysin were evaluated in the same samples by optimized assays based on the biological activity, giving results according to the Pharmacopoeial specifications. The combination of assays is necessary to assure the quality, and to improve the methods that can be applied for the characterization of streptokinase, by ensuring batch-to-batch consistency of the bulk and finished biopharmaceutical products. / A estreptoquinase é clinicamente utilizada como agente trombolítico para o tratamento de pacientes com infarto agudo do miocárdio e trombose arterial e venosa. Neste trabalho, validouse o ensaio com substrato cromogênico para a determinação de potência de formulações biofarmacêuticas. O método foi validado, avaliando-se os parâmetros de linearidade (r² = 0,9988), precisão intra e inter-dias, exatidão e robustez; o método mostrou resultados adequados, os quais cumpriram os requisitos preconizados, com limite de quantificação de 2,50 UI/mL e limite de detecção de 1,10 UI/mL. O ensaio biológico foi realizado pelo método de ponto final cromogênico, fornecendo potências entre 98,42% e 108,97%. Os resultados demonstraram a validade do ensaio cromogênico para a determinação de potência de estreptoquinase nas formulações biofarmacêuticas. Além disto, as atividades de estreptodornase e estreptolisina foram avaliadas nas mesmas amostras, por métodos otimizados baseados na atividade biológica, dando resultados de acordo com as especificações farmacopeicas. A combinação de ensaios é necessária para assegurar a qualidade e aprimorar os métodos que podem ser aplicados para a caracterização de estreptoquinase, assegurando a consistência entre lotes de soluções concentradas e de produtos biofarmacêuticos acabados.

Mast cells are key players of the innate immune system due to their location at the interface of host and pathogen encounters, such as on mucosal surfaces or the skin. Secreting a variety of compounds, they communicate with other immune cells in a highly specific manner. Subsequently, reinforcements against foreign invaders are recruited while also defending the host, using bacteriolytic effector molecules. One type of pathogens which are competent challengers of the host’s immune system are Streptococci, causing a burden for humans and animals. Streptococcus equi subspecies equi is one example, a highly contagious horse pathogen with a silent carrier subset, causing “strangles”, a disease resulting in equine morbidity and mortality all over the world. The present study aimed to explore the virulence factors in S. equi responsible for immune system activation, represented by mast cells. Knockout mutants of the genes aroB, hasA, pyrC, recA, sagA and a combination of those, including a deletion strain of all superantigens (seeHILM), were co-cultivated with murine bone-marrow-derived mast cells (BMMCs). Mast cells alone and S. equi strain 4047 (wild-type) were used as controls. It was shown that 4 h after encounter of the bacteria, BMMCs responded with IL-6, TNF-α and MCP-1 secretion, indicating an inflammatory response to all strains except against the sagA mutant (ΔsagA) or the multi-deletion strain, the latter lacking several virulence factors including sagA. These results were confirmed at the mRNA-level where IL-6, TNF-α and Nr4a3 gene expression was significantly upregulated in BMMCs after 4 h incubation with wild-type S. equi. In contrast, when BMMCs were co-cultivated with sagA-deficient S. equi, no detectable upregulation was seen. These results were further confirmed in peritoneal-derived mast cells. After 24 h no secretion of cytokines was detected in response to ΔsagA mutants, in contrast to the strong cytokine output in response to wild-type S. equi. To elucidate the role of SagA, the precursor of streptolysin S (SLS), lysis was determined, and it was observed that ΔsagA does not lyse mast cells in contrast to wild-type with intact SLS. Transwell-based experiments indicated a partially contact-dependent response of mast cells to bacteria. Taken together, this study shows for the first time that SLS is the major mast cell activator produced by S. equi. I suggest the possible mechanism of cell death by lysis and reprogrammed signaling pathways of the host by sublytic concentrations of SLS, resulting in damage associated pattern-mediated signaling as well as auto- and paracrine amplification by inflammatory cytokines and other messenger molecules.