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Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia trachomatis is an obligate intracellular bacterial pathogen that can infect the eyes, genital tract, and disseminate to lymph nodes in humans. Many C. trachomatis infections are clinically asymptomatic and can become chronic if left untreated. When humans are infected with C. trachomatis, a cytokine that is produced is interferon-gamma (IFN-γ). In vitro, IFN-γ stimulates expression of the host enzyme indoleamine 2,3-dioxygenase. This enzyme converts free intracellular tryptophan to N-formylkynurenine. Tryptophan starvation induces C. trachomatis to enter a viable-but-nonculturable state termed persistence, which has been proposed to play a key role in chronic Chlamydial disease. To circumvent host induced tryptophan depletion, urogenital strains of C. trachomatis encode a functional tryptophan synthase (TS). TS synthesizes tryptophan from indole and serine, allowing Chlamydia to reactivate from persistence. Transcriptomic analysis revealed C. trachomatis differentially regulates hundreds of genes in response to tryptophan starvation. However, genes that mediate entry, survival, and reactivation from persistence remain largely unknown. Using a forward genetic screen, we identified six Susceptible to IFN-γ mediated Persistence (Sip) mutants that have diminished capacities to reactivate from persistence with indole. Mapping the deleterious persistence alleles in three of the Sip mutants revealed that only one of the mutants had a mutation in TS. The two other Sip mutants mapped had mutations in CTL0225, a putative integral membrane protein, and CTL0694, a putative oxidoreductase. Neither of these genes plays a known role in tryptophan synthesis. However, amino acid (AA) competitive inhibition assays suggest that CTL0225 may be involved in the transport of leucine, isoleucine, valine, cysteine, alanine, and serine. Additionally, metabolomics analysis indicates that all free amino acids are depleted in response to IFN-γ, making this amino acid transporter essential during persistence. Taken together we have identified two new chlamydial persistence genes that may play a role in chronic chlamydial disease.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25333号 / 農博第2599号 / 新制||農||1106(附属図書館) / 学位論文||R6||N5505 / DFAM / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Das Enzym Tryptophan Synthase ist ein ausgezeichnetes Beispiel einer molekularen Fabrik auf der Nanoskala mit zwei katalytischen Zentren. Der katalytische Zyklus des Moleküls beruht zudem auf zahlreichen allosterischen Wechselwirkungen sowie der Übertragung des Intermediats Indol durch einen intramolekularen Tunnel. In dieser Arbeit wird das erste kinetische Modell eines einzelnen Tryptophan Synthase Moleküls konstruiert und analysiert. Simulationen zeigen starke Korrelationen zwischen den Zuständen der Katalysezentren sowie die Ausbildung von Synchronisation. Mit stochastischer Thermodynamik wird die experimentell unzugängliche Reaktionskonstante für die Rückübertragung des Indols aus Messdaten rekonstuiert. Methoden, die den Informationsaustausch in bipartiten Markovnetzwerken charakterisieren, werden auf beliebige Markovnetzwerke verallgemeinert und auf das Modell angewendet. Der abschließende Teil befasst sich mit chemischen Reaktionsnetzwerken von Metaboliten und Enzymen. Es werden algebraische Modelle (Halbgruppen) konstruiert, welche aufeinanderfolgende und simultane katalytische Funktionen von Enzymen und von Unternetzwerken erfassen. Diese Funktionen werden genutzt, um eine natürliche Dynamikum sowie hinreichende und notwendige Bedingungen für seine Selbsterhaltung zu formulieren. Anschließend werden die algebraischen Modelle dazu genutzt, um eine Korrespondenz zwischen Halbgruppenkongruenzen und Skalenübergängen auf den Reaktionsnetzwerken herzustellen. Insbesondere wird eine Art von Kongruenzen erörtert, welche dem Ausspuren der globalen Struktur des Netzwerkes unter vollständiger Beibehaltung seiner lokalen Komponenten entspicht. Während klassische Techniken eine bestimmte lokale Komponente fixieren und sämtliche Informationen über ihre Umgebung ausspuren, sind bei dem algebraischen Verfahren alle lokalen Komponenten zugleich sichtbar und eine Verknüpfung von Funktionen aus verschiedenen Komponenten ist problemlos möglich. / The channeling enzyme tryptophan synthase provides a paradigmatic example of a chemical nanomachine with two distinct catalytic subunits. It catalyzes the biosynthesis of tryptophan, whereby the catalytic activity in a subunit is enhanced or inhibited depending on the state of the other subunit, gates control the accessibility of the reactive sites and the intermediate product indole is directly channeled within the protein. The first single-molecule kinetic model of the enzyme is constructed. Simulations reveal strong correlations in the states of the active centers and the emergent synchronization. Thermodynamic data is used to calculate the rate constant for the reverse indole channeling. Using the fully reversible single-molecule model, the stochastic thermodynamics of the enzyme is closely examined. The current methods describing information exchange in bipartite systems are extended to arbitrary Markov networks and applied to the kinetic model. They allow the characterization of the information exchange between the subunits resulting from allosteric cross-regulations and channeling. The final part of this work is focused on chemical reaction networks of metabolites and enzymes. Algebraic semigroup models are constructed based on a formalism that emphasizes the catalytic function of reactants within the network. A correspondence between coarse-graining procedures and semigroup congruences respecting the functional structure is established. A family of congruences that leads to a rather unusual coarse-graining is analyzed: The network is covered with local patches in a way that the local information on the network is fully retained, but the environment of each patch is not resolved. Whereas classical coarse-graining procedures would fix a particular patch and delete information about the environment, the algebraic approach keeps the structure of all local patches and allows the interaction of functions within distinct patches.

The growing prevalence of antibiotic-resistant bacteria is a global health crisis that threatens the effectiveness of antibiotics in medical treatment. Increases in the number of antibiotic-resistant bacteria and a drop in the pharmaceutical development of novel antibiotics have combined to form a situation that is rapidly increasing the likelihood of a post-antibiotic era. The development of antibiotics with novel enzymatic targets is critical to stall this growing crisis. In silico methods of molecular modeling and drug design were utilized in the development of novel tryptamine analogs as potential antibiotics through their inhibition of the bacterial enzyme tryptophan synthase. Following the creation of novel tryptamine analogs, the molecules were analyzed in silico to determine their binding affinity to human MAOB and the E. coli α-subunit, E. coli β2-dimer and the M. tuberculosis β2-dimer of tryptophan synthase. Ten tryptamine analogs displayed significant increases in tryptophan synthase binding affinity and show promise as potential antibiotics and antibiotic adjuvants. Further in silico modeling determined that the binding sites of the tryptamine analogs were similar to wild-type tryptamine in the E. coli β2-dimer, the M. tuberculosis β2-dimer and human MAOB, while the analogs’ binding sites to the E. coli α-subunit differed. Although no tryptamine analogs increased tryptophan synthase binding affinity while decreasing human MAOB binding affinity, related increases in MAOB binding affinity warrants further research into the analogs’ potentials as MAO inhibitors. Given the increases in tryptophan synthase binding affinity and similar β2-dimer binding sites, a provisional patent was filed on the ten identified tryptamine analogs. Moving forward, we recommend the synthesis of the ten identified tryptamine analogs. Following synthesis, further research should be conducted to determine the in vitro and in vivo antibiotic properties of the ten tryptamine analogs.