京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17977号 / 医博第3841号 / 新制||医||1001(附属図書館) / 80821 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 稲垣 暢也, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As doenças tropicais têm um grande impacto sobre a saúde em países de baixa renda, estando relacionadas com condições de pobreza e desigualdade. A tripanossomíase africana é uma infecção parasitaria negligenciada incluída na agenda da Organização Mundial de Saúde. Esta enfermidade é causada pelo Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense, sendo transmitida pela mosca tsé-tsé (Glossina sp.) e geralmente fatal se não tratada. Os fármacos usados no seu tratamento são ineficazes, difíceis de administrar e causam severas reações adversas. Portanto, existe a necessidade do desenvolvimento de alternativas quimioterápicas eficazes e seguras. Assim, a enzima fosfoglicerato mutase (PGAM) surge como um importante alvo molecular. Esta enzima esta envolvida no metabolismo de glicose, sendo necessária para a viabilidade do parasito. Somado a isso, ela difere da enzima dos hospedeiros permitindo a identificação de inibidores específicos. Não obstante, esforços têm sido realizados para identificar inibidores da PGAM, bem como para elucidar sua estrutura e mecanismo de reação. Nosso propósito é obter o modelo de alta resolução desta macromolécula sem ligantes e conseqüentemente analisar a mudança de conformação que esta sofre ao se ligar ao seu substrato natural. A PGAM de Trypanosoma brucei obtida na expressão e purificação mostrou-se cataliticamente ativa nos ensaios cinéticos. Por experimentos de cromatografia de exclusão molecular observamos que a amostra purificada se comportava na forma de monômero. Dados de difração de raios-X foram coletados para cristais da macromolécula obtidos na ausência de ligantes. A estrutura cristalográfica foi resolvida a 2.3 Å, apresentando um dímero na unidade assimétrica. Ambas as moléculas do dímero estavam na forma livre e apresentava grande diferença conformacional se comparadas com as PGAMs de estruturas conhecidas que estão ligadas ao substrato ou produto natural. Por espalhamento de raios-X a baixos ângulos confirmamos que a enzima é monomérica em condições que mimetizam a fisiológica. A mudança conformacional induzida pelo ligante não afeta a topologia dos dois domínios da PGAM. Entretanto, há mudanças nos ângulos torcionais da cadeia principal dos laços que conectam os domínios da proteína. Além disso, o metal cobalto parece estar envolvido na estabilização da estrutura terciária da PGAM na conformação livre. Finalmente, este novo modelo estrutural pode contribuir para o esforço internacional de desenvolver fármacos tripanocidas / Tropical diseases represent a major burden on population health in low-incoming countries, being related to poverty and social disadvantage. African trypanosomiasis is a neglected parasitic infection on the agenda of World Health Organization. This disorder is caused by Trypanosoma brucei gambiensis and Trypanosoma brucei rhodesiensis, transmitted by the tsetse fly (Glossina sp.), and usually fatal if untreaded. The drugs used in the treatment are ineffective, difficult to administer, and cause severe adverse reactions. Therefore, there is a need to develop effective and safe chemotherapies. Thus, the enzyme phosphoglycerate mutase (PGAM) emerges as an important molecular target. This enzyme is involved in glucose metabolism, and is necessary for viability of the parasite. Moreover, it differs from the host enzyme allowing the identification of specific inhibitors. Nevertheless, efforts have been made in identifying PGAM inhibitors and to elucidate their structure and mechanism of reaction. Our purpose is to obtain the high resolution model of the macromolecule free from ligands and consequently to analyze the change in conformation that undergoes upon binding to its natural substrate. Trypanosoma brucei PGAM obtained in the expression and purification was shown to be catalytically active in the kinetics assays. In the size exclusion chromatography we observed that the purified sample behaves as a monomer. X-ray diffraction data were collected for crystals of the macromolecules obtained in the absence of ligands. The crystal structure was solved to 2.3 Å, showing a dimmer in the asymmetric unit. Both molecules of the dimmer were in free form, and had a large conformational difference compared with those of know PGAM structures that are connected to the natural substrate or product. Small angle X-ray scattering confirm that the enzyme is monomeric under conditions that mimic the physiological. Ligand-induced conformational change does not affect the topology of the two domains of the PGAM. However, there are changes in torsional angles of the main chain of the loops that connect the protein domains. Additionally, the metal cobalt seems to be involved in stabilizing the tertiary structure of PGAM in the free conformation. Finally, this new structural model may contribute to the international effort to develop trypanocidal drugs.

As doenças tropicais têm um grande impacto sobre a saúde em países de baixa renda, estando relacionadas com condições de pobreza e desigualdade. A tripanossomíase africana é uma infecção parasitaria negligenciada incluída na agenda da Organização Mundial de Saúde. Esta enfermidade é causada pelo Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense, sendo transmitida pela mosca tsé-tsé (Glossina sp.) e geralmente fatal se não tratada. Os fármacos usados no seu tratamento são ineficazes, difíceis de administrar e causam severas reações adversas. Portanto, existe a necessidade do desenvolvimento de alternativas quimioterápicas eficazes e seguras. Assim, a enzima fosfoglicerato mutase (PGAM) surge como um importante alvo molecular. Esta enzima esta envolvida no metabolismo de glicose, sendo necessária para a viabilidade do parasito. Somado a isso, ela difere da enzima dos hospedeiros permitindo a identificação de inibidores específicos. Não obstante, esforços têm sido realizados para identificar inibidores da PGAM, bem como para elucidar sua estrutura e mecanismo de reação. Nosso propósito é obter o modelo de alta resolução desta macromolécula sem ligantes e conseqüentemente analisar a mudança de conformação que esta sofre ao se ligar ao seu substrato natural. A PGAM de Trypanosoma brucei obtida na expressão e purificação mostrou-se cataliticamente ativa nos ensaios cinéticos. Por experimentos de cromatografia de exclusão molecular observamos que a amostra purificada se comportava na forma de monômero. Dados de difração de raios-X foram coletados para cristais da macromolécula obtidos na ausência de ligantes. A estrutura cristalográfica foi resolvida a 2.3 Å, apresentando um dímero na unidade assimétrica. Ambas as moléculas do dímero estavam na forma livre e apresentava grande diferença conformacional se comparadas com as PGAMs de estruturas conhecidas que estão ligadas ao substrato ou produto natural. Por espalhamento de raios-X a baixos ângulos confirmamos que a enzima é monomérica em condições que mimetizam a fisiológica. A mudança conformacional induzida pelo ligante não afeta a topologia dos dois domínios da PGAM. Entretanto, há mudanças nos ângulos torcionais da cadeia principal dos laços que conectam os domínios da proteína. Além disso, o metal cobalto parece estar envolvido na estabilização da estrutura terciária da PGAM na conformação livre. Finalmente, este novo modelo estrutural pode contribuir para o esforço internacional de desenvolver fármacos tripanocidas / Tropical diseases represent a major burden on population health in low-incoming countries, being related to poverty and social disadvantage. African trypanosomiasis is a neglected parasitic infection on the agenda of World Health Organization. This disorder is caused by Trypanosoma brucei gambiensis and Trypanosoma brucei rhodesiensis, transmitted by the tsetse fly (Glossina sp.), and usually fatal if untreaded. The drugs used in the treatment are ineffective, difficult to administer, and cause severe adverse reactions. Therefore, there is a need to develop effective and safe chemotherapies. Thus, the enzyme phosphoglycerate mutase (PGAM) emerges as an important molecular target. This enzyme is involved in glucose metabolism, and is necessary for viability of the parasite. Moreover, it differs from the host enzyme allowing the identification of specific inhibitors. Nevertheless, efforts have been made in identifying PGAM inhibitors and to elucidate their structure and mechanism of reaction. Our purpose is to obtain the high resolution model of the macromolecule free from ligands and consequently to analyze the change in conformation that undergoes upon binding to its natural substrate. Trypanosoma brucei PGAM obtained in the expression and purification was shown to be catalytically active in the kinetics assays. In the size exclusion chromatography we observed that the purified sample behaves as a monomer. X-ray diffraction data were collected for crystals of the macromolecules obtained in the absence of ligands. The crystal structure was solved to 2.3 Å, showing a dimmer in the asymmetric unit. Both molecules of the dimmer were in free form, and had a large conformational difference compared with those of know PGAM structures that are connected to the natural substrate or product. Small angle X-ray scattering confirm that the enzyme is monomeric under conditions that mimic the physiological. Ligand-induced conformational change does not affect the topology of the two domains of the PGAM. However, there are changes in torsional angles of the main chain of the loops that connect the protein domains. Additionally, the metal cobalt seems to be involved in stabilizing the tertiary structure of PGAM in the free conformation. Finally, this new structural model may contribute to the international effort to develop trypanocidal drugs.

Indiana University-Purdue University Indianapolis (IUPUI) / Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, <i>Stenotrophomonas maltophilia</i> and <i>Pseudomonas aeruginosa</i>, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in<i> S. maltophilia</i>, isogenic deletion of phosphoglycerate mutase (<i>gpmA</i>) and two chaperone-usher pilin subunits, <i>S. maltophilia</i> fimbrae-1 (<i>smf-1</i>) and<i> cblA</i>, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δ<i>smf-1</i> and Δ<i>cblA</i> showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in <i>P. aeruginosa</i>, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when <i>polB</i>, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in <i>S. maltophilia</i> and genetic switches during the acute infection lifestyle in <i>P. aeruginosa</i>.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world’s human population. Despite the multiplicity of antimicrobial mechanisms mounted by its host, M. tuberculosis shows a remarkable ability to survive either by evoking survival strategies or by interference with critical macrophage functions that are required to successfully respond to the infection. It has been postulated that the outcome of exposure to M. tuberculosis (in terms of disease symptoms) largely depends upon the selective gene expression of tuberculosis bacilli along with activation of specific signaling pathways in the infected host cells during different phases of infection. In this perspective, determination of the complete genome sequence of Mycobacterium tuberculosis has provided crucial information with respect to the physiology of this bacterium and the pathogenesis of tuberculosis. However, putative functional annotation to all hypothetical proteins coded by M. tuberculosis genome remains complex. One important outcome of the genome-sequencing project was the discovery of two new multigene families designated PE and PPE. About 10% of the M. tuberculosis coding capacity is devoted to the PE and PPE genes, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N terminus of their gene products. In addition to these motifs, proteins of PE family share N-terminal domains of approximately 100 amino acids, whereas the PPE proteins possess an N-terminal domain of about 180 amino acids. Many PE and PPE proteins are composed only of these N-terminal homologous domains. However, other members possess an additional C-terminal segment of variable length, often composed of multiple copies of polymorphic GC rich sequences (PGRS). The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. In perspective of above-mentioned observations, we have chosen Rv0754 as a representative PE family gene. Rv0754 was shown to be upregulated in tubercle bacilli upon infection of bone marrow derived macrophages as well as in M. tuberculosis isolated from alveolar macrophages of infected mice. In the current investigation, we demonstrate that Rv0754 is hypoxia responsive gene based on promoter or transcript expression analysis. Further, extensive bioinformatics analysis predicated that Rv0754 posses possible Phosphoglycerate Mutase domain, an enzyme known for its significant role not only in the glycolytic pathway of the carbohydrate metabolism, but also for the crucial cell fate decision during conditions like oxidative stress as well as infection. Experimental data clearly suggests that hypoxic environment dependent expression of Rv0754 imparts resistance to macrophages from oxidative stress. These findings could be attributed to the presence of catalytically active Phosphoglycerate Mutase domain of Rv0754. More often, sophisticated regulation/modulation of key signaling events regulate the critical cell fate decisions during oxidative stress. In this context, TLR2 dependent triggering of PI3K-ERK1/2- NF-κB signaling axis by Rv0754 may be operative in imparting resistance to oxidative stress. Further, Rv0754 triggers COX-2 expression by activating PI3K-ERK1/2-NF-κB cascade in mouse macrophages. These observations are of relevance as Rv0754 is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. Additionally, Rv0754 elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0754 is an immunodominant antigen demonstrating significant T cell and humoral reactivity. These observations clearly advocate that Rv0754 protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0754 is immunogenic. Taken together, our findings suggest that Rv0754 is a novel PE_PGRS protein with unique features which could generate conditions that favor survival of the mycobacteria.