A cana-de-açúcar (Saccharum sp.) é uma planta que ocupa posição de destaque entre as culturas de importância econômica no cenário internacional, principalmente no Brasil, que é o maior produtor mundial. O estudo da diversidade microbiana associada a plantas, principalmente aquelas com interesse comercial, apresenta-se como importante alternativa para melhorar as características e a sustentabilidade destas culturas. Neste sentido, bactérias endofíticas e rizobactérias tem sido alvo de muitos estudos, pois apresentam efeitos benéficos para as plantas, como promoção de crescimento e inibição de patógenos. Um dos grupos de bactérias que colonizam a cana-de-açúcar é composto por espécies do gênero Burkholderia. Este gênero é composto de bactérias que podem ser encontradas em diferentes nichos ecológicos, como o solo, a água, ou em associação com plantas, fungos, e outros animais, além de humanos. Na interação bactéria-planta, as espécies de Burkholderia podem colonizar a rizosfera e o interior das raízes hospedeiro, onde podem estimular o crescimento do vegetal, contribuir para sua nutrição, como também protegê-lo da ação de fitopatógenos. Sendo assim, isolados de Burkholderia spp. do interior das raízes (endofíticos) e da rizosfera de cana-de-açúcar foram avaliados quanto à capacidade de fixar nitrogênio, produzir AIA, sideróforos, enzimas de interesse biotecnológico, solubilizar fosfato inorgânico e inibir patógenos desta cultura. Estes isolados também foram caracterizados geneticamente por análise de restrição e seqüenciamento dos genes 16S rDNA e gyrB, além da caracterização genotípica por BOX-PCR. Os resultados indicam que os isolados possuem potencial para promoção de crescimento vegetal, inibição de patógenos e produção de lipases. Filogeneticamente, a maioria dos isolados pertencem ao complexo Burkholderia cepacia com similaridade à B. cepacia e B. cenocepacia. Considerando a ocorrência dos isolados como endófitos ou rizosféricos, as metodologias fenotípicas e genotípicas não foram capazes de distinguir os membros componentes destas comunidades. Este trabalho evidencia a ampla associação deste grupo com cana-de-açúcar, e destaca as possíveis aplicações que tais bactérias podem ter no cultivo e sustentabilidade desta cultura. / Sugarcane (Saccharum spp.) occupies a position of prominence among the economically important crops in the international scene, mainly in Brazil, which is the world\'s largest producer. The study of microbial diversity associated with plants, especially those with commercial interest, presents itself an important alternative to improve the performance and sustainability of these crops. In this sense, endophytic bacteria and rhizobacteria has been target of many studies, since they have beneficial effects for plants, such as plant growth promotion and inhibition of pathogens. One of the bacterial groups that colonize sugarcane is composed of species of the genus Burkholderia. This genus is composed of a bacterium that can be found in different ecological niches, such as soil, water, or in association with plants, fungi and other animals, as well as in humans. In the association bacterium-plant, the species of Burkholderia can colonize the rhizosphere and the inside of the host roots, which can stimulate the growth of the plant, contributing to its nutrition, but also protects it from the action of phytopathogens. Thus strains of Burkholderia spp. isolated from the inside of the roots (endophytic) and from the rhizosphere of sugarcane were evaluated for the ability to fix nitrogen, produce IAA, siderophores, enzymes of biotechnological interests, solubilize inorganic phosphate and inhibits pathogens of the same crop. These strains were also genetically characterized by the analysis of enzymatic restriction and sequencing of 16S rDNA and gyrB genes, in addition to the characterization by genotypic technique BOX-PCR. The results indicate that the strains have potential for plant growth promotion, inhibition of pathogens and production of lipases. Phylogenetically, the isolates were affiliated to Burkholderia cepacia complex, with mainly similarity to B. cepacia and B. cenocepacia. Considering the occurrence of isolated as endophytes or rhizosphere, the genotypic and phenotypic methods were not able to distinguish the members of these communities. This research work demonstrates the broad association that this group has with sugarcane, and highlights the possible applications that these bacteria may have in cultivation and sustainability of this crop.

A linhagem queratinolítica Chryseobacterium sp. Kr6 mostrou-se com possibilidade de aplicação em processos envolvendo queratinólise, principalmente na hidrólise de penas de frango e depilação de couro bovino. No presente trabalho avaliou-se o efeito da composição do meio sobre o crescimento e atividade proteolítica deste isolado e uma protease queratinolítica (queratinase) foi purificada e caracterizada. O microrganismo mostrou-se adaptado à utilização de queratina como substrato durante o crescimento, produziu diferentes proteases dependendo do meio utilizado e a maior atividade proteolítica foi atingida quando utilizado meio de cultivo com penas como única fonte de carbono e nitrogênio. A adição de fonte extra de nutrientes resultou em uma parcial repressão catabólica. Uma protease extracelular (Q1) foi purificada cerca de 14 vezes utilizando cromatografia de interação hidrofóbica em Phenyl-Sepharose CL 4B e gel filtração em Superose H12R. Q1 mostrou ser uma proteína monomérica com peso molecular de 64 KDa determinado por SDS-PAGE e pH e temperatura ótimos de 8,5 e 50°C respectivamente. O perfil de inibição indica tratar-se uma metaloprotease e as seqüências internas dos peptídeos resultantes de digestão tríptica mostraram homologia ao sítio ativo e de ligação ao Zn da família M14 (Carboxipeptidase). A atividade proteolítica foi estimulada pela presença de íons Ca2+ e Mg2+ e inibida por Cu2+, Zn2+, Al2+, Hg 2+ e agentes redutores. Q1 apresentou atividade queratinolítica sobre o substrato keratin azure, mas não foi capaz de hidrolisar penas de frango sugerindo a necessidade de outras enzimas durante o processo de degradação de penas. Utilizando os iniciadores degenerados desenhados com base na seqüência dos peptídeos, foi amplificado um fragmento de 470 pb correspondente a uma região do possível gene desta metaloproteína utilizando DNA e cDNA como molde. A seqüência do fragmento pode estar sendo expressa, mas não apresentou similaridade e homologia a proteínas conhecidas e portando, indicativa de uma nova metaloprotease. / The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of media composition on the protease production and growth by this strain was studied and a keratinolytic protease (keratinase) was purified and characterized. The strain was adapted to use keratin as substrate to growth, produced different proteases in different media composition and the higher proteolytic activity was reached when used feather as only source of carbon and nitrogen. The addition of sources of nutrients has resulted in partially repressed catabolism. An extracellular protease Q1) was purified 14-fold by chromatography using the hydrophobic interaction Phenyl-Sepharose CL 4B column and gel filtração in Superose 12HR. SDS-PAGE indicated that the Q1 is a monomeric protein with molecular mass of 64 KDa. and optima pH and temperature were 8,5 e 50°C, respectively. The inhibition profile indicates to be a Zn-metalloprotease and analysis of tryptic peptides sequence revealed sequence homology to the conserved active site and Zn binding site, which may characterize keratinase Q1 as a member of M14 metalloprotease family (Carboxipeptidase). The activity was stimulated by of Ca2+ and Mg2+ and inhibited by Cu2+, Zn2+, Al2+, Hg 2+ and reducing agents. Q1 presented keratinolytic activity under substrate keratin azure, but was unable to hydrolyze poultry feather, suggesting the requirement by other enzymes in the feather hydrolysis mechanism. Degenerate primers amplified a 470 bp, corresponding to a probable gene region of this metalloprotein, with DNA and cDNA. The sequence is being expressed but do not showed similarity and homology to known proteins, thus indicating a new metalloprotease.

Este trabalho descreve a construção de plasmídeos para expressão e ancoragem de proteínas de alta afinidade a íons Hg2+ e As5+. Os genes merR e arsR de C. metallidurans foram inseridos no vetor que contém o sistema para expressão e ancoragem de proteínas heterólogas em bactérias Gram-negativas originando os plasmídeos pCM-Hg e pCM-As. MerR e ArsR foram produzidas sob comando do promotor pan. E. coli recombinantes apresentaram resistência 100% superior a Hg2+ e As5+. C. metallidurans/pCM-As apresentou MIC > Na3As02 1000 mM sendo a Gram-negativa com maior capacidade de sobrevivência a íons As5+. Os plasmídeos elevaram a sobrevivência das bactérias estudadas, podendo ser usados para aumentar índices de sobrevivência e fornecer viabilidade a outras cepas. Células recombinantes apresentaram capacidade de adsorver Hg2+ ou As5+ do meio em níveis superiores às linhagens selvagens. As bactérias descritas são excelentes candidatas para biorremediação. Este trabalho apresenta pela primeira vez a ancoragem da proteína ArsR na superfície celular de um micro-organismo. / This work describes the construction of plasmids for expression and anchoring of high affinity proteins to Hg2+ or As5+ ions. C. metallidurans merR and arsR genes were inserted into the vector which contains the system for expression and anchoring of heterologous proteins in Gram-negative bacterias origining the plasmids pCM-Hg and pCM-As. MerR and ArsR were produced under pan promoter command. Recombinant E. coli showed resistance 100% higher to Hg2+ and As5+. C. metallidurans/pCM-As showed MIC > Na3As02 1000 mM being the most resistance Gram-negative able to survive in As5+ sites. The plasmids increased the studied Gram-negatives bacterial surviving and they can be utilized in other strains to increase the surviving levels and supply viability. Recombinant cells showed ability for adsorption of Hg2+ or As5+ from the media in enhanced levels as compared to the wild type. The described bacterias are excellent candidates for bioremediation. This work presents for the first time the cell surface display of ArsR protein on a microorganism.

Made available in DSpace on 2014-07-29T15:25:21Z (GMT). No. of bitstreams: 1 TESE SILVANA LV SANTOS 2012 - texto.pdf: 1699937 bytes, checksum: 1ea3a901a7686050491e179ef0b83b40 (MD5) Previous issue date: 2012-03-30 / This cross-sectional study was performed in the dressing rooms of the primary healthcare network of Goiânia, Goiás, Brasil, with the following objectives: to identify the prevalence of Gram-negative bacteria (GNB) in venous ulcers (VU) with clinical signs of infection; analyze the susceptibility profile of the isolates; detect the production of AmpC &#946;-lactamases and metallo-beta-lactamases, and extended spectrum beta-lactamase (ESBL); describe the clinical signs and symptoms of infection in VU; evaluate the wounds clinical stage of infection and its relationship with the presence of GNB. The data were analyzed by means of descriptive statistics procedures, proportion and Chi-square tests (p<0.05). All ethical aspects were followed. The participants were 69 patients with venous ulcers, with or without arterial complication, totaling 98 wounds. It was verified that 74.5% of the wounds showed GNB growth, particularly enterobacteria (53.8%) and non-fermenting gram-negative bacteria (46.2%). The prevalent species among the enterobacteria was Escherichia coli (24.5%), followed by Enterobacter aerogenes, Pantoea agglomerans and Proteus mirabilis (12.2% each). Regarding the non-fermenting gram-negative bacteria, the prevalent genre was Pseudomonas (66.6%), particularly the species P. aeruginosa (59.5%), present in 25.5% of the analyzed wounds. Regarding the susceptibility profile of the enterobacteria, the highest resistance rates were to tetracycline (38.8%) and amoxicillin-clavulanic acid (26.5%). Among P. aeruginosa, the highest resistance was observed for cefoxitin (100%). Regarding the production of the AmpC enzyme, 30% of the microorganisms in the CESP (Citrobacter spp., Enterobacter spp., Serratia spp. and Providencia spp.) group, and 100% of the P. aeruginosa were resistant to cefoxitin. The remaining microorganisms of the CESP group (70%) that were sensitive to cefoxitin were subjected to a confirmatory test, and 37.5% were found to be positive for the production of the AmpC enzyme. Regarding metallo-beta-lactamase, 23.8% of the non-fermenting gram-negative bacteria showed reduced sensitivity to imipenem, meropenem or ceftazidime. When subjected to the confirmatory test, 8% of the P. aeruginosa were positive for the MBL enzyme. Regarding the clinical signs and symptoms of infection, the highlighted results with >70% frequency are: opaque and/or reddish brown discoloration; increase in exudate volume and pain. Stage-three infection was the most prevalent (71.4%). An association was found between cellulitis and friable granulation tissue that bleeds easily and the culture for GNB. In conclusion, the presence of gram-negative pathogens with resistance profiles in primary healthcare patients suggests the need to implement microbiological surveillance for patients with VU experiencing a prolonged or difficult healing process, and that the identification VU infection should be guided by knowledge regarding the etiology, classic characteristic and clinical stages of infection. / Estudo transversal realizado nas salas de curativos da atenção primária a saúde em Goiânia, Goiás, Brasil, cujos objetivos foram identificar a prevalência de bastonetes Gram-negativos (BGN) em úlceras venosas (UV) com sinais clínicos de infecção; analisar o perfil de suscetibilidade dos isolados; detectar a produção de &#946;-lactamases tipo AmpC, metalo-beta-lactamase e ESBL; descrever os sinais e sintomas clínicos de infecção em UV; avaliar o estágio clínico de infecção das lesões e a relação destes com a presença de BGN. Utilizou-se de análise descritiva, teste de proporções e Qui-quadrado (p<0,05). Os aspectos éticos foram observados. Participaram 69 pacientes com úlceras venosas com ou sem complicações arteriais, totalizando 98 lesões. Verificou-se que em 74,5% das lesões houve crescimento de BGN. Foram identificadas Enterobactérias (53,8%) e bastonetes Gram-negativos não fermentadores (46,2%). Dentre as enterobactérias prevaleceu a espécie Escherichia coli (24,5%), seguida de Enterobacter aerogenes, Pantoea aglomerans e Proteus mirabillis com 12,2% cada um. Em relação aos bastonetes Gram-negativos não fermentadores, destacou-se a prevalência do gênero Pseudomonas com 66,6% e em especial a espécie P. aeruginosa com 59,5%, presente em 25,5% das feridas analisadas. Quanto ao perfil de suscetibilidade das enterobactérias, destacaram-se a resistência à tetraciclina (38,8%), à amoxacilina-ácido clavulânico (26,5%). Entre as P. aeruginosa, a maior resistência foi observada para cefoxitina (100%). Quanto à produção de enzima AmpC, 30% dos micro-organismos do grupo CESP e 100% das P. aeruginosa foram resistentes a cefoxitina. Os demais micro-organismos do grupo CESP (70%) sensíveis à cefoxitina foram submetidos ao teste confirmatório e 37,5% apresentaram-se positivos a produção de enzima AmpC. Em relação a metalo-beta-lactamase, 23,8% dos bastonetes Gram-negativos não fermentadores apresentaram sensibilidade reduzida ao imipenem, meropenem ou ceftazidima. Ao serem submetidos ao teste confirmatório, observou-se que 8% das P. aeruginosa foram positivas para a enzima MBL. Não foi identificada ESBL. Quanto aos sinais e sintomas clínicos de infecção na lesão, destacaram-se, com freqüência >70%: descoloração do tipo opaca e/ou tijolo vermelho escuro; aumento do volume do exsudato e dor. O estágio três de infecção foi o mais prevalente (71,4%). Houve relação entre celulite e tecido de granulação friável que sangra facilmente e o resultado de cultura para BGN. Conclui-se que a presença de patógenos Gram-negativos resistentes em pacientes na atenção primária a saúde sugere a necessidade de instituir-se vigilância microbiológica para os pacientes com UV com processo cicatricial de difícil evolução e que a identificação de infecção nas UV deve ser norteada pelo conhecimento acerca da etiologia, características clássicas e estágios clínicos de infecção.

O presente trabalho teve como objetivo realizar um estudo de vigilância epidemiológica de bactérias MR em isolados obtidos de amostras de búfalo de bubalinocultura e em animais de estimação apresentando sinais e sintomas clínicos de infecção urinária. O estudo relata resultados inéditos referentes à disseminação de bactérias MR, com alto índice de resistência a antimicrobianos de uso clínico e do agronegócio, constituindo o primeiro reporte mundial da emergência de cepas de Escherichia coli produtoras de <font face=\"Symbol\">b-lactamases de amplo espectro (ESBL) do tipo CTX-M-8 e AmpC plasmidial (pAmpC) CMY-2 na bubalinocultura e a presença de cepas de E. coli produtoras de ESBL do tipo CTX-M-15, CTX-M-8 e CTX-M-2, e pAmpC CMY-1, CMY-2 e DHA-1 em animais de companhia é relatada pela primeira vez no Brasil. Nos isolados de E. coli ESBL positivos, não foi constatada relação clonal. As cepas isoladas de búfalos pertencem aos grupos A e B1 e em animais de companhia foram identificados predominantemente os grupos filogenéticos de alta virulência B2 e D. / This study aimed to conduct an epidemiological surveillance on MDR among Gram-negative bacilli recovered from samples from buffalo and in pets exhibiting signs and symptoms related to urinary tract infection. The study reports the spread of MDR bacteria exhibiting a high resistance profile to veterinary- and human-use <font face=\"Symbol\">b-lactams and quinolones, in livestock of buffalos and in pets, constituting the first worldwide report of CTX-M-8-type extended-spectrum <font face=\"Symbol\">b-lactamase (ESBL)- and CMY-2-type plasmid AmpC (pAmpC)-producing E. coli strains in buffalo. Moreover, to the best of knowledge, this is the first report of CTX-M-15-, CTXM-8-, CTX-M-2, CMY-1, CMY-2- and DHA-1-producing E. coli strains in pets in Brazil. With respect to the origin of resistance, we found no clonal relatedness among MDR. E. coli isolates from buffalos belonging to groups A and B1 and in companion animals, the phylogenetic analysis of virulence in E. coli denoted the predominance of the highly virulent phylogenetic groups B2 and D.

Les protéines synthétisées et sécrétées par les bactéries jouent des rôles importants pour leur survie. Les bactéries à Gram négatif ont développé des voies de sécrétion en tant qu'armes principales pour transporter des facteurs de virulence dans l'environnement extracellulaire ou dans des cellules hôte. L'un de ces systèmes, le T9SS a été principalement étudié chez l'agent pathogène oral Porphyromonas gingivalis et chez la bactérie mobile Flavobacterium johnsoniae. Un autre complexe, le T2SS est le principal déterminant de la virulence de la bactérie Pseudomonas aeruginosa, un agent pathogène de la fibrose kystique. Dans le cadre de ma thèse, j'ai résolu la structure atomique de plusieurs composants centraux du T9SS et du T2SS. Concernant le projet T9SS, j'ai essayé de cristalliser le domaine cytoplasmique de GldL de F. johnsoniae. La co-cristallisation de GldL avec des Nbs a été réalisée sans succès. Néanmoins, les structures cristallines de deux nanobody contre GldL ont été résolues par remplacement moléculaire. De plus, j'ai également travaillé sur la protéine PG1058 de P. gingivalis. J'ai résolu sa structure par diffraction anomale à la longueur d’onde du selenium. Concernant le projet T2SS, je me suis concentré sur la partie N-terminale de XcpQ, une sous-unité de la sécrétine. J'ai résolu la structure cristalline de XcpQN012 seul et en complexe avec le nanobody vhh04 à une résolution de 2,98 Å et de 2,9 Å, respectivement. Enfin, j'ai participé à la détermination structurale de TssK, un composant de plaque de base du système de T6SS et déterminer la structure cristalline d'un nanobody contre le domaine périplasmique de PorM. / Proteins synthesized and secreted by bacteria serve many important roles in their survival. In particular, Gram-negative bacteria have evolved secretion pathways as the main weapons for transporting virulence factors into target cells or into the extracellular environment. One of these systems, the type IX secretion system (T9SS) or the Por secretion system, has been studied mainly in the oral pathogen Porphyromonas gingivalis and the gliding bacterium Flavobacterium johnsoniae. Another complex, the type II secretion system (T2SS) is the main determinant of the virulence of Pseudomonas aeruginosa, a cystic fibrosis pathogen. In my PhD thesis, I solved the atomic structure of several core components of both T9SS and T2SS.For the T9SS project, I tried to crystallize the cytoplasmic domain of GldL from F. johnsoniae. The co-crystallization of GldL with Nbs was unsuccessfull. The crystal structures of two nanobodies against GldL were solved by molecular replacement. I also worked on the PG1058 protein of P. gingivalis. I obtained crystals of the selenomethionine-derivatized PG1058 OmpA_C-like domain that diffracted up to 1.55 Å, and solved its structure by single-wavelength anomalous diffraction. For the T2SS project, I focused on the N-terminal part of XcpQ, a subunit of the secretin. I solved the crystal structure of XcpQN012 alone and in complex with nanobody vhh04 at a resolution of 2.98 Å and 2.9 Å, respectively. In addition, I also took part in the structural determination of the base plate component TssK of the T6SS and determined the crystal structure of one nanobody (vhh19) against the periplasmic domain of PorM.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance.  In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on  in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with examples advocating regimens that (i) promote bacterial killing and reduce risk for toxicity in preterm and term newborn infants receiving gentamicin, (ii) achieve a fast initial bacterial killing and reduced resistance development of colistin in critically ill patients by application of a loading dose, and (iii) overcome existing meropenem resistance by combining colistin and meropenem

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.

<p>Nitric oxide (NO) is an endogenous gaseous signaling molecule produced by three NO synthase isoforms (NOS1, 2, 3) and important in host defense. The induction of NOS2 during bacterial sepsis is critical for pathogen clearance but its sustained activation has long been associated with increased mortality secondary to multiple organ dysfunction syndrome (MODS). High levels of NO produced by NOS2 incite intrinsic cellular dysfunction, in part by damaging macromolecules through nitration and/or nitrosylation. These include mitochondrial DNA (mtDNA) and enzymes of key mitochondrial pathways required for maintenance of normal O2 utilization and energy homeostasis. However, animal studies and clinical trials inhibiting NOS2 have demonstrated pronounced organ dysfunction and increased mortality in response to live bacterial infections, confirming that NOS2 confers pro-survival benefits. Of particular interest here, the constitutive NOS1 and NOS3 have been linked to the up-regulation of nuclear genes involved in mitochondrial biogenesis but no comparable role has been described for NOS2. <italic> Therefore, I hypothesized that NOS2 is indispensible for host protection but must be tightly regulated to ensure NO levels are high enough to activate mitochondrial and other pro-survival genes, but below the threshold for cellular damage.</italic></p><p>This hypothesis was explored with two major Aims. The <italic>first Aim</italic> was to define the role of NOS2 in the activation of mitochondrial biogenesis in the heart of <italic>E. coli</italic>-treated mice. The <italic>second</italic> was to investigate the ability of NOS2 to be transcriptionally regulated by an enzyme previously shown to induce mitochondrial biogenesis, heme oxygenase-1 (HO-1). This hypothesis was tested using an <italic>in vivo</italic> model of sublethal heat-killed <italic>E. coli</italic> (<italic>HkEC</italic>) peritonitis in C57B/L6 (Wt), NOS2-/-, and TLR4-/- mice. Additionally, <italic>in vitro</italic> systems of mouse AML-12 or Hepa 1-6 cells pretreated with HO-1 activators or <italic>Hmox1</italic> shRNA prior to inflammatory challenge with lipopolysaccharide (LPS) +/- tumor necrosis factor-&alpha; (TNF-&alpha;). For the first Aim, Wt, NOS2-/-, and TLR4-/- mice were treated with (<italic>HkEC</italic> and cardiac tissue analyzed for mitochondrial function, expression of nuclear and mitochondrial proteins needed for mitochondrial biogenesis, and histological expression of NOS2 and TLR4 relative to changes in mitochondrial mass. For the second Aim, Wt mice were pretreated with hemin or carbon monoxide (CO) to activate HO-1 prior to <italic>HkEC</italic>-peritonitis. Liver tissue in these animals was evaluated at four hours for HO-1 induction, <italic>Nos2</italic> mRNA expression, cytokine profiles, and nuclear factor (NF)-&kappa;B activation. Liver cell lines were pretreated with hemin, CO-releasing molecule (CORM), or bilirubin one hour before LPS exposure and the <italic>Nos2</italic> transcriptional response evaluated at two and 24 hours. The MTT assay was used to confirm that <italic>in vitro</italic> treatments were not lethal. </p><p>These studies demonstrated that <italic>HkEC</italic> induced mtDNA damage in the heart that was repaired in Wt mice but not in NOS2-deficient mice. In KO mice, sustained mtDNA damage was associated with the reduced expression of nuclear (NRF-1, PGC-1&alpha;) and mitochondrial (Tfam, Pol-&gamma;) proteins needed for mitochondrial biogenesis. The findings thus supported that NOS2 is required for mitochondrial biogenesis in the heart during Gram-negative challenge. Evaluation of the relationship between HO-1 and NOS2 in murine liver was more complex; HO-1 activation in <italic>HkEC</italic>-treated Wt mice attenuated 4-hour <italic>Nos2</italic> gene transcription. In liver cell lines, hemin, CORM, and bilirubin were unable to suppress <italic>Nos2</italic> expression at the time of maximal induction (2 hours). <italic>Nos2</italic> was, however, suppressed by 24 hours, suggesting that the regulatory impact of HO-1 induction was not engaged early enough to reduce <italic>Nos2</italic> transcription at 2 hours. It is concluded that NOS2 induction in bacterial sepsis optimizes the expression of the mitochondrial biogenesis transcriptional program, which subsequently can also be regulated by HO-1/CO in murine liver. This provides a potential new mechanism by which immune suppression and mitochondrial repair can occur in tandem during the acute inflammatory response.</p> / Dissertation

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the aldol condensation of the five-carbon sugar phosphate, arabinose 5-phosphate (A5P), and phosphoenol pyruvate (PEP) to give the eight-carbon phosphorylated sugar, KDO8P. It is the second committed step in the synthesis of KDO, a necessary component of the cell wall of Gram-negative bacteria. This thesis describes the design, synthesis and evaluation of a number of inhibitors of KDO8P synthase that utilise the functionality of one or both substrates. The KDO8P synthase family can be divided based on the requirement of a divalent metal ion. Chapter 2 describes the growth, purification and characterisation of an example from both the metal-independent KDO8P synthases (Neisseria meningitidis, Nme) and metal-dependent KDO8P synthases (Acidithiobacillus ferrooxidans, Afe) in order to utilise these enzymes for the inhibition studies described in this thesis. In Chapter 3, a number of small molecule PEP analogues were selected as mimics of KDO8P synthase reaction intermediates and tested as inhibitors of KDO8P synthase from N. meningitidis and A. ferrooxidans. Glyphosate, (E)-vinyl phosphonate and the fluorinated analogue of (E)-vinyl phosphonate were selected as mimics of the high-energy oxocarbenium intermediate through which the KDO8P synthase reaction is thought to occur. The two enantiomers of phospholactate were selected in order to investigate the chirality of the tetrahedral intermediate and determine the importance of this chirality for inhibition of KDO8P synthase. All five inhibitors were found to be moderate to poor inhibitors of both the KDO8P synthase from N. meningitidis and A. ferrooxidans. Chapter 4 describes the design and synthesis of inhibitors that incorporated structural features of the second substrate, A5P, in order to improve inhibition from that observed for the PEP analogues investigated in Chapter 3. A bisphosphate inhibitor was designed that incorporated a terminal phosphate moiety, representative of the phosphate of A5P. A large increase in inhibition was found, compared to the phospholactates from which it was derived. A structure-activity-relationship study was undertaken on this compound by design of compounds that lacked one of the two phosphate moieties of the bisphosphate inhibitor, in order to determine their relative importance. The inhibition results indicate that the primary terminal phosphate, thought to bind in the A5P phosphate binding site, is more important for inhibition of KDO8P synthase than the secondary phosphate. In Chapter 5 these investigations into the inhibition of KDO8P synthase are discussed in detail, and interpreted using the aid of computational studies. In addition several approaches are described for the completion and advancement of the studies presented here in this thesis.