Orientador: João Pessoa Araújo Júnior / Resumo: O Brasil é o segundo maior produtor de carne bovina do mundo e para se manter competitivo tem objetivado melhorias em higiene e segurança alimentar. Micro-organismos deteriorantes diminuem a vida de prateleira dos produtos e podem viabilizar patógenos em biofilmes predominantemente heterogêneos. Pseudomonas aeruginosa são bactérias mais comuns em carnes, e tem como característica a alta capacidade de produção de biofilme. Sendo ambientais, a contaminação da carne é facilitada por falhas de higiene e boas práticas, sendo relevante estudos sobre a sua presença em produtos cárneos. Neste trabalho, objetivou-se estudar a intensidade de produção de biofilme e sua expressão gênica por cepas de P. aeruginosa isoladas em planta de processamento bovino. As amostras foram obtidas por suabes de carcaças e superfícies em planta de processamento bovino totalizando sete coletas, divididas em dois dias, amostrando-se 22 pontos em cada coleta. Os isolados foram confirmados físico-quimicamente. A produção de biofilme por espectrofotometria classificou a intensidade em fortes, moderadas, fracas e não produtoras. Foram isoladas 32 cepas, das quais 4 demonstraram forte produção de biofilme, 3 moderadas, 4 fracas e 8 não produtoras. Foi predominante a contaminação em carcaças recém abatidas, anteriormente à refrigeração. As amostras foram confirmadas usando o alvo oprL em análise por qPCR, e a comparação da expressão de alginato, algU e algD posteriormente normalizados pelo gene 16S foi realizada... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil is the second largest beef producer in the world and in order to remain competitive, it has aimed at improving hygiene and food safety. Damaging microorganisms decrease the shelf life of products and may render pathogens viable in predominantly heterogeneous biofilms. Pseudomonas aeruginosa are the most common bacteria in meats, characterized by high biofilm production capacity being environmental, meat contamination is facilitated by good practices hygiene faults being relevant studies on their presence in meat products. The objective of this study was to determine the intensity of biofilm production and its gene expression by strains of P. aeruginosa isolated from a bovine processing plant. Samples were obtained by carcass swabs and surfaces in a bovine processing plant, totaling seven samples, divided in two days, sampling 22 points in each collection. The isolates were physicochemically confirmed. The biofilm production by spectrophotometry classified the intensity as strong, moderate, weak and non-producing. Thirty-two strains were isolated, 4 of which showed strong biofilm production, 3 moderate, 4 weak and 8 non-producing. Contamination was predominant in freshly slaughtered carcass prior to refrigeration. All samples were confirmed in qPCR analysis with the oprL target, and gene expression of strong and weak samples were developmented with alginate genes, algU and algD, normalized by 16S gene. Expression analyzes of the algU and algD genes did not demonstrate s... (Complete abstract click electronic access below) / Mestre

Pseudomonas aeruginosa é um importante patógeno humano oportunista que causa graves infecções em pacientes imunocomprometidos e em pacientes portadores de fibrose cística (FC). O objetivo deste trabalho foi avaliar a capacidade de nanopartículas de albumina sérica bovina associadas aos antígenos totais de P. aeruginosa em proteger camundongos do desafio pela via nasal por este patógeno. Camundongos foram imunizados pela via subcutânea usando antígenos totais de P. aeruginosa, nanopartículas vazias ou nanopartículas associadas aos antígenos totais de P. aeruginosa nos dias 0, 7 e 14. A produção de anticorpos IgG total e os subtipos IgG1 e IgG2a foram avaliados por ELISA. Os camundongos imunizados foram desafiados com P. aeruginosa e os pulmões foram coletados para estudos histopatológicos e detecção da bactéria no pulmão. Nossos resultados mostram que camundongos imunizados com nanopartículas associadas aos antígenos totais de P. aeruginosa produziram altos títulos de anticorpos IgG1 anti-P. aeruginosa e títulos significativos de IgG2a. Os dados também mostram que camundongos imunizados com nanopartículas associadas aos antígenos totais de P. aeruginosa e desafiados com a bactéria apresentaram diminuição dos sinais inflamatórios, com uma redução significativa na intensidade e concentração de células inflamatórias, diminuição da hemorragia, sinais de edema e hiperemia nos pulmões se comparado aos outros grupos. A bactéria não foi detectada nos pulmões dos animais imunizados e infectados nos tempos analisados. Portanto, essa formulação é capaz de induzir uma resposta protetora nos animais infectados, sendo portanto, uma promissora plataforma para vacinas contra P. aeruginosa. / Pseudomonas aeruginosa is an important opportunistic human pathogen that causes severe infections in immunocompromised patients and also in cystic fibrosis patients. The aim of this work was to evaluate the ability of bovine serum albumin nanoparticles with entrapped antigens extracted from P. aeruginosa to protect mice from intranasal challenge with this pathogen. Mice were immunized via the subcutaneous route using P. aeruginosa antigens, empty nanoparticles or nanoparticles with entrapped P. aeruginosa antigens on days 0, 7 and 14. The total IgG antibody production and specific IgG1 and IgG2a titer were measured by ELISA. Immunized mice were challenged with live P. aeruginosa and their lungs were collected for histopathology studies and for detection of bacteria in their lungs. Our data showed that NPPa-vaccinated mice presented a high anti-Pseudomonas IgG1 and a low IgG2a antibody titles and decreased inflammatory signs, with significant reduction in intensity and concentration of inflammatory cells, lower hemorrhagic, edema and hyperemia signs in the lungs of challenge mice with live P. aeruginosa if compared to the other groups. The bacteria was not detected in the lungs from infected mice in the analyzed time. Therefore, this formulation is able to induce a functional response in an animal model of infection and thereby it is a promising platform for P. aeruginosa vaccines. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Neste trabalho foram preparados materiais híbridos do tipo silicatos organicamente modificados (Ormosils) contendo fosfotungstato, [PW12O40]-3 e dopados com nanopartículas sintetizadas core@shell SiO2@TiO2. Os objetivos a serem atingidos com estes materiais são obter filmes com alta sensibilidade à radiação UV e capazes de fotossintetizar nanopartículas de Ag no seu interior e superfície visando revestimentos antibacterianos. O pigmento fotocrômico nos filmes é o fosfotungstato e o core@shell irá atuar como agente potencializador da resposta fotocrômica dele. Dessa maneira variou-se a quantidade de volume da suspensão das partículas core@shell tentando maximizar a resposta fotocrômica com a menor quantidade possível de partículas adicionadas. Observa-se uma resposta não linear da sensibilidade ao UV observada pelo aumento no fotocromismo com a quantidade de suspensão de partículas adicionada. Ormosils com nanopartículas de Ag fotossintetizadas foram preparados com intuito de verificar a inibição desses na formação de biofilmes de bactérias patogênicas, ou seja, Staphylococcus aureus e Pseudomona aeruginosa. Os materiais foram caracterizados por espectroscopias vibracionais, espectroscopia de fotoelétrons de raios X (XPS), Difração de Raios X (DRX), Fluorescência de Raios X, Microscopia de Força Atômica(AFM) e Microscopia Eletrônica de Varredura (MEV). Nos testes de inibição da formação de biofilme notou-se uma maior inibição frente às bactérias S. aureus usando filmes de ormosils core@shell. Entretanto, para a inibição de P. aeruginosa a presença de core@shell não de maneira significativa. Na presença de NP de Ag ambas as bactérias foram inibidas totalmente. Os testes microbiológicos foram caracterizados através de contagem de placa, microscopia de Epifluorescencia, teste de reaproveitamento, teste do halo de crescimento, Microscopia Eletrônica de Varredura e espectroscopia de fotoelétrons de raios X (XPS). / In this project, hybrid materials based on organo modiefied silicates (Ormosils) containing phosphotunsgtic acid (HPW) and doped with SiO2@TiO2 core@shell nanoparticles were prepared. The objectives of the project was to obtain films of these hybrid ormosil-HPW-SiO2@TiO2 materials with high sensitivity to UV radiation and, which after coating with silver nanoparticles, could be used for antibacterial applications. The HPW in these films serves as the photochromic component while the role of SiO2@TiO2 particles was to enhance the innate phototromic reponse of the HPW. The prepared hybrid materials and hybrid films were characterized by vibrational spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction analysis, X-ray fluoresence, atomic force microscopy and scanning electron microscopy. The effect of addition of SiO2@TiO2 particles on the photochromic reponse was systematically studied in order to obtain films with maximum photochromic response. The UV sensitity or the photochromic response showed a non-linear increase as function of the amount of SiO2@TiO2 particles added. The hybrid ormosil-HPW-SiO2@TiO2 films modified with silver nanoparticles were studied for their antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa as model bacteria. The ormosil-HPW-SiO2@TiO2 films showed a good degree of inhibition against S. aureus, while the presence of SiO2@TiO2 in the ormosil-HPW films had no significant effect on the inhibition ability of the hybrid films against P. aeruginosa. The ormosil-HPW-SiO2@TiO2 films modified with Ag totally inhibited the growth of both the model bacteria studied. These photochromic and antibacterial hybrid films can find useful applications in UV-sensing devices and antibacterial coatings.

Evalúa la eficacia de dos desinfectantes de uso hospitalario (Dezavid® y Supersafe-D®) frente a biopelículas de Pseudomonas aeruginosa y Staphylococcus aureus formadas sobre acero inoxidable. Adicionalmente se evaluó la eficacia del hipoclorito de sodio (lejía tradicional) de uso doméstico. Se formó durante 6 días una biopelícula sobre discos de acero inoxidable de 1 cm de diámetro y 1 mm de espesor, posteriormente se les trató con concentraciones de 0,05 %, 0,10 % y 1,00 % de Dezavid®; Supersafe-D® y lejía tradicional, dejando actuar el desinfectante durante el tiempo recomendado por el fabricante. Posteriormente se cuantificó las UFC/disco en comparación con un grupo de discos que no fueron sometidos a ningún desinfectante. Los desinfectantes con mayor eficacia fueron Dezavid® 1,00 %; 0,10 % y Supersafe-D® para Staphylococcus aureus ATCC y cepa clínica. En el caso de Pseudomonas aeruginosa ATCC y cepa clínica los desinfectantes con mayor eficacia fueron Dezavid® 1,00 % y Supersafe-D®. La lejía (1:20) tuvo igual eficacia que Dezavid® 1,00 %; 0,10 % y Supersafe-D® para Staphylococcus aureus ATCC y cepa clínica. En el caso de Pseudomonas aeruginosa ATCC y cepa clínica, la lejía (1:20) tuvo igual eficacia que Dezavid® 0,10 %. / Tesis

Pseudomonas aeruginosa (Pa) est un pathogène opportuniste responsable d’infections pulmonaires aigues graves chez les malades prédisposés. Devant l’émergence croissante de la résistance aux antibiotiques, le développement de thérapeutiques alternatives adjuvantes est indispensable et nécessite la compréhension des interactions hôte-pathogènes au cours de l’infection. La voie métabolique de dégradation du tryptophane appelée voie des kynurénines produit chez l’hôte des métabolites aux propriétés immunomodulatrices connues. Récemment, l’existence de cette voie a été mise en évidence chez Pa, bien que la nature et la quantité de métabolites produits ne soient pas parfaitement connus. La production bactérienne de kynurénines pourrait interférer avec la mise en place de la réponse immunitaire de l’hôte et sa régulation au cours des différentes phases de l’infection, altérant la balance immunitaire pulmonaire au profit du pathogène. A ce titre, la voie des kynurénines de Pa constituerait une cible thérapeutique potentielle. L’objectif de ce travail de thèse est d’étudier l’implication de la voie des kynurénines de Pa dans la virulence bactérienne et la réponse immune de l’hôte dans un modèle murin d’agression respiratoire aiguë. Pour cela, les souris sont infectées avec des souches sauvages de Pa, avec des souches mutantes ΔkynA, non productrices de kynurénines, et des souches ΔkynU, surproductrices de kynurénines. Les interactions potentielles avec la voie des kynurénines de l’hôte sont explorées en inhibant la première enzyme de la voie métabolique, l’indoleamine-2,3-dioxygenase (IDO). Enfin, le rôle du récepteur arylhydrocarbone (AhR), récepteur connu des kynurénines et impliqué dans l’immunité pulmonaire, est exploré en comparant la réponse à l’infection de souris AhR KO à celle des souris sauvages. Dans ce travail, nous décrivons tout d’abord la production des différents métabolites de la voie des kynurénines de Pa in vitro et in vivo dans le modèle d’infection respiratoire aigue, en décrivant pour la première fois la production d’acide kynurénique et de 3-hydroxy-kynurénine pour cette bactérie. Ensuite, nous montrons que les kynurénines bactériennes interfèrent avec la réponse immune de l’hôte, en majorant le recrutement cellulaire alvéolaire, tout en atténuant le niveau d’inflammation et l’activation des cellules présentatrices d’antigènes. Enfin, nous rapportons que l’IDO et l’AhR sont impliqués dans cette immunomodulation, faisant des kynurénines bactériennes des agents du dialogue hôte-pathogène au cours de l’infection respiratoire aigue. A la lumière de ces résultats, la voie des kynurénines pourrait constituer une cible thérapeutique d’intérêt dans les infections respiratoires à P. aeruginosa. / Pseudomonas aeruginosa (Pa) is a Gram-negative bacteria frequently involved in healthcare-associated pneumonia and considered as a « problem-pathogen ». To face the announced post-antibiotic era due to increasing resistance and lack of new antibiotics, new treatment strategies have to be developed. During pneumonia, lung injury results from both bacterial-mediated virulence and host response. Modulation of an overreacting host response could be an alternative therapeutic target in Pa-induced lung infection. Kynurenines are small molecules resulting from tryptophan degradation with reported immunomodulatory properties. Pa is known to produce kynurenine, but the functional enzymes, types and amounts of secreted metabolites are poorly known. Interestingly, many host cells also possess the kynurenine pathway, whose metabolites are known to control immune system homeostasis. The following experiments aim to determine whether bacterial metabolites can interfere with the host’s immune response, leading to a possible immunomodulatory interplay between bacteria and host kynurenine pathways, impacting on the pathophysiology of P. aeruginosa infection. To that goal, we use a murin model of acute lung injury. Mice were infected with WT strain of Pa, compared to mutant strains unable to produce kynurenine (ΔkynA), and mutant strains overproducing them (ΔkynU). Moreover, we studied the interactions between bacterial and host kynurenine pathways by inhibiting the first enzyme of the host pathway called indoleamine-2,3-dioxygenase (IDO). Finally, we assessed the role of the arylhydrocarbon receptor (AhR), a known receptor to kynurenine involved in lung immunity, using AhR KO mice. First, we assess types and levels of metabolites produced by Pa in an in vitro model, and the relevance of this production in vivo. We show for the first time that Pa is able to secrete kynurenine at clinically relevant levels, and other metabolites such as kynurenic acid and 3 OHkynurenine, what was unknown to date. Second, we show that bacterial metabolites were able to modulate the host innate immune response, by increasing alveolar recruitment of neutrophils, associated with decreased inflammatory cytokines levels and impairment of antigen-presenting cells activation. Finally, we report that IDO and AhR are involved in this kynurenine-mediated immunomodulation. These data suggest that pulmonary infection with a bacteria highly expressing the kynurenine pathway enzymes could lead to an imbalance in the immune response to infection, thus constituting a potential therapeutic target to improve Pa-induced pneumonia outcome.

Um estudo de coorte prospectivo foi realizado com o objetivo de comparar a eficácia da polimixina B à de outros antibióticos. Foram estudados pacientes com pneumonia ou traqueobronquite associadas à ventilação mecânica causadas por Pseudomonas aeruginosa ou Acinetobacter baumannii. Critérios de inclusão para este estudo foram idade igual ou superior a dezoito anos e uso de terapia antimicrobiana apropriada por período igual ou superior a 48 horas. O desfecho primário avaliado foi mortalidade em 30 dias. Variáveis clínicas foram comparadas entre os pacientes que utilizaram polimixina B e os que utilizaram outras drogas. O modelo de regressão de Cox foi realizado. Um total de 67 episódios ocorridos em 63 pacientes foi analisado: 45(67,2%) foram tratados com polimixina B e 22 (32,8%) com comparadores. A maior parte dos comparadores (72,7%) era de beta-lactâmicos. A maioria dos episódios foi de pneumonia associada à ventilação mecânica (PAV). As infecções foram causadas por P. aeruginosa em 28 casos (41,8%), por A. baumannii em 35 casos (52,2%) e por ambos em 4 casos (6%). A mortalidade geral em 30 dias foi de 44,8% (30 de 67): 53,3% (24 de 45) no grupo da polimixina B e 27,3% (6 de 22) no grupo dos comparadores (p=0,08). A taxa de mortalidade no grupo da polimixina e comparadores foi de 65,6 e 12,0 por 1000 pacientes-dia, respectivamente (p=0,02). A análise multivariada mostrou que o uso de polimixina B foi fator independente associado à mortalidade em 30 dias (Hazard Ratio ajustada, 4,3; Intervalo de Confiança de 95%, 1,39-13,03), após ajuste para tempo de internação hospitalar antes da infecção e aumento > 100% da creatinina em relação ao valor basal durante o tratamento. O escore APACHE II no inicio da infecção foi mantido no modelo final, embora não tenha atingido significância estatística, para ajuste de possível fator confundidor residual. Não houve diferenças significativas nos desfechos secundários, incluindo tempo de ventilação mecânica após terapia adequada, superinfecção e erradicação bacteriana nas secreções respiratórias. A terapia com polimixina B foi inferior comparada a outras drogas na pneumonia e traqueobronquite associadas à ventilação mecânica causadas por P. aeruginosa e A. baumannii. / To compare the efficacy of polymyxin B with other antimicrobials in the treatment of ventilator-associated pneumonia (VAP) and tracheobronchitis (VAT) by Pseudomonas aeruginosa or Acinetobacter baumannii, a prospective cohort study was performed. Patients who received appropriate therapy for ≥48h were analyzed. The primary outcome was 30-day mortality. A total of 67 episodes were analyzed: 45 (67.2%) treated with polymyxin B and 22 (32.8%) with comparators. Thirty-day mortality was 44.8%: 53.3% (24 of 45) in the polymyxin B group and 27.3% (6 of 22) in the comparator group, P=0.08. The mortality rates in the polymyxin B and comparator group were 65.6 and 12.0 per 1000-patients-day, respectively (P=0.02) Treatment with polymyxin B was independently associated with 30-day mortality in multivariate model, with similar results in the subgroup of patients with VAP, suggesting that this antibiotic may be inferior to other drugs in the treatment of VAP and VAT by these organisms.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.

Publicación a texto completo no autorizada por el autor / Busca purificar exoproteasas de Pseudomonas sp. por el sistema acuoso bifásico PEG/citrato para obtener hidrolizados proteicos de las semillas de Lupinus mutabilis “Tarwi”. Busca purificar parcialmente las exoproteasas de Pseudomonas sp. mediante extracción líquido-líquido aplicando estudios de diseño factorial de las variables: peso molecular de PEG, concentración de PEG, concentración de citrato y pH. Evalúa la adición de 1% (w/w) de líquidos iónicos formados por 1-butil-3-metil imidazol con diferentes aniones y determinar su efecto en la eficiencia de extracción de exoproteasas de Pseudomonas sp. por sistemas acuosos bifásicos. Realiza la hidrolización de las proteínas de Lupinus mutabilis “Tarwi” con exoproteasas de Pseudomonas sp. purificada por ATPS. / Tesis

Pseudomonas aeruginosa is a gram-negative pathogen that causes opportunistic infections in immunocompromised individuals. Whereas clinical isolates from acute infections are characterized by host cell cytotoxicity and motility, isolates from chronic infections are characterized by biofilm formation and persistence. The type III secretion system (T3SS) causes cytotoxicity by injecting effectors into host cells. T3SS gene expression is activated by ExsA, an AraC family transcriptional regulator. Transcription of exsA is controlled by two promoters, PexsC and PexsA, which are regulated by ExsA and the cAMP-Vfr system, respectively. Additional global regulatory systems also influence T3SS including the second messenger signaling molecule c-di-GMP and the RsmAYZ regulatory system. c-di-GMP signaling increases biofilm production and decreases acute virulence factor expression. A previous study found that c-di-GMP alters cAMP levels and affect cAMP-Vfr signaling. Other studies found that c-di-GMP signaling alters expression of the small non-coding regulatory RNAs, rsmY and rsmZ. The RsmAYZ post-transcriptional regulatory system regulates ExsA translation. We hypothesize that c-di-GMP regulates T3SS expression by altering exsA transcription through the cAMP-Vfr dependent PexsA promoter. Overexpression of YfiN, a c-di-GMP synthase, decreases T3SS reporter activity in PA103 and requires a functional GGDEF active site for full inhibition. Inhibition by YfiN does not require rsmYZ. YfiN expression decreases cAMP-Vfr signaling and coordinately inhibits PexsA-lacZ reporter activity. Consistent with the proposed model, YfiN expression in a vfr mutant does not further decrease T3SS reporter activity. These data indicate that the YfiN alters T3SS expression through transcriptional control of the cAMP-Vfr dependent PexsA promoter.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes acute nosocomial infections as well as chronic infections in cystic fibrosis (CF) patients. P. aeruginosa utilizes a type III secretion system (T3SS) during acute infections to promote host cell cytotoxicity and inhibit phagocytosis. Regulation of T3SS expression can be classified into two distinct categories: intrinsic and extrinsic. T3SS intrinsic regulation involves the well-characterized ExsECDA cascade that controls T3SS gene transcription. Extrinsic regulation involves global regulatory systems that affect T3SS expression. Despite general knowledge of global regulation of T3SS expression, few specific mechanisms have been elucidated in detail. The overall goal of my thesis work was to provide clarity to global regulatory mechanisms controlling T3SS expression. One well-documented observation is that P. aeruginosa isolates from CF patients commonly have reduced T3SS expression. In chapter II, I describe how the MucA/AlgU/AlgZR system, commonly activated in CF isolates through mutation of the mucA gene, inhibits T3SS gene expression. My experiments demonstrate that the AlgZR two-component system inhibits ExsA expression through two separate global regulatory systems. First, as previously described, AlgZR inhibits ExsA expression by reducing activity of the cAMP/Vfr signaling pathway. Vfr, a homolog of Escherichia coli Crp, regulates T3SS gene expression through an unknown mechanism. Second, AlgZR alters the activity of the RsmAYZ system to specifically reduce ExsA expression. The RNA-binding protein RsmA, a homolog of E. coli CsrA, activates ExsA expression at a post-transcriptional level. Previous studies in our laboratory identified several transposon insertion mutants that appeared to be novel extrinsic regulators of T3SS gene expression. One of those candidates, named DeaD, is a putative ATP-dependent RNA helicase. My experiments in chapter III reveal that DeaD regulates T3SS expression by directly stimulating exsA translation. Mutants lacking deaD have reduced exsA translational reporter activity and ExsA expression in trans fails to complement a deaD exsA double mutant for T3SS gene expression. I demonstrate that purified DeaD stimulates ExsA expression in a coupled in vitro transcription/translation assay, confirming our in vivo findings. In chapter II, I observed that RsmA activates the transcription of RsmY and RsmZ, two small non-coding RNAs that act to sequester RsmA from target mRNAs. My experiments in chapter IV begin to dissect the RsmA-activation mechanism of RsmY/Z expression. I show that RsmA activation requires the previously described Gac/Lad/Ret system that controls RsmY/Z expression. RsmA, however, does not alter Gac/Lad/Ret gene transcription or translation. Interestingly, an RsmA variant deficient in RNA-binding, RsmA R44A, was able to complement an rsmA mutant for RsmY/Z expression. I hypothesized that RsmA interacts with an unknown protein to activate RsmY/Z expression and identified several potential interaction partners using co-purification assays. Together, my combined experiments elucidate novel global regulatory pathways controlling T3SS gene expression during acute and chronic P. aeruginosa infections, and provide a foundation towards the goal of developing future treatment options.