Nosocomial infections associated with implanted medical- devices are on the rise due to a growing immunocompromised patient population. The organisms of interest in this study are Pseudomonas aeruginosa and Candida albicans. These organisms are opportunistic pathogens and are frequently implicated as the cause of infection and colonization of medical devices. P. aeruginosa is a motile gram-negative bacterium that is able to suppress the growth of C. albicans. Quourm sensing mimicry and biofilm formation on the hyphal surface of C. albicans by P. aeruginosa aids in suppression. C. albicans is a dimorphic fungus capable of quorum sensing with E,E-farnesol and is a central focus in this work. The goal of this project is to determine changes in protein expression when P. aeruginosa is exposed to E,E,-farnesol using 2D DIGE®. Changes in the cytosolic proteome of P. aeruginosa expose metabolic shifts that result in suppression of C. albicans. This work summarizes the effect of growth phase and concentration of E,E-farnesol on P. aeruginosa PAO1 and GSU3. Preliminary results reveal a general response of P. aeruginosa to C. albicans as changes in relevant metabolic nodes that affect pyocyanin production and the induction of virulence factors that lead to the killing of C. albicans. The overall goal of this study was to generate a profile of protein expression where a variety of conditions to further characterize the response could be easily assayed.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT).Nous avons observé une protéine précédemment identifiée, PsrA, nécessaire pour la pleine activation de l'expression du SSTT chez P. aeruginosa. Les analyses par retard de migration électrophorétique de fragments du promoteur de l'operon régulateur exsCEBA ont montré que la protéine recombinante PsrA pourrait se fixer sur celui-ci. Le mutant DpsrA a montré une diminution marquée de la sécrétion des effecteurs de type III et une faible résistance à la bactéricidie par des cellules de type phagocytaires, PLB-985. L'ensemble des résultats suggèrent que PsrA est un nouvel activateur qui est impliqué dans l'expression du SSTT en augmentant le niveau de la transcription d'exsCEBA.Dans un second temps, nous avons mis en évidence qu'un signal inhibiteur, de type quorum sensing inconnu et produit dans la phase stationnaire de la culture, peut réprimer l'expression du SSTT in vitro. L'analyse de milliers de mutants de transposition a montré que la production de ce signal dépend du tryptophane, qui est le précurseur de nombreux métabolites dont l'acide d'indole-3-acétic (IAA). IAA-Na et un autre membre de cette famille de molécules, le acide 1-naphthalenacétique (NAA-K) aux concentrations millimolaires peuvent en effet inhiber l'expression et la sécrétion du SSTT. L'identification précise de ce signal nécessite des investigations plus poussées.

The opportunistic pathogen P. aeruginosa is a leading cause of hospital-accquired infections, and is also the primary cause of morbidity and mortality in patients with cystic fibrosis (CF). In this thesis, I describe the identification and characterization of a novel LysR-type transcription regulator (LTTR) of P. aeruginosa named BexR. I show that BexR exhibits reversible ON/OFF bistable expression, which leads to the bistable expression of several genes including one encoding a virulence factor. I present results suggesting that this bistable expression depends on positive feedback of BexR. This work illuminates the simplicity with which a transcription regulatory network can exhibit a complex behavior and generate phenotypic diversity in a clonal population.

The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-­lactams, (2) expression of PBPs with low-affinity for β-­lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-­lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance. The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-­lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-­lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-­lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction. The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.

Neutrophils play an important, yet poorly understood role, in complement mediated pathologies. Here we identified that neutrophils contain key components from the complement alternative pathway: properdin (CFP), complement component 3 (C3), complement factor B (CFB), and complement factor H (CFH). Activation of neutrophils resulted in secretion of these complement components. When neutrophils are further activated to form neutrophil extracellular traps (NETs), CFP is deposited onto the surfaces of the NETs. In addition, CFP is able to bind to Pseudomonas aeruginosa, an opportunistic bacterium which can activate neutrophils to form NETs. Furthermore, NETs activate complement and increase formation of the terminal complement complex. The activation of complement on NETs can be initiated through multiple pathways, however, activation of the alternative pathway is dependent on CFP. This mechanism, potentially required for effective host defense, may also contribute to complement activation and disease.

Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

Neutrophils play an important, yet poorly understood role, in complement mediated pathologies. Here we identified that neutrophils contain key components from the complement alternative pathway: properdin (CFP), complement component 3 (C3), complement factor B (CFB), and complement factor H (CFH). Activation of neutrophils resulted in secretion of these complement components. When neutrophils are further activated to form neutrophil extracellular traps (NETs), CFP is deposited onto the surfaces of the NETs. In addition, CFP is able to bind to Pseudomonas aeruginosa, an opportunistic bacterium which can activate neutrophils to form NETs. Furthermore, NETs activate complement and increase formation of the terminal complement complex. The activation of complement on NETs can be initiated through multiple pathways, however, activation of the alternative pathway is dependent on CFP. This mechanism, potentially required for effective host defense, may also contribute to complement activation and disease.

Azithromycin improves clinical outcomes in patients with cystic fibrosis (CF), specifically in patients infected with Pseudomonas aeruginosa. Azithromycin shifts macrophage programming away from a pro-inflammatory classical (M1) phenotype, and towards an anti-inflammatory alternative (M2) phenotype; however, little is known about this mechanism, nor of its impact upon immune response to pulmonary infection. We set out to determine the mechanism by which azithromycin is able to alter macrophage phenotype, and assess the effect of azithromycin induced macrophage polarization on inflammation during pulmonary infections. Utilizing macrophage cell culture, we found that azithromycin increased IKKβ, a signaling molecule in the NFκB pathway, which likely is altering macrophage programming. Using a Pseudomonas infection model in mice that lack physiologic alternative macrophage activation, we showed that azithromycin’s ability to alter macrophage function and decrease lung damage was independent of interleukin control of macrophage programming. Azithromycin increased fibrotic protein production both in vivo and in vitro, but blunted immune-driven fibrotic damage. We extended our study to patients with CF, describing gene expression in macrophages isolated from sputum samples. We found markers consistent with a shift toward M2 polarization in these patients. These data suggest potential mechanisms by which azithromycin benefits patients with CF.

Pseudomonas aeruginosa causes various infections in humans, animals and plants. Type III secretion system (T3SS) is one of the essential virulence factors used by P. aeruginosa. In this study, a previously uncharacterized gene PA0466 and its role in T3SS regulation have been examined. The results indicate that PA0466 is a novel T3SS regulator. It regulates T3SS directly through an unknown pathway and has a minor effect on the GacA-RsmA pathway. Besides the role in the interaction between the pathogen and the host, T3SS may also play a role in the interspecies interaction. A real-time PCR based Competitive Index (CI) assay was used to compare the wild type and T3SS mutant with and without the presence of Staphylococcus spp.. The results indicate that PAO1 was more competitive than exsA mutant and the difference was even bigger in the presence of Staphylococcus, suggesting T3SS may play a significant role in bacterium-bacterium interaction.