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Hewinson, R. G. "#BETA#-Lactamase-mediated resistance to '#beta#-lactamase-stable' cephalosporins in Pseudomonas aeruginosa." Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382716.
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Silistre, Hazel. "Riboregulation in Pseudomonas aeruginosa." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32634/.
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The opportunistic human pathogen Pseudomonas aeruginosa controls virulence, production of secondary metabolites, motility, biofilm formation, growth in anaerobic conditions, intracellular and intercellular signalling and the switch from an acute to a chronic mode of infection at the transcriptional and post-transcriptional levels by modulation of the Gac/Rsm system. Cell density-dependent signal accumulation and environmental stimulators such as pH changes and ion limitation activate the GacS/GacA two-component system which in turn triggers transcription of the small regulatory RNAs RsmY and RsmZ. These sRNAs sequester multiple copies of the RNA-binding protein RsmA, antagonising its function. The RsmA/CsrA proteins act as translational repressors by binding to the GGA-motifs in the untranslated region of target mRNAs and blocking ribosome binding. In this study, the biological function of RsmN, an RsmA homologue with a conserved RNA-binding pocket but a distinct protein folding, the predicted autoregulatory mechanism of RsmN, the nature of target transcripts of RsmN, and the cross-regulation between the two Rsm proteins were investigated. The positive control of proteolytic and elastinolytic activities and swarming motility by RsmN has been demonstrated using single and inducible double deletion mutants of rsmN. Furthermore, rsmN deletion increased microcolony formation during biofilm formation. Regulation by RsmN was most apparent in the absence of RsmA, when rsmN expression was induced via a multicopy plasmid and at temperatures lower than 37°C. The double deletion of rsmA and rsmN affected growth, diminished proteolytic and elastinolytic activities, triggered autolysis and led to the increased secretion of the type VI secretion system protein Hcp1. Moreover, the double deletion of rsmA and rsmN altered the colony morphology of P. aeruginosa. Mutagenesis of the functionally critical, conserved RNA-binding residue which is identified as Arg44 in RsmA and Arg62 in RsmN resulted in the loss of RsmN function. In a genome-wide analysis by RNASeq, target transcripts were co-purified with RsmN from 37°C and 34°C cultures of a wild-type strain expressing rsmN in multicopy numbers. RNASeq results indicated that small regulatory RNAs such as CrcZ, RsmY and RgsA are common targets of RsmN and RsmA, whereas PhrS is a target of RsmN only. Other common RsmA and RsmN targets included transcriptional regulators, heat shock proteins, proteases, starvation response proteins, components of the denitrification pathway, outer membrane proteins required for pore formation, type III and type VI secretion system proteins and RsmA. Transcripts of heat shock proteins, the tss operon genes and rsmA were enriched by RsmN at 37°C but not at 34°C whereas the lasB transcript was enriched by RsmN at 34°C but not at 37°C. Based on the list of common targets of RsmA and RsmN and the results obtained from phenotypic assays, induction of the lytic Pf4 prophage, accumulation of alkyl quinolone or c-di-GMP signalling molecules, imbalanced redox state, carbon starvation, increased membrane permeability, and aggregation of misfolded proteins are suggested as possible mechanisms triggering the excessive autolysis of the rsmNind ΔrsmA mutant under uninducing conditions. The data gathered so far suggests that rsmN is differentially expressed, with increased RsmN activity at temperatures below 37°C in comparison with RsmA, and, RsmA and RsmN collectively contribute to the regulation of secondary metabolite production, motility and microcolony formation in P. aeruginosa.
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Janssen, Kayley Hope. "Post-transcriptional regulation of virulence factors in Pseudomonas aeruginosa." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5952.
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Pseudomonas aeruginosa is a Gram-negative bacterium capable of causing infections in immunocompromised individuals. The CsrA family of RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the post-transcriptional level. P. aeruginosa has a canonical CsrA family member (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for both RsmA and RsmF. The initial target aptamer was a 57 nt RNA transcript containing a central core randomized at 15 sequential positions. Most of the selected aptamers were the expected size and shared a common consensus sequence (CAnGGAyG). Longer aptamers (80-140 nts) containing two consensus-binding sites were also identified. Representative short (single consensus site) and long (two consensus sites) aptamers were tested for RsmA and RsmF binding. Whereas RsmA bound the short aptamers with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long aptamers with high affinity. Mutation of either consensus GGA site in the long aptamers reduced or eliminated RsmF binding, suggesting a requirement for two binding sites. Based on our observations that high affinity binding by RsmF appears to require two binding sites, we used an in-silico approach to search for candidate RsmF targets in the P. aeruginosa genome. We queried a library of 5’ UTRs (untranslated regions) for potential targets of RsmF based on the number and positions of GGA motifs, and secondary structure. Experimental validation of potential targets yielded few direct targets for both RsmA and RsmF indicating that additional factors contribute to differential binding in vivo.
P. aeruginosa has distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA activity is controlled by two small, non-coding regulatory RNAs (RsmY and RsmZ). In addition, we recently identified a sRNA (RsmV) that also contributes to RsmA and RsmF activity. Bioinformatic analyses suggest that these sRNAs each have 3-4 putative RsmA/RsmF bindings sites. Each site contains a GGA motif presented in the loop portion of a predicted stem-loop structure. These sRNAs regulate RsmA activity, and possibly RsmF, by sequestering RsmA and/or RsmF from target mRNAs. We characterize the contribution of each GGA site in RsmV, RsmY, and RsmZ using functional assays. We provided evidence that RsmF has more restrictive binding preferences compared to RsmA.
The type III secretion system (T3SS) is an important virulence factor that contributes to P. aeruginosa pathogenesis. Production of the T3SS is activated by host-associated signals and is tightly controlled at several levels. Global regulators including cAMP-Vfr signaling and Hfq contribute to tight regulation of the T3SS. Vfr (virulence factor regulator) is a transcription factor that responds to increased intracellular levels of cAMP. Vfr directly activates exsA transcription. ExsA activates transcription of the entire T3SS regulon. Hfq is an RNA chaperone that stabilizes sRNA and/or facilitates their binding to mRNA targets. Hfq is found in many bacteria and regulates stress responses, metabolism, and virulence. P. aeruginosa Hfq regulates about 5% of the genome and has a role in post-transcriptional control of T3SS in many Gram-negative bacteria. The mechanism of Hfq control of P. aeruginosa T3SS remains to be described. To better understand how Hfq regulates the T3SS we sought to identify mRNA targets of Hfq. Utilizing several reporters to genes involved in T3SS gene expression, we found that exsA transcription is decreased by Hfq activity. ExsA translation is also decreased by Hfq in conjunction with sRNA 179. Our findings show that Hfq may indirectly and directly regulate exsA translation.
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Vareechon, Chairut Charles. "Host-Pathogen Interaction in Pseudomonas aeruginosa Infection." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1499427972888735.
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Nelson, Shona Margaret. "Studies on biofilm growth of Pseudomonas aeruginosa PA01." Thesis, Aston University, 1993. http://publications.aston.ac.uk/12581/.
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The development of in vitro techniques to model the surface-associated mode of growth is a prerequisite to understanding more fully the physiological changes involved in such a growth strategy. Key factors believed to influence bacterial persistence in chronic infections are those of the biofilm mode of growth and slow growth rate. Methods for controlling Pseudomonas aeruginosa biofilm population growth rates were investigated in this project. This microorganism was incompatible with the in vitro 47mm diameter membrane filter-based biofilm technique developed for the study of Escherichia coli and Staphylococcus epidermidis by Gilbert et al (Appl. Environ. Microbiol. 1989, 55, 1308-1311). Two alternative methods were designed. The first comprised a 25mm diameter cellulose acetate membrane filter supported in an integral holder. This was found to be limited to the study of low microbial population densities with low flow rates. The second, based on a cylindrical cellulose fibre depth filter, permitted rapid flow rates to be studied and allowed growth rate control of biofilm and eluted cells. Model biofilms released cells to the perfusing medium as they grew and divided. The viability of released cells was reduced during, and shortly after, inclusion of ciprofloxacin in the perfusate. Outer membrane profiles of biofilm populations exhibited at least two bands not apparent in planktonic cells grown in batch and chemostat culture, and LPS profiles of biofilm populations showed variation with growth rate. Cell surface hydrophobicity of resuspended biofilm cells varied little with growth rate, whilst it decreased markedly for cells released from the biofilms as growth rate increased. Cells released from the biofilm were more hydrophilic than their sessile counterparts. Differing growth rates, LPS profiles and hydrophobicity are proposed to have a bearing on the release of cells from the adherent population.
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Lam, Howard Andrew. "Cranberry derived materials modulate quorum sensing in Pseudomonas aeruginosa." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114290.
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Cranberry-derived materials (CDMs) have recently been shown to block the group associated swarming motility in Pseudomonas aeruginosa without blocking swimming or twitching motilities. However, their mechanism of action is as of yet unknown. We hypothesized that these CDMs interfere with communication pathways important in swarming called quorum sensing (QS) and measured the impact of the CDMs on QS at three levels: QS signalling gene transcription, QS signal production, and activity/production of QS-controlled virulence factors. Crude cranberry powder extract (CP) repressed the las and rhl systems, the two main QS systems in P. aeruginosa, – as measured by the use of a lacZ bioreporter and β-galactosidase assay. CP also reduced the accumulation of both las and rhl QS signals in the medium. Exposure to 5 mg/mL or 10 mg/mL CP stimulated cell growth but still resulted in the reduction of the growth-normalized activity of key virulence factors. Cranberry-derived proanthocyanidins (cPACs) had little direct effect on the regulatory QS gene transcription or QS signal production in P. aeruginosa but reduced the activity/production of key virulence factors. Compounds found in cranberries may be clinically useful; however, further research is required to isolate the active compound(s) from CP and overcome the associated increased cell growth.Les matériaux dérivés des canneberges (MDC) ont été récemment démontré ayant la capacité de bloquer le mode de déplacement communautaire de type swarming au Pseudomonas aeruginosa sans bloquer les modes de déplacement individuels de type swimming et twitching. Pourtant, leur mode d'action est inconnu. Nous avons supposé que ces MDC interférent avec une voie de communication essentiel pour le mode de déplacement de type swarming appelé quorum sensing (QS), et nous avons mesuré l'impact des MDC sur QS à trois niveaux : la transcription des gènes de signalisation QS, la production des signaux QS, et l'activité/production des facteurs de virulence contrôlées par le QS.L'extrait de canneberges en poudre brut (CP) a réprimé la transcription des gènes de signalisation dans les systèmes las et rhl, les deux systèmes de QS majeurs au P. aeruginosa, utilisant un rapporteur biologique lacZ et un essai β-galactosidase. Le CP a aussi causé une réduction de l'accumulation des signaux pour les systèmes las et rhl dans le médium. L'addition du CP aux concentrations de 5 mg/mL ou 10 mg/mL a stimulé la croissance des cellules mais a aussi réduit l'activité et la production de plusieurs facteurs de virulence. Les proanthocyanidines d'origine de canneberges (cPACs) ont eu peu d'effet sur les systèmes QS de P. aeruginosa mais ont quand même réduit l'activité et la production de plusieurs facteurs de virulence, normalisée pour la croissance des cellules. Les MDC pourraient un jour être cliniquement utiles, mais des recherches supplémentaires sont nécessaires pour isoler le(s) composé(es) actif(s) du CP et de surmonter le problème de la croissance cellulaire augmenté.
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Qureshi, Asif Mahmood. "Studies of mutability in Methylophilus methylotrophus and Pseudomonas aeruginosa." Thesis, University of Kent, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328558.
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Beaudoin, Trevor Wayne. "Investigating the role of ndvB in Pseudomonas aeruginosa biofilms." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28352.
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Pseudomonas aeruginosa is an opportunistic pathogen prevalent in nosocomial infections and patients with cystic fibrosis. P. aeruginosa shows a high degree of antibiotic tolerance which in part can be attributed to the formation of biofilms. The increased antibiotic resistance seen in biofilms can be attributed to several factors including differential gene expression within biofilms that can lead to biofilm specific mechanisims of antibiotic resistance.
The ndvB gene is important for biofilm specific antibiotic resistance in P. aeruginosa. It is important in signaling and regulation of gene expression in other pathogens. Microarray analysis comparing gene expression between wildtype and a ndvB deletion mutant was performed to identify genes that might be regulated by the gene product, believed to be cyclic glucans, that contribute to biofilm specific antibiotic resistance. The array analysis identified 24 genes that were differentially regulated by ndvB, including a response regulator, agmR, as well as most of the genes which it regulates. Quantitative real-time polymerase chain reactions using primers specific to agmR and its associated genes confirmed that they were expressed in a ndvB related manner. Minimal bactericidal concentration assays were performed and confirmed that these genes are important in resistance to tobramycin and ciprofloxacin.
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Fu, Yinan. "Structure and dynamics of Pseudomonas aeruginosa ICP." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/723/.
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Pseudomonas aeruginosa inhibitor of cysteine peptidases (PA-ICP) is a potent protein inhibitor of papain-like cysteine peptidases (CPs) identified in Pseudomonas aeruginosa, an opportunistic pathogenic bacteria that can cause severe infections in human. It belongs to the newly characterized natural CP inhibitors of the I42 family, designated the ICP family. The members of this family are present in some protozoa and bacterial pathogens. They can inhibit both parasite and mammalian CPs with high affinity and specificity. Whether the main biological function of the proteins in the pathogens is to regulate the hydrolytic activity of the organisms’ endogenous CPs or exogenous CPs so as to facilitate the pathogens’ invasion or survival is still under investigation. Although Pseudomonas aeruginosa contains a CP inhibitor, no CP genes are found in its genome, suggesting that the targets of PA-ICP may be exogenous. This hypothesis is supported by the presence of a putative secretion signal peptide at the N-terminus of PA-ICP which may be involved in exporting the protein to target exogenous CPs. In order to shed light on the biological function and inhibitory specificity of PA-ICP, the structure and backbone dynamics of this protein were characterised using NMR spectroscopy. In this project, the inhibitory activity of PA-ICP to a range of mammalian model CPs was also studied. Like its previously studied homologs, PA-ICP adopts an immunoglobulin fold comprised of seven β-strands. Three highly conserved sequence motifs located in mobile loop regions form the CP binding site. The inhibitor exhibits higher affinity toward the mammalian CP cathepsin L than cathepsins H and B. Homology modelling of the PA-ICP-cathspin L interaction based on the crystal structure of the chgasin-cathpsin L complex shows that PA-ICP may inhibit the peptidases by blocking the enzyme’s active site and that the interactions between chagasin and CPs may be conserved in PA-ICP-peptidase complexes. The specificity of the inhibitors may be determined by the relative flexibility of the loops bearing the binding site motifs and the electrostatic properties of certain residues near the binding sites.
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Lindestam, Arlehamn Cecilia Sofie. "Intracellular triggering of inflammation by the extracellular bacterium Pseudomonas aeruginosa." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1401/.
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Abstract P.aeruginosa is an extracellular, Gram-negative opportunistic pathogen. One of the most important virulence factors during infection is the type III secretion system (T3SS). This system is found exclusively in Gram-negative bacteria and it forms a conduit between the bacteria and the host cell through which effector molecules can be translocated. These effectors alter the function of the host cell to promote survival of the bacterium. Infections are detected initially by the innate immune system via germ-line encoded receptors, pathogen recognition receptors (PRRs). These receptors recognise conserved microbial patterns, known as pathogen-associated molecular patterns and molecules which signal danger, danger-associated molecular patterns. PRRs are both membrane bound, such as Toll-like receptors (TLRs), and cytosolic, such as Nod-like receptors (NLRs). Some NLRs are involved in the formation of multimeric protein complexes, the Nod-signalosome and inflammasomes. These lead to the activation of NF-κB and the activation of caspase-1 and subsequent proteolytic processing of interleukin-1β (IL-1β) into its mature form. Both processes contribute to the inflammatory response following infection. In this study we sought to elucidate whether P.aeruginosa is able to trigger cytosolic PRRs and the mechanism of this activation. Initially we studied inflammasome activation by P.aeruginosa. We demonstrated that P.aeruginosa is able to activate the NLRC4/ASC-inflammasome complex. This was found to be dependent on a functional T3SS, but independent of any effectors passing through the system. The activation was discovered by detection of processed, and thus active caspase-1 fragments, as well as by secretion of mature IL-1β. The mechanism of the inflammasome activation was then investigated. We found that the NLRC4-dependent inflammasome activation is also dependent on extracellular potassium. An increase of extracellular potassium leads to a complete abrogation of inflammasome activation by P.aeruginosa and Salmonella. To further elucidate this finding, we investigated the leakiness of the pore formed by the T3SS in the host cell membrane. No flux of ions or small molecules could be detected in the host cell membrane following infection. However, host-membrane repair mechanisms were triggered, which could be detected by lysosomal-associated membrane protein (LAMP)-1-specific staining of the plasma membrane. We hypothesize a role for membrane potential in triggering of inflammasome activation by bacteria possessing a secretion system. Potassium-efflux has previously been identified as a activator of the NLRP3 inflammasome, but no changes in intracellular potassium could be found during this study. The activation of the NLRC4 inflammasome by the Pseudomonal strain PA103 was shown, in this study, to be independent of flagellin. Instead, the bacterial molecule responsible for inflammasome activation was shown to be pilin. Pilin is important for attachment to the host cell and the function of the T3SS. We showed that a strain lacking pilin were still able to translocate effectors through its T3SS. However, it was unable to activate the inflammasome complex. Transfection of purified pilin into cells was shown to trigger inflammasome activation. This was found to be dependent on caspase-1 but independent of NLRC4 and ASC, which is not in agreement with the results found for live bacteria. We hypothesised that the reason for this is the delivery method used, since a T3SS and infection delivers proteins and molecules differently compared to a transfection reagent. Finally, the role for Nod1 in infection by P.aeruginosa was explored. We could not identify Nod1-dependent NF-κB-activation using luciferase reporter gene assays. We therefore hypothesise that Nod1 does not have a role in the innate immune response to P.aeruginosa. In conclusion, we have identified NLRC4- and ASC-dependent inflammasome activation by P.aeruginosa. This activation was shown to be dependent on a functional T3SS and the surface protein pilin, as well as extracellular potassium. This describes a novel NLRC4-activation mechanism dependent on potassium and identifies pilin as a PRR-trigger for the first time.
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Shand, Geoffrey H. "Antibiotic resistance and outer membrane protein antigens of Pseudomonas aeruginosa." Thesis, Aston University, 1985. http://publications.aston.ac.uk/12475/.
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Turner, Keith Holte. "Bistability in Pseudomonas aeruginosa." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10159.
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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.
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Pandey, Sundar. "Novel Role of Pseudomonas Aeruginosa LptD Operon." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3734.
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Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.
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Daniel, Frank Hubert. "Extended-spectrum #beta#-lactamases from pseudomonas aeruginosa isolates collected in Turkey." Thesis, Queen Mary, University of London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267679.
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El-Falaha, B. M. A. "Resistance to antibacterial agents : studies with Escherichia coli and Pseudomonas aeruginosa." Thesis, Cardiff University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304154.
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Sherwin, Suzanna Jane. "Persister cell-mediated antimicrobial tolerance in Pseudomonas aeruginosa biofilm populations." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/334072/.
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Bacteria preferentially live in biofilms; complex structures of single- or multiple- species of bacteria, surrounded by a self-produced polymeric extracellular matrix and attached to a surface. In this state, they are extremely recalcitrant to antibiotics, in part due to the presence of dormant or very slowly replicating cells called persisters. While lacking specific antibiotic resistance genes, persister cells are able to survive lethal stress due to their slow replication state, and once the antibiotic challenge has been removed, can seed fresh populations of surviving bacteria. Despite recent progress in understanding molecular determinants of persister cell formation, few studies have examined in vitro the evolutionary and ecological drivers that sustain their presence. A functional role for persister cells is hypothesized in deferring replication within bacterial populations that are too crowded to sustain growth. This model predicts that slow-growing lineages with increased persister allocations will arise among density dependent bacterial populations, and that frequent exposure to lethal and non-lethal stresses will impact persister allocation. In support of the model, it was found that flow-cell biofilm culture rapidly resulted in new P. aeruginosa lineages with increased persistence, and that exposure to antibiotics can enhance this process. These observations have important clinical implications where chronic biofilm infections are treated with long-term antibiotic exposure. The second results chapter examines the hypothesis that hypermutable phenotypes of P. aeruginosa are a characteristic of persister cell biology. Recent research has shown that key features of the development of bacterial biofilms include increasing mutation rates and increasing numbers of persister cells and the present work addresses whether there may be a relationship between these phenomena. A detailed examination of mutation levels and SOS response-mediated gene expression was carried out in persister cells. Persisters that had been isolated using both DNA damaging (ofloxacin) and non-DNA damaging (ethanol) antimicrobial treatments exhibited significantly increased frequencies of mutation to rifampicin resistance. Gene expression analysis of persister cells isolated following ethanol treatment demonstrated up-regulation of mutS, a DNA mismatch repair gene, suggesting that persister cells may undergo elevated DNA damage and mutation that can occur independently of the SOS response. The third chapter aimed to study the role of persisters in determining the spatial and temporal pattern of biofilm formation following antibiotic treatment. A key feature of biofilms thought to play a role in antimicrobial tolerance is their ability to develop discrete, differentiated microcolony structures during colonization of a surface - these foci within biofilms are highly recalcitrant towards antimicrobials yet the factors that determine their differentiation and growth are poorly understood. This chapter therefore aimed to study the role of persisters in the initiation of microcolony foci and in mediating regrowth of biofilms. In this work, biofilm initiation was studied under a variety of conditions including with or without exposure to lethal or sub-lethal antibiotic challenge and as expected persister cell populations were able to generate significantly more biomass than in biofilms formed from non-persister populations. Dual labelling experiments were also carried out, where mixed persister and non-persister populations were tagged with either red or green fluorescent proteins. These experiments demonstrated that persister-mediated regrowth predominantly occurs as discrete microcolony foci that do not mix with the surviving non-persister cell population, suggesting that persisters are the progenitors of clonal microcolony foci within biofilms that are structurally distinct from the rest of the population of cells within the biofilm. In summary, this thesis provides new information on the biology of persister cells in P. aeruginosa biofilms, and contributes to scientific understanding of the emergence of antibiotic tolerance in bacterial biofilm populations.
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Allaway, David. "Transposon Tn5 mutagenesis in Pseudomonas aeruginosa : a study of #Beta#-lactamase expression." Thesis, Oxford Brookes University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330311.
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Bertrand, Jacob Joseph. "Genetic analysis of the Chp chemosensory system of Pseudomonas aeruginosa." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378483.
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Speaks, Tyler. "AlgR Directly Controls rsmA in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etd/2570.
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Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.
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Marwan, Mohammed Adbusalam. "Pseudomonas aeruginosa alginate and the evasion of the immune response in cystic fibrosis." Thesis, Brunel University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282915.
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Green, Angharad. "Investigating the genetic basis of preservative resistance in an industrial Pseudomonas aeruginosa strain." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/108193/.
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Pseudomonas aeruginosa is a common industrial contaminant associated with costly recalls of home and personal care(HPC) products. Preservation systems are used to prevent bacterial contamination and protect consumers, but little is known about the mechanisms of preservative resistance in P. aeruginosa. The aim of this research was to map genetic and metabolic pathways associated with preservative resistance and bacterial growth in HPC products. The genome of the industrial strain P. aeruginosa RW109 was sequenced, functionally annotated, and compared to other strains of the species. This revealed the first complete genome of a P. aeruginosa isolate from the HPC industry. Comparative analysis with 102 P. aeruginosa strains from various sources, showed industrial strains’ genomes to be significantly larger than clinical and environmental strains and RW109’s genome was the largest of the species (7.8 Mbp) and included two plasmids. Identification of differentially expressed genes by RNA-Seq (more informative than mini-Tn5-luxCDABE mutagenesis), revealed complex genetic networks utilised by RW109 when exposed to benzisothiazolone(BIT), phenoxyethanol (POE) and a laundry detergent formulation. Differential expression of five sets of genes was consistently observed in response to these industry relevant conditions - MexPQ-OpmE efflux pump, sialic acid transporter and isoprenoid biosynthesis (gnyRDBHAL) genes were frequently upregulated; whereas phnBA and pqsEDCBA genes encoding PQS production and quorum-sensing, respectively, were consistently down-regulated. Genome-scale metabolic network reconstruction of RW109, the first with a P. aeruginosa industrial strain, along with integration of transcriptomic data, predicted essential pathways for RW109’s preservative resistance (e.g. cell membrane phospholipid biosynthesis as a key pathway for POE resistance). This study highlights the utility of integrating genomic, transcriptomic and metabolic modelling approaches to uncover the basis of industrial bacterial resistance to preservative and product formulations. The ability to predict the metabolic basis of P. aeruginosa preservative resistance will inform the development of targeted industrial preservation systems, enhancing product safety and minimising future resistance development.
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Vakulskas, Christopher Anthony. "Mechanism of transcriptional activation by Pseudomonas aeruginosa ExsA." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/612.
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ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.
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Ashish, Abdul. "Impact of Pseudomonas aeruginosa Liverpool epidemic strain (LES) on cystic fibrosis patients." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2026659/.
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Cystic Fibrosis (CF) is the commonest life limiting inherited disease illness in the western world. Over the last few decades there have been many advances in the diagnosis and management of this condition. Patients born with the disease now are living into their fourth decade, which is a statement to the progress made over time. Along with the progress there have been new challenges in the world of CF. In the last two decades there have been several studies reporting the presence of transmissible Pseudomonas Aeruginosa (Psa) strains in CF clinics worldwide. The first one to be reported in UK Liverpool paediatric clinic was later identified as the Liverpool Epidemic Strain (LES). Previous studies have demonstrated chronic infection with LES can result in accelerated fall in lung function, increased hospitalisation and antibiotic requirements. This thesis looks at the effect of chronic infection of adults with CF and its implications on healthcare institutions caring for such patients. In particular on an individual level i investigated the health related quality of life associated with patients chronically infected with LES and compared it those with unique or no Psa strains. I have demonstrated that chronic infection with LES strain significantly worsens health related quality of life compared to those with unique or no Psa strains. Patients infected by transmissible Psa strains had worse physical functioning, respiratory symptoms, treatment burden, vitality, role, health perception and emotion than those with unique Psa strains (p < 0.01), and significantly poorer physical functioning, respiratory symptoms, treatment burden, body image, weight, role, and emotion than those without any Psa infection (p < 0.05). To understand the susceptibility of LES strains to common anti-pseudomonal antibiotics I studied the antibiograms of patients infected with LES strain over a 5-year period and compared the change in susceptibility to those infected with unique Psa strain. LES exhibited significantly more resistant isolates in 2004 (p < 0.0001). There was an increase in antibiotic resistance in both LES and other Psa strains over time (p < 0.001). Cox proportional hazards analysis of both unmatched (n=125) and matched (n=56) patients in 2004 revealed that LES infected patients were more likely to develop antibiotic resistant isolates over time (hazard ratio 8.1, p < 0.001). Fewer LES isolates were classed as fully sensitive in both matched and unmatched groups at the end of study period (p < 0.001). I then looked at the phenotypic characteristics exhibited by LES strain in comparison with unique Psa strain during an infective episode to elucidate whether it is a specific character of LES assisting to survive in harsh CF environment. We analyzed sets of 40 sputum samples isolates from five CF patients each chronically infected with a different non-LES strain of P. aeruginosa. For each sample (two per patient), diversity was assessed by characterising nine phenotypic traits. All P. aeruginosa populations were highly diverse. The majority of phenotypic variation found was due to within-sample variation. I demonstrated that maintenance of diverse populations in the CF lung is a general feature of P. aeruginosa infections rather than a unique characteristic of LES. To assess the healthcare resource implication on institutions caring for such patients we carried out an economic analyses of healthcare utilisation of patients chronically infected with LES to those with unique Psa strain over a four-year period. Ascertainable costs were correlated in these two groups of patients. The mean cost per patient per year was higher for LES patients for inpatient care (£4393.37 v £1817, p=0.0006), outpatient attendance (£3764 v £2515.91, p=0.0035) and also hospital antibiotic therapy (£980 v £ 505, p=0.001). Regular prescription costs were similar in both groups. Overall, the healthcare cost of caring for an adult CF patient with LES chronic infection was significantly more (1.6 times) than that for a matched patient with unique Psa strain chronic infection. Finally, I looked the effects of segregation policies instituted at Liverpool adult Cf centre in limiting cross infection. Regular genotypic surveillance of sputum samples of all patients was instituted to monitor cross infection rates. This study elucidated the cross infection policies over a 7-year period and looked at the yearly number of patients with LES and other strains. There was a decline in the proportion of patients with LES (71% to 53%) and an increase in those with unique strains (23% to 31%) and without Psa infection (6% to 17%) over the study period. Areas of potential future research based on this thesis are also outlined.
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Miller, Ian. "Post-transcriptional Regulation of RsmA In Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etd/3481.
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Pseudomonas aeruginosa is a Gram-negative bacillus found in numerous environments. Gene regulatory mechanisms such as; Two-Component Systems, transcriptional and post-transcriptional regulators, and small non-coding RNAs control the expression of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection. A significant post-transcriptional regulator involved in these regulatory networks is the Regulator of Secondary Metabolites (RsmA). In this study, we investigated the contribution of a putative stem-loop on expression of RsmA. We constructed rsmA leader fusions to measure translation with and without the stem-loop present. Secondly, we introduced point mutations to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo by western blot analysis using an HA-tagged rsmA. Our data suggests that the segment of RNA that contains the putative stem-loop structure serves some function in post-transcriptional regulation of RsmA.
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Haney, Carl Edwin. "Effects on Iron Nanoparticles on Pseudomonas Aeruginosa Biofilms." University of Dayton / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1324058048.
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Moosavi-Nasab, Marzieh. "Identification of low molecular weight compounds produced or utilized by pychrotrophic meat spoilage organisms." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27892.
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Meat Juice Medium (MJM), an aqueous extract of meat, was inoculated with Pseudomonas aeruginosa and incubated for 7 d at 4$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). Two predominant compounds produced during spoilage of MJM were detected using HPLC. These compounds with retention times (RT) of 21.48 and 32.04 min were tentatively identified as acetic and butyric acids, respectively. These compounds were also produced when MJM was replaced with Brain Heart Infusion Broth medium. In later experiments, the effect of glucose supplementation on the rate of MJM spoilage was examined. Glucose 0.5% (wt/vol) was added to the MJM, inoculated with P. aeruginosa and incubated at 30$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). HPLC of samples after 1d of incubation indicated the presence of 8 predominant compounds including acetic and butyric acids. Their concentrations were, in general, higher in control samples of MJM without added glucose. Using HPLC, TLC, Pyrolysis/GC/MS, FTIR and GC-MS methodologies, the compounds with RT of 8.91, 9.67, 11.96, 13.33, 17.74, 21.48, 26.07 and 32.04 min were tentatively identified as cadaverine, 2-keto gluconic acid, fructose, lactic acid, acetic acid, methanol and butyric acid. In contrast to the results of previous researchers, cadaverine was produced in large amounts while no putrescine was produced by P. aeruginosa. During the spoilage period, the levels of glucose, fructose and total carbohydrate were monitored. Addition of glucose to MJM delayed slime production by 4 days and increase to maximum pH of 8.3 by 7 days. Results suggest that addition of glucose to MJM delays spoilage by P. aeruginosa.
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Al-Qassemi, Rasha A. "Aspects of behaviour of Pseudomonas aeruginosa associated with water supplies." Thesis, University of Reading, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288687.
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Lee, Pei-Chung. "Effector Secretion Control by the Pseudomonas aeruginosa Type III Secretion System." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1301596980.
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Chand, Nikhilesh. "The Two-Component Sensor KinB Regulates Pseudomonas aeruginosa Virulence." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10113.
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Bacteria commonly use two-component sensors to sense and respond to their environment. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its ability to adapt to diverse environments, including the human host. Several of these sensors such as GacS have been shown to play a role in the regulation of virulence in this pathogen. However, the role of the majority of sensors remains unknown. In this thesis I show that the two-component sensor KinB is required for full P. aeruginosa virulence in the recently characterized model host Danio rerio. I found that KinB regulates several virulence-associated phenotypes in P. aeruginosa including pyocyanin and elastase production and motility. I show that KinB regulates these phenotypes through the global sigma factor AlgU, which plays a critical role in the repression of P. aeruginosa acute virulence factors and through its cognate response regulator, AlgB, albeit in a non-canonical manner. KinB’s primary role in the regulation of acute virulence is to act as a phosphatase to dephosphorylate AlgB.
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Niazy, Abdurahman. "Virulence Factor Production in PyrE Mutants of Pseudomonas Aeruginosa." Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc28458/.
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It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.
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Gooderham, William James. "Regulation of virulence and antimicrobial peptide resistance in Pseudomonas aeruginosa." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1014.
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Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA regulated genes as well as other genes consistent with its virulence phenotypes.
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Khakimova, Malika. "The role of the stringent response in the regulation of anti-oxidant defenses in «Pseudomonas aeruginosa»." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114581.
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The stringent response (SR) is a conserved bacterial stress response mechanism that allows bacteria to survive during stress and starvation. This response is mediated by (p)ppGpp, a hyperphosphorylated guanosine signal that regulates expression of genes to inhibit cellular proliferation and induce genes important for survival and adaptation. This study investigates the role of the SR in regulating oxidative stress pathways in Pseudomonas aeruginosa (PA), a human opportunistic pathogen that can cause infections in immunocompromised patients, and patients suffering from cystic fibrosis. Our results demonstrate that inactivation of the SR significantly increased susceptibility to killing by multiple oxidants, impaired anti-oxidant defenses, and increased the levels of endogenous reactive oxygen species (ROS). In addition, we demonstrated that overexpressing KatA, the primary catalase in PA, protected the ΔSR mutant from hydrogen peroxide killing as well as several antibiotics. Our results also suggested that hydroxyalkyl quinolone molecules were responsible for increased intracellular levels of ROS but did not impair catalase activity. In summary, we found that the SR was required for tolerance against oxidative stress, and regulated expression of both catalases katA and katB. Finally, overexpression of the KatA catalase was sufficient to protect against hydrogen peroxide killing, and partially protected against antimicrobial killing.La réponse stringente (RS) est un mécanisme commun aux bactéries qui leur permet de répondre et survivre aux stress nutritionels et environmentaux. Cette réponse est médiée par le signal (p)ppGpp, une petite molécule de guanosine hyperphosphorylée. Celle-ci régule l'expression des gènes afin d'inhiber la prolifération cellulaire et d'induire des gènes importants pour la survie et d'adaptation au stress. Cette étude porte sur le rôle de la RS dans la régulation des voies de stress oxydatif chez Pseudomonas aeruginosa (PA), un pathogène humain opportuniste qui peut causer des infections chez les patients immunosupprimés et les patients souffrant de fibrose kystique. Nos résultats démontrent que l'inactivation de la RS augmente grandement la susceptibilité aux oxidants, diminue les défenses anti-oxidantes, et induisent une production endogènes dérivés réactifs de l'oxygène (DRO) plus élevée. Nous avons également démontré que la sur-expression de KatA, l'enzyme catalase principale chez PA, protège le mutant ΔRS contre le peroxyde d'hydrogène ainsi que plusieurs antibiotiques bactériocides. Finalement, nos résultats suggèrent aussi que les molécules hydroxyalkyl quinolones sont responsables de l'augmentation des niveaux intracellulaires de DRO, mais n'altèrent pas l'activité des superoxide dismutases ou catalases. En résumé, la RS est nécessaire à la tolérance contre le stress oxidatif, et régule l'expression des deux catalases katA et katB. De plus, l'activité de la catalase KatA est suffisante pour protéger le mutant RS contre le peroxide d'hydrogène, et partiellement protéger contre l'effect bactericide des antibiotiques.
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Jurgens, Dawn Julie. "Pseudomonas aeruginosa biofilms in drinking water and the evolution of antibiotic resistance." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27594.
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In nature, bacteria grow attached to surfaces in communities called biofilms. Biofilm bacteria are phenotypically distinct from free-floating planktonic bacteria, and one of the phenotypic characteristics of biofilms is that they are highly resistant to antimicrobials. Because of this, biofilms growing inside water distribution systems are not killed by the residual water disinfectant. Bacteria that live inside drinking water distribution system biofilms are under stress due to the constant exposure to the residual disinfectant and the nutritional stress of living in an oligotrophic environment. These stresses can activate the bacterial stress response mechanisms, resulting in increased mutation rates. The mutations that occur are random, but the possibility exists for a mutation to arise that confers increased resistance to antibiotics.
The objective of this study was to determine if biofilm bacteria acquire antibiotic resistance when exposed to the residual water disinfectant chloramine. I grew biofilms of Pseudomonas aeruginosa in drinking water bioreactors and treated them with chloramine. The bacteria were sampled over time for isolates that were resistant to ciprofloxacin; isolates were obtained from both the chloramine treated and control biofilms. The ciprofloxacin-resistant isolates had some phenotypic differences from the wildtype P. aeruginosa, as well as being resistant to rifampicin and chloramphenicol, but not resistant to tobramycin and gentamicin.
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Pathak, Aditi. "New vaccines for infectious diseases : immunological targeting of the quorum sensing system of Pseudomonas aeruginosa." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12632/.
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Pseudomonas aeruginosa is an opportunistic pathogen of animals and humans causing medical complications in burns, wounds, and cystic fibrosis. P. aeruginosa is efficient at adapting its virulence phenotype depending on the site of infection. Emerging multi-drug resistant strains and a limited number of effective anti-pseudomonal antibiotics renders P. aeruginosa infections increasingly difficult to treat. To address this need, this thesis considers targeting bacterial quorum sensing, which regulates the production of virulence factors, as an alternative prophylactic strategy. The P. aeruginosa quorum sensing system is compromised of three interlinked but independent systems, Las, Rhl and Pqs, which produce and utilise quorum sensing system molecules, 3OC12-HSL, C4-HSL and PQS respectively. Immunological targeting of the quorum sensing system molecule 3OC12-HSL, through active immunisation (vaccines), inhibits the Las system, resulting in a longer life expectancy in mice infected with P. aeruginosa in vivo. However, P. aeruginosa has the capacity to develop resistance, through compensatory mechanisms, towards quorum sensing inhibition that targets the Las system only. This emphasises the need to target all three quorum sensing systems, Las, Rhl, and the Pqs, in order to inhibit quorum sensing. The present study focuses particularly on the development of a multi-component anti-quorum sensing system vaccine that would target the three main quorum sensing system molecules, effectively compromising the quorum sensing system and minimising compensatory mechanisms. This involved the synthesis of haptens based on the quorum sensing system molecules, which were used to haptenise the immunogenic carrier keyhole limpet haemocyanin. Syntheses of the haptens, AP1 (derivative of 3OC8-HSL) and AP2 (derivative of PQS), were conducted using adapted published methods. The resulting conjugates, AP1-keyhole limpet haemocyanin and AP2-keyhole limpet haemocyanin were immunogenic in mice and rabbits. The specific anti-hapten polyclonal antibodies that were generated demonstrated cross-reactivity with the natural quorum sensing system molecules of P. aeruginosa that translated in significant and specific anti-quorum sensing system molecule activity in bioluminescence reporter assays. Anti-AP1 polyclonal antibodies were able to reduce biofilm formation at high concentrations, however, significant reduction of biofilm formation was seen when the anti-hapten antibodies were used in combination. In this study, it has been demonstrated that the inhibition of the quorum sensing system should include the three systems, Las, Rhl and PQS, and that this can be done by a multi-component anti-quorum sensing system vaccine. These data suggest that a multi-component anti-quorum sensing system vaccine takes us a step forward to a viable prophylaxis against P. aeruginosa for susceptible patients.
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Wragg-Légaré, Stéphanie. "Characterization of the regulatory region of the Mu-like Pseudomonas aeruginosa transposable phage D3112." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59979.
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Bacteriophage D3112 is a Mu-like transposable bacteriophage of Pseudomonas aeruginosa. D3112 phage has a double-stranded DNA genome of approximately 38 kb with a genetic organization similar to that of coliphages Mu and D108. Cloning and DNA sequencing of the D3112cts15 left end has revealed a unique large open reading frame spanning nucleotides 892 to 170 of the lower strand. A polypeptide of an apparent molecular weight of 19.6 kDa was visualized by SDS-PAGE when this ORF was cloned and overexpressed in Escherichia coli.By an in vivo functional assay, it was shown that the presence of 1174 bp of the D3112 left end on an E. coli-P. aeruginosa shuttle vector in P. aeruginosa prevented lytic development of superinfecting D3112 phage, indicating that the cloned fragment encodes the D3112 (c) repressor. By an in vitro functional assay, it was shown that a crude extract, containing overexpressed repressor (c), appeared to bind specifically to a 277 bp fragment of the D3112 left end. This fragment was also bound specifically by purified E. coli IHF protein. These protein-DNA interactions, and their location, suggest a role in transcription and regulation of the repressor (c) gene for this fragment. Preliminary purification steps of the D3112 repressor (c) show no interaction with weak ion exchange and precipitation by 60% (w/v) ammonium sulfate.
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Stacey, Sean D. "Regulating rsmA Expression in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etd/1232.
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Pseudomonas aeruginosa, a Gram-negative bacillus, commonly infects immunocompromised individuals and uses a variety of virulence factors to persist in these hosts. The posttranscriptional regulator, RsmA, plays a role in the expression of many virulence factors in P. aeruginosa. RsmA up regulates virulence factors used in colonizing hosts. However, regulation of rsmA is not well elucidated. Transposon mutagenesis was performed on P. aeruginosa containing a transcriptional rsmA-lacZ fusion to answer this question. Mutants were screened via β-galactosidase assay and transposon insertions identified via arbitrary PCR. A probable MFS transporter, we named mtpX, was one significant transposon mutant identified. A ΔmtpX mutant containing the rsmA-lacZ transcriptional fusion was constructed to confirm our results. Further analysis of rsmA, looking at RNA and protein levels, revealed varying results in nonmucoid versus mucoid backgrounds. Phenotypic assays were performed to characterize this unknown transporter and develop a putative mechanism as to how MtpX affects rsmA expression.
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McDaniel, Cameron T. "Characterization of the Response of Pseudomonas Aeruginosa to the Novel Bactericidal Agent AB569 and its use as a Model Organism in Microbial Fuel Cells." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1535377019879586.
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McCaughey, Laura C. "The development of pyocins as novel antimicrobials for the treatment of Pseudomonas aeruginosa lung infection." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/6497/.
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Diaz, Manisha Regina. "Regulation of virulence gene expression by Rsm homologs in Pseudomonas aeruginosa." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4612.
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Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression and usually function to inhibit translation of target genes, but in some cases can also exert positive regulatory effects. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. Expression of the T3SS is under the direct control of ExsA, a transcriptional activator. Previous microarray analyses showed that exsA transcript levels were reduced two-fold in an rsmA mutant. In chapter II I examine the role of RsmA in regulating ExsA expression. I demonstrate that expression of a ExsA-LacZ translational fusion was reduced two-fold in an rsmA mutant suggesting a specific effect of RsmA on ExsA expression. The effect of RsmA on ExsA expression occurs at a post-transcriptional level and is independent of mRNA and protein stabilization mechanisms. RsmA directly interacts with the exsCEBA transcript at multiple sites. Truncation analyses indicate that the -37 to +85 region (relative to the ATG start codon) is necessary and sufficient for RsmA-dependent control. I identified two binding sites, BS1 (-25 bp) and BS2 (+84), involved in the interaction of RsmA with the exsA transcript using sequence analysis, site-directed mutagenesis, EMSA assays, RNase footprints, and RNaseH cleavage assays. Mutagenesis of both binding sites results in an RsmA-independent phenotype. I further demonstrate that RsmA is able to activate ExsA expression. I propose a model wherein RsmA relieves a block on ExsA translation. Collectively, this work shows that RsmA directly binds and activates ExsA expression at the post-transcriptional level.
Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. Through the course of other projects in the lab, we observed several phenotypes that could not be accounted for by a single copy of RsmA. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. RsmF is dimeric in solution. The structure of RsmF differs substantially from other CsrA homologs by having alpha-helices located between the beta-2 and beta-3 strands. In chapter III I examine the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I show that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the beta-1 and beta-5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes. In chapter IV I identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-througput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5'-RUACARGGAC-3', with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.
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Evans, Joanna. "The prevalence of IS6100 and its association with hypermutability in cystic fibrosis isolates of pseudomonas aeruginosa from local hospitals." Master's thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/2707.
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O'Reilly, Amy. "Assay development towards the characterisation of the bifunctional activity of P. aeruginosa and E. coli Class A PBPs." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/93414/.
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Pseudomonas aeruginosa is a Gram-negative species of bacteria that is of high clinical importance and is on the ESKAPE list of pathogens, causing the most serious nosocomial infections World-wide. Current antibiotics in clinical use against P. aeruginosa include carbenicillin, cefepime, ceftazidime, and the fluoroquinoline ciprofloxacin. The multi-drug resistant (MDR) phenotype exhibits widespread resistance to β-lactams and antibiotics from multiple classes often need to be administered in conjunction with β-lactamase inhibitors. Penicillin binding proteins (PBPs) are essential for cell viability and are important antibiotic targets, with the Class A bifunctional PBPs synthesising the principle component of the bacterial cell wall, peptidoglycan. The focus of this project was to investigate the kinetics of transglycosylation and transpeptidation mechanisms with a view to improving our understanding of bacterial cell wall synthesis, maintenance and regeneration. In this project, the function of Class A PBPs 1A and 1B from Escherichia coli and P. aeruginosa, with particular focus on PBP1A has been investigated. Transglycosylation and transpeptidation have been probed from multiple angles using orthogonal assays and fluorescently-labelled and native lipid substrates, as well as Lipid II structural variants. P. aeruginosa PBP1A transglycosylase and transpeptidase activities are demonstrated continuously, and compared to the model organism equivalent, E. coli PBP1A. The continuous monitoring of activity gave insights into the possibility of two catalytic sites for transglycosylation: a donor and an acceptor site. Data was fitted to different kinetic models to elucidate the best fit for extracting meaningful kinetic parameters. The data acquired over the course of this project suggests that P. aeruginosa PBP1A and E. coli PBP1A exhibit similar functional behaviour to each other, with E. coli PBP1B showing distinct activity from PBP1A. PaPBP1B activity was not detected at a level required to kinetically characterise the enzyme and it is possible that this PBP has an as yet undetermined stimulatory cofactor. The assays developed and optimised in this thesis will pave the way for further in-depth studies of P. aeruginosa PBP1A, which could be used to screen for putative inhibitor compounds and potentially identify certain characteristics required for antimicrobial efficacy. Over the past 4 years, major leaps in our knowledge of assay development, the transglycosylase and transpeptidase activities of Class A PBPs and substrate features and requirements for transglycosylase and transpeptidase donors and acceptors have thus far and will continue to unravel the unique and fascinating mechanistic intricacies of the bifunctionality of PBPs.
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Hughes, Abigail. "PA2771 Affects algZ expression and AlgZ/R Phenotypic Outputs in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etd/3462.
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Pseudomonas aeruginosa is a central nosocomial pathogen that can infect any tissue in the human body. A two-component system in P. aeruginosa that regulates many virulence factors is the AlgZ/R system. A previously unidentified regulator of algZ, that does not affect algR, has been identified via transposon mutagenesis, ‘PA2771’. The mechanism of regulation has not been previously studied, and novel evidence of PA2771 functioning as a diguanyalate cyclase was observed. When PA2771 is active, cyclic di-GMP levels are high, promoting the upregulation of the fimU operon and Type VI pili. In the PA2771 mutant, an upregulation in the expression of the flagellar genes and swarming phenotype was observed, and restored via complementation. PA2771's function in regulating algZ expression, is likely indirect and alters virulence gene regulation and phenotypic outputs in P. aeruginosa in the switch between twitching and swimming motility, and appears to be specific to PA2771.
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Hall, Clayton Wallace. "Analysis of Transcriptional Regulators Involved in Pseudomonas aeruginosa Antibiotic Resistance and Tolerance." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39483.
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Cystic fibrosis (CF) is the most common fatal genetic disorder that afflicts young Canadians. The major cause of morbidity and mortality in patients with CF is chronic pulmonary infection with the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. Once established, P. aeruginosa lung infections cannot be cleared despite sustained and aggressive antimicrobial therapy. Treatment failure of P. aeruginosa lung infections is caused by a combination of antibiotic resistance and tolerance mechanisms. Antibiotic resistance is mainly mediated by multidrug efflux pumps such as MexAB-OprM. Antibiotic tolerance has been attributed to biofilms and to nutrient starvation. In this thesis, I present an analysis of three transcriptional regulators (PA3225, RpoS, and RpoN) and their contributions to resistance and tolerance in P. aeruginosa. PA3225 is a transcriptional regulator that I initially identified as a candidate regulator of a type VI secretion system (T6SS) that had been previously implicated in biofilm tolerance. While a ΔPA3225 deletion mutant did not, unfortunately, have dysregulated expression of the T6SS, I fortuitously discovered that the mutant displayed increased resistance to various antibiotics from different functional classes. I linked the increased antibiotic resistance of ΔPA3225 to upregulation of MexAB-OprM and provided evidence that PA3225 may be a direct repressor of mexAB-oprM. Next, I sought to identify a transcriptional regulator of ndvB, which is another gene that plays a role in biofilm tolerance. I found that the stationary phase sigma factor, RpoS, was essential for expression of ndvB in stationary phase and biofilm cells. Moreover, RpoS was important for tolerance of stationary phase cells to tobramycin (TOB), an aminoglycoside antibiotic that is used to treat CF patients. In recent years, several groups have sought to identify novel treatments to combat antibiotic tolerance in P. aeruginosa. A popular strategy is metabolic potentiation, which involves co-administration of an antibiotic with a metabolite to reverse tolerance due to nutrient starvation. For example, one group found that fumarate (FUM) combined with TOB (TOB+FUM) was highly effective at killing tolerant P. aeruginosa. FUM uptake depends on C4-dicarboyxlate transporters, which are transcriptionally regulated by the alternative sigma factor, RpoN. Importantly, rpoN loss-of-function mutations are a recognised mechanism of pathoadaptation in CF clinical isolates. I demonstrated that TOB+FUM was unable to kill ΔrpoN stationary phase and biofilm cells due to loss of FUM uptake and that rpoN alleles from CF clinical isolates were unable to complement the ΔrpoN mutant. These findings could have important implications for TOB+FUM as a treatment modality in CF patients with a high burden of rpoN mutants. Overall, my work has provided interesting and, in the case of RpoN, clinically relevant insights into the regulatory networks that determine antibiotic susceptibility in P. aeruginosa.
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Intile, Peter J. "Global regulation of the Pseudomonas aeruginosa type III secretion system." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1638.
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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.
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Irvine, Sharon C. "The phenotypic, genotypic and transcriptomic characterisation of a novel Pseudomonas aeruginosa small colony variant isolated from a chronic murine infection model." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8211/.
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Mathias, Elizabeth. "Exopolysaccharides of the Pseudomonas aeruginosa Biofilm Matrix." Ohio University Honors Tutorial College / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1400069245.
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Jabir, Majid Sakhi. "The interactions between inflammasome activation and induction of autophagy following Pseudomonas aeruginosa infection." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5331/.
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Introduction Autophagy is a cellular process whereby elements within cytoplasm become engulfed within membrane vesicles and trafficked to fuse with lysosomes. This is a common cellular response to starvation, allowing non-essential cytoplasmic contents to be recycled in times of energy deprivation. However, autophagy also plays an important role in immunity and inflammation, where it promotes host defence and down-regulates inflammation. A specialised bacterial virulence mechanism, the type III secretion system (T3SS) in Pseudomonas aeruginosa (PA), an extracellular bacterium, is responsible for the activation of the inflammasome and IL-1β production, a key cytokine in host defence. The relationship between inflammasome activation and induction of autophagy is not clear. Hypothesis and aims The central hypothesis is that induction of autophagy occurs following PA infection and that this process will influence inflammasome activation in macrophages. Our aims were to determine the role of the T3SS in the induction of autophagy in macrophages following infection with PA, and to investigate the effects of autophagy on inflammasome activation and other pro-inflammatory pathways following infection with these bacteria. Materials and methods Primary mouse bone marrow macrophages BMDMs were infected with PA, in vitro. Induction of autophagy was determined using five different methods: - electron microscopy, immunostaining of the autophagocytic marker LC3, FACS, RT-PCR assays for autophagy genes, and post-translational conjugation of phosphatidylethanoloamine (PE) to LC3 using Western blot. Inflammasome activation was measured by secretion of active IL-1β and caspase-1 using ELISA and Western blot. Functional requirements of proteins were determined using knockout animals or SiRNA mediated knockdown. Result and Conclusions PA induced autophagy that was not dependent on a functional T3SS but was dependent on TLR4 and the signaling molecule TRIF. PA infection also strongly induced activation of the inflammasome which was absolutely dependent on a functional T3SS. We found that inhibition of inflammasome activation increased autophagy, suggesting that the inflammasome normally inhibits this process. Further experiments showed that this inhibitory effect was due to the proteolytic action of caspase-1 on the signaling molecule TRIF. Using a construct of TRIF with a mutation in the proteolytic cleavage site, prevented caspase-1 cleavage and increased autophagy. TRIF is also involved in the production of interferon-β following infection. We also found that caspase-1 cleavage of TRIF down-regulated this pathway as well. Caspase-1 mediated inhibition of TRIF-mediated signaling is a novel pathway in the inflammatory response to infection. It is potentially amenable to therapeutic intervention. Recognition of a pathogen infection is a key function of the innate immune system that allows an appropriate defensive response to be initiated. One of the most important innate immune defences is provided by a multi-subunit cytoplasmic platform termed the inflammasome that results in production of the cytokine IL-1β. The human pathogen Pseudomonas aeruginosa activates the inflammasome following infection in a process that is dependent on a specialized bacterial virulence apparatus, the type III secretory system (T3SS). Here, we report the novel finding that this infection results in mitochondrial damage and release of mitochondrial DNA into the cytoplasm. This initiates activation of an inflammasome based on the protein NLRC4. Autophagy induced during infection removes damaged mitochondria and acts to down-regulate NLRC4 activation following infection. Our results highlight a new pathway in innate immune activation following infection with a pathogenic bacterium that could be exploited to improve outcomes following infection.
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Patel, Neha N. "A Cationic Zn-Containing Porphyrin Exhibits Potent Antibiotic Activity Against Cells and Biofilms of the ESKAPE Pathogen Pseudomonas aeruginosa: A Mechanistic Study." University of Dayton / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1618481857088121.
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Marsden, Anne Elizabeth. "Characterization of ExsA self-association and DNA-binding as therapeutic targets to prevent expression of the Pseudomonas aeruginosa type III secretion system." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1879.
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Regulation of the Pseudomonas aeruginosa type III secretion system (T3SS) is controlled by the transcriptional activator ExsA, a member of the AraC family of regulators. Members of this family are characterized by a conserved DNA-binding domain, which contains two helix-turn-helix DNA-binding motifs and is generally located in the carboxy-terminal domain (CTD). Previous work from our lab has characterized the interaction between promoter DNA and the ExsA CTD. Two monomers of ExsA bind promoter DNA at binding sites 1 and 2 (centered at -41 and -65 relative to the transcriptional start site, respectively) to recruit RNA polymerase and activate transcription of all T3SS promoters. This interaction is required for T3SS gene expression, making it an attractive target for inhibitors designed to disrupt T3SS activity. In this study, I have characterized a group of N-hydroxybenzimidazole compounds that disrupt the ExsA-DNA interaction, leading to decreased gene expression and T3SS-mediated cytotoxicity. Furthermore, N-hydroxybenzimidazoles interact with the ExsA DNA-binding domain, and due to the conserved nature of this domain, these compounds have broad-spectrum activity against ExsA homologs.
The amino-terminal domain (NTD) of AraC family proteins is poorly conserved at the primary amino acid sequence level; however, oligomerization and/or ligand binding is commonly mediated by the NTD of AraC family proteins. The ExsA NTD is required for both self-association and interaction with the anti-activator protein ExsD. In addition to DNA-binding by the ExsA CTD, I have shown that ExsA self-association is required for maximal activation of T3SS promoters, providing another target for ExsA inhibitors. In the current model for ExsA interactions with promoter DNA, ordered occupation of binding site 1 followed by occupation of binding site 2 is facilitated by an interaction between ExsA monomers. In this study, I identified an α-helix required for ExsA self-association and showed that ExsA self-association serves two distinct roles to promote occupation of binding site 2 by a second ExsA monomer: (1) self-association relieves NTD-mediated inhibition of site 2 occupation, and (2) self-association facilitates occupation of low-affinity binding sites.
Lastly, the interaction between ExsD and ExsA prevents both ExsA self-association and DNA-binding, resulting in loss of activation at T3SS promoters. An understanding of the molecular basis for ExsA inhibition by ExsD is unknown but could provide valuable insight for the development of ExsA inhibitors. I performed site-directed and random mutagenesis to identify ExsA residues involved in the ExsA-ExsD interaction. Residues required for this interaction were identified within the ExsA self-association helix and elsewhere in the NTD; therefore, ExsD appears to prevent ExsA self-association by contacting multiple regions of ExsA, including the self-association helix. As a whole, these studies were performed to further our understanding of the protein-protein and protein-DNA interactions that are integral to the regulatory activity of ExsA and to provide direction for the development of therapeutic strategies that prevent expression of the P. aeruginosa T3SS.
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Limoli, Dominique H. "Investigating the host and microbial determinants of Pseudomonas aeruginosa mucoid conversion." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406024226.
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