Le monitoring des écosystèmes terrestres nécessite une connaissance approfondie des interactions entre microorganismes, minéraux et métaux dans les sols. Afin d'évaluer in vivo la disponibilité de métaux tel que le fer dans des systèmes bactéries-minéraux, une approche basée sur l’utilisation de biosenseurs bactériens fluorescents et d’une analyse spectroscopique non-invasive a été explorée. Ce travail a notamment conduit à la construction chez Pseudomonas aeruginosa de fusions génétiques couplant des promoteurs régulés par le fer à des rapporteurs fluorescents multicolores. Les souches obtenues ont été utilisées comme senseur de la disponibilité du fer constitutif de différents minéraux (Nontronites). La réponse de ces biosenseurs bactériens a été étudiée en couplant la spectroscopie de fluorescence à balayage synchrone (SFS) à la décomposition canonique polyadique Candecomp / Parafac (CP). Avec des plans d’expérience privilégiant la diversité des réponses, le couplage SFS-CP garantit une estimation conjointe et rapide de l’expression de plusieurs promoteurs d’intérêts, y compris dans des milieux auto-fluorescents. Cette méthode originale permet, entre autres, de s’affranchir des problèmes liés aux recouvrements spectraux des protéines fluorescentes et fournit une estimation robuste et précise de la réponse des biosenseurs. Appliquée à d’autres plans d’expériences, elle démontre également que la bio-dissolution des nontronites par P. aeruginosa est assurée par la production de sidérophores et contrôlée par la cristallochimie des feuillets des smectites, notamment par la distribution des atomes de fer(III) entre les tétraèdres et les octaèdres / Monitoring terrestrial ecosystems requires a better understanding of the interactions between microorganisms, minerals and metals in the environment. To assess in vivo availability of metals such as iron in bacteria-mineral system, an approach based on whole-cell fluorescent biosensors and non-invasive spectroscopy was explored. This work led to the construction in Pseudomonas aeruginosa of a set of gene fusions coupling iron-regulated promoters to multicolour fluorescent reporters. The recombinant strains were used as sensors of structural iron availability in nontronites NAu-1 and NAu-2. The response of these biosensors was studied by coupling synchronous fluorescence spectroscopy (SFS) with canonical polyadic Candecomp/Parafac (CP) decomposition. On the basis of experimental designs favouring response diversity, the coupled SFS-CP method guarantees a joint estimate of gene expression from multiple promoters, even in highly fluorescent media. This novel method can solve the issue of spectral bleed-through of fluorescent proteins and provides a means to integrate multiple signals from combinations of whole-cell fluorescent bioreporters. In addition, we could show using SFS-CP that P. aeruginosa indirectly mobilize Fe(III) from nontronites primarily through the production of pyoverdine siderophore. The structural Fe(III) present on the edges of NAu-2 rather than NAu-1 particles appears to be more bioaccessible, suggesting that the distribution of Fe, in the tetrahedron and/or in the octahedron sites, governs the solubilization process

Kött är skelettmuskler från olika djur som till exempel gris, nötdjur eller får. På kött kan många bakterier tillväxa. Pseudomonas är ett släkte bakterier som vanligen orsakar att mat blir dålig. De finns i vår omgivning och kan ge problem bland annat på grund av biofilmbildning inom sjukvård och på livsmedelsindustrier. Slakteriet KLS Ugglarps har tidigare sett att vissa styckningsdetaljer av gris blivit missfärgade med en blå färg och ville ta reda på orsaken till detta. Berörda delar var främst karré och kotlett. Pseudomonas aeruginosa hade tidigare hittats i lokalerna och misstänktes även i detta fall.Kött, både med och utan missfärgningar, undersöktes med hjälp av olika mikrobiologiska metoder för att se vilken bakterie som var orsaken till den blå färgen. Renodling och isolering utfördes och analyser gjordes med hjälp av API 20NE, gramfärgning och oxidastest för att kunna identifiera bakterien och ett referensisolat användes som kontroll. Det visade sig att det inte var P. aeruginosa utan istället en Pseudomonas fluorescens och denna kunde säkerställas som orsaken till blåfärgen genom att den isolerade bakterien från köttet ympades till sterilt kött och då gav en blå färg igen efter inkubering. En smittspårning utfördes i produktionslokalen för att se om bakterien kunde hittas innan uppstart samt under produktionens gång. Proverna visade vid odling att det fanns ytor i lokalen som var odlingspositiva för Pseudomonas. / Meat is skeletal muscle from different animals, such as pigs, cattle or sheep. Pseudomonas are bacteria that may cause food spoilage. The bacteria live in our environment and can cause problems due to biofilm formation in hospitals and industries. The slaughterhouse KLS Ugglarps has found that some pig cuttings have become discolored with at blue color and they wanted to find out what caused it. Pseudomonas aeruginosa had previously been found in the production area and was suspected as the cause.Meat, with and without discoloration, was investigated using various microbiological methods to see which bacterium cause the blue color. Different colonies were isolated and identified. The methods used were API 20NE, gram staining and oxidation test. A reference isolate was used as control. It was found that it was not P. aeruginosa but instead Pseudomonas fluorescens that caused the blue color. This was confirmed by applying the isolated bacteria to sterile meat and the blue color did appear after incubation. A screening for the source of contamination was performed in the production area to see if the bacterium could be found before start-up and during production. The samples taken showed that there were bacteria at both time points and that the production surfaces at the beginning of the production line were more prone to contamination than the other surfaces.

Please read the abstract in the section 00front of this document / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted

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. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

La phospholipase A2 sécrétée de type IIA (sPLA2-IIA) est une protéine possédant une activité antibactérienne très élevée envers les bactéries à Gram positif. La mucoviscidose (MV) est une maladie génétique résultant de la mutation du gène Cftr. L'altération de la fonction du CFTR dans les poumons favorise la colonisation par des pathogènes bactériens, dont les plus retrouvés, S. aureus et P. aeruginosa, colonisent les voies aériennes de manière séquentielle: S. aureus est principalement retrouvé chez les jeunes patients et P. aeruginosa chez les adultes. Les mécanismes impliqués dans ce changement de prévalence restent cependant mal connus. Dans ce travail, nous démontrons que l'expression de la sPLA2-IIA est plus importante dans les poumons de patients MV que dans les poumons de patients non MV et augmente avec l'âge des patients. La sPLA2-IIA présente dans les expectorations de patients MV est capable de tuer spécifiquement S. aureus. L'utilisation de modèles animaux nous a permis de démontrer sa spécificité d'action contre S. aureus et l'induction de son expression par P. aeruginosa contribuant à l'élimination de S. aureus des voies respiratoires. Enfin, nous avons identifié les cellules épithéliales comme une source majeure de sPLA2-IIA chez les patients MV. Dans ces cellules, P. aeruginosa induit l'expression de la sPLA2-IIA par un mécanisme dépendant de l'injection de la toxine ExoS et du facteur de transcription KLF2. L'ensemble de ces résultats indique i) que la sPLA2-IIA induite par P. aeruginosa, participe à l'élimination de S. aureus dans les voies aériennes des patients MV et ii) qu'une bactérie élimine une autre bactérie en utilisant la défense de l'hôte. / The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host defense protein endowed with antibacterial activity, especially against Gram positive bacteria. Cystic fibrosis (CF) is a genetic disease due to mutations of Cftr gene. In the lungs, CFTR mutation favored bacterial colonization by bacterial pathogens, of which S. aureus and P. aeruginosa are the most isolated. These two bacterial species sequentially colonized airways of CF patients: S. aureus is predominant in young patients and P. aeruginosa in adults. But the mechanisms involved in this switch of bacterial prevalence are still unknown. In this work we showed that sPLA2-IIA levels were increased in lungs of CF patients compared to lungs of non-CF patients and that sPLA2-IIA levels increased with age of patients. sPLA2-IIA recovered from CF patients expectorations was active and killed specifically S. aureus. Using animal models of lung infection, we demonstrated the selectivity of sPLA2-IIA against S. aureus and that P. aeruginosa induced sPLA2-IIA expression, the latter contributed to S . aureus elimination from the airways. Finally, we identified epithelial cells as a major source of sPLA2-IIA in CF airways. In these cells, P. aeruginosa induced sPLA2-IIA expression through injection of ExoS toxin and activation of KLF2 transcription factor. Taken together, these results indicate that i) P. aeruginosa-induced sPLA2-IIA expression in CF airways participated to S. aureus elimination and ii) a bacteria eliminate another bacteria by manipulating host innate immunity.

Extended-spectrum β-lactamases (ESBLs) constitute a major problem given their broad substrate specificity and ability to hydrolyse many of the extended-spectrum third-generation cephalosporins currently in use in hospital settings. Guiana extended-spectrum-type (GES-1 – GES-9) ESBL enzymes have mainly been found in Pseudomonas aeruginosa (P. aeruginosa) and only at a limited number of geographical sites, mainly France, Greece and South Africa. Detection of GES-type ESBL-producing P. aeruginosa isolates in the clinical microbiology laboratory using conventional methods is problematic with molecular methods yielding better results. The aim of this study was to utilise various molecular techniques to determine the prevalence of GES-type ESBLs, characterise their genetic determinants and determine their clonal relatedness. The study further aimed to apply a sequence-selective, competitive PNA-based multiplex PCR in real-time for the identification and differentiation of GES-type enzymes. The prevalence of GES-type ESBLs was determined successfully through DNA sequencing. An increase in GES-2 prevalence since 2000 was noted which emphasised the importance of constant surveillance to monitor antibiotic determinants, their spread and overall prevalence. The knowledge on prevalence could be used in turn to monitor the efficacy of infection control measures and antibiotic regimens. Repeated sequencing confirmed the presence of blaGES-5 in P. aeruginosa isolates. As far as could be established, this study reported the first occurrence of GES-5 in South Africa and was the second description of GES-5 in P. aeruginosa. Application of a sequence-specific, competitive PNA-based multiplex PCR in real-time utilising SYBR Green was not suitable for the identification and differentiation of the blaGES genes. Although the method achieved different melting temperatures for the bla<GES genes tested, these temperatures were not suitable for accurate differentiation. Melting temperatures obtained for the same blaGES gene varied and those for different genes overlapped. An approach exploiting the high temperature shift caused by the PNA-probe rather than its competitive nature might be more successful. Random amplified polymorphic DNA typing has been described as a fast and simple method with high discriminatory power for the typing of P. aeruginosa and was thus used to determine the clonal relatedness of the bla<GES positive P. aeruginosa isolates. The occurrence of identical or similar P. aeruginosa isolates producing ESBLs in a single hospital setting emphasised the importance of constant surveillance. The study further identified identical P. aeruginosa clones that occurred in different hospitals indicating spread from a common external reservoir into these hospitals. The occurrence of highly drug-resistant P. aeruginosa in the environment has serious implications in a country with an ever increasing immune-compromised population. These finding were of concern since they demonstrated that acquired GES ESBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. The information obtained in this study should be used to create awareness of the potential ESBL problem threatening current antimicrobial regimens in South Africa. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2008. / Medical Microbiology / unrestricted

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

Pseudomonas aeruginosa is an opportunistic pathogen that express a plethora of virulence components controlled through two-component regulatory systems that allow for sensing and responding to environmental stimuli. This study was aimed at identifying transcription regulators of algZ that encodes the histidine sensor kinase (AlgZ) of the AlgZR two-component regulatory system. To understand how the algZ gene is transcriptionally controlled, transposon mutagenesis was used to create a mutant library with varying algZ expression based on their b-Galactosidase activity. The gene PA3327 was identified as a potential regulator of algZ expression using arbitrary PCR. This gene encodes a probable non-ribosomal peptide synthetase responsible for the biosynthesis of secondary metabolites such as antibiotics. Further experiments are required to understand how PA3327 transcriptionally regulates algZ expression and its physiological role in the organism. Because the AlgZ/R system regulates virulence, it is possible to attenuate virulence by targeting the expression of algZ gene.

Please read the abstract in the front section of this document / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2005. / Medical Microbiology / unrestricted