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Title: Immunochemical diagnostic strategies based on Quorum Sensing
Author: Montagut Cañete, Enrique José
Director/Tutor: Marco Colás, Ma. Pilar
Keywords: Immunoquímica
Expressió gènica
Gene expression
Issue Date: 4-Feb-2021
Publisher: Universitat de Barcelona
Abstract: [eng] The final goal of the thesis was to improve the diagnostic of infections caused by P. aeruginosa and S. aureus through a better understanding of the bacterial communication process known as Quorum Sensing (QS) and its implication on the pathogenesis. Subsequently, this objective was based on the development of immunochemical methods to profile the expression of different QS signalling molecules, as tools to investigate the mechanisms involved in the pathogenesis of S. aureus and P. aeruginosa and also to assess the potential role of these molecules as biomarkers of infection. In this thesis, it was synthetized, for the first time, haptens against non-immunogenic molecules from the pqs system of P. aeruginosa (2-heptyl-4-quinolone (HHQ), 2-heptyl-3-hydroxy-(1H)-4- quinolone (PQS) and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO)). Regarding S. aureus, two different hapten synthesis strategies using AIP-IV as a model for other AIPs were explored based on the stability of the native molecule: one of the haptens (AIP4S) contained the original thiolactone cycle found in AIP-IV, yet it was suspected to caused stability problems during the subsequent immunization process, and the other hapten (AIP4NH) was a heterologous structure forming a lactam cycle, more stable versus hydrolysis. By means of bio-conjugation techniques all the haptens were covalently linked to highly immunogenic proteins with excellent yields. Specific polyclonal antibodies were obtained against the aforementioned QS molecules. In the case of S. aureus, the preliminary experiments using the As produced against AIP4S demonstrated higher avidity versus AIP-IV than the one produced against AIP4NH and thus, they were selected for developing the definitive immunochemical assay. Consequently, the preferred strategy for the generation of antibodies against the rest of AIPs (I to III) was the one including the original thiolactone ring. The obtained As were used to develop competitive indirect ELISAs for the quantification of the previously mentioned QS molecules. The detectability achieved in all cases was in the low-nM range, which is under the range of concentrations found in bacterial cultures. In the case of the HHQ, PQS and HQNO assays from P. aeruginosa, the detectability achieved is on the same range that the ones found in clinical samples such as sputum, allowing to continue in the near future their evaluation as biomarkers of infection in such type of clinical samples. Despite the structural similarities between the three quinolone metabolites, the generated antibodies demonstrated greater affinity versus their respective targets, which will allow to differentiate between them in biological samples where the three might be present. In the case of S. aureus, the antibodies raised against AIP4S-HCH also showed greater specificity versus AIP-IV than for any other AIP produced by this pathogen, even though the difference between AIP-I and IV was just one amino acid. As a pilot study on the evaluation of the QS molecules as biomarkers of infection, the developed ELISAs were implemented for the measurement of the mentioned QS molecules in culture broth samples from clinical isolates with different infection types and/or severity. In the case of P. aeruginosa, the assays allowed to stratify patients with distinct severity of infection (acute/chronic) relying on the amount of all three measured quinolones produced by the clinical isolates at 8h of growth. It is noteworthy that these molecules have different biological roles at signalling or physiological level and their concentration in clinical samples might be relevant for understanding the pathogenesis of a P. aeruginosa. Regarding S. aureus, the assay was able to measure AIP-IV produced by agr-IV type strains and different production profiles were observed, nonetheless a higher number of clinical isolates would have to be measured in order to obtain adequate conclusions.
[spa] El objetivo final de la tesis fue mejorar el diagnóstico de infecciones causadas por P. aeruginosa y S. aureus mediante una mejor comprensión del proceso de comunicación bacteriana conocido como Quorum Sensing (QS) y su implicación en la patogénesis. Para ello, este objetivo se basó en el desarrollo de métodos inmunoquímicos para evaluar la expresión de diferentes moléculas de señalización QS, como herramientas para investigar los mecanismos implicados en la patogénesis de S. aureus y P. aeruginosa. Del mismo modo, se evaluó en estudios piloto el papel potencial de estas moléculas como biomarcadores de infección. La tesis se dividió en dos apartados principales: herramientas inmunoquímicas para la determinación de moléculas de P. aeruginosa QS como biomarcadores de infección y, por otro lado, herramientas inmunoquímicas para la evaluación de moléculas del QS de S. aureus. Estas secciones están precedidas de capítulos introductorios basados en una revisión bibliográfica sobre el sistema de QS y su detección en ambos patógenos. En los capítulos experimentales se explica: Primero, el diseño y síntesis de haptenos para las moléculas, no inmunogénicas, del QS producidas por P. aeruginosa (2-heptil-4-quinolona (HHQ), 2-heptil-3-hidroxi-(1H)-4-quinolona (PQS) y 2-heptil-4-hidroxiquinolina N-óxido (HQNO)) y S. aureus (AIP-I a IV). En segundo lugar, la síntesis y generación de bioconjugados de anticuerpos policlonales específicos contra las moléculas QS de las dos bacterias patógenas antes mencionadas. En tercer lugar, el desarrollo de herramientas de análisis inmunoquímico para la cuantificación de estas moléculas de QS bacterianas. Posteriormente, se realizaron estudios preliminares sobre el valor potencial de moléculas QS seleccionadas como biomarcadores de infección utilizando las herramientas de análisis inmunoquímico desarrolladas.
Appears in Collections:Tesis Doctorals - Departament - Química Orgànica

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