{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/383905"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/383905","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Towards Understanding the Genetic Basis of Virulence in Pseudomonas aeruginosa","abstract":"Pseudomonas aeruginosa is an exceptionally diverse and adaptable Gram-negative bacterial pathogen, with the ability to cause acute infections at multiple sites within human hosts. It is a leading cause of healthcare-associated infections worldwide, and is a leading cause of pulmonary infections in people with cystic fibrosis (CF). Despite the impact of P. aeruginosa infections in the CF population, the underlying mechanisms that govern its pathoadaptiveness to the CF lung, and the full extent to which this contributes to changes in virulence and other biological processes, remains poorly understood. I therefore set out to investigate the effects of gene disruption on P. aeruginosa virulence traits associated with infections of the CF lung, to gain a deeper understanding of genotype-phenotype relationships, uncover potential genes for future investigation, and discover new functions of previously unannotated genes. First, I designed and then conducted high throughput phenotyping assays using an arrayed P. aeruginosa transposon mutant library covering approximately 4000 non-essential genes. Mutants were phenotypically screened using agar-based assays for six virulence factors that are associated with CF lung infections and are frequently observed to be variable in clinical isolates from people with CF (swimming and twitching motility, secretion of gelatinase and caseinase, and production of rhamnolipids and siderophores). Photographic images of phenotyping plates were analysed using machine learning analysis methods. Across all six phenotypes studied, I identified more than 500 hits in 449 genes that resulted in a change in phenotype when disrupted. Of these, 280 were genes that do not currently have functional annotations. Functional enrichment analysis of genes highlighted the importance of amino acid biosynthesis and metabolism, nucleotide biosynthesis and metabolism, and biogenesis of motility related structures. This study has laid the foundations for understanding how gene mutations contribute to changes in P. aeruginosa virulence and describes new methods for high throughput screening of arrayed mutant libraries and automated analysis of image-based data which could be transferred to the study of other pathogens and other phenotypes. Next, I employed the high throughput sequencing based method, TraDIS (Transposon Directed Insertion-site Sequencing) to simultaneously screen pooled P. aeruginosa transposon mutants for genes that are essential for metabolism and biosynthesis of essential nutrients. Pooled mutants were cultured in the presence of overlapping nutrient pools and relative abundance analysis was used to deconvolute the results to the individual substrate level. From this study, I identified 79 genes that contribute to metabolism or biosynthetic pathways of which included 25 with no current functional annotation. Functional enrichment analysis revealed the themes of bacterial motility and biofilm formation to be significant, indicating a possible relationship between changes in the expression of these two virulence behaviours and alterations to pathways involved in biosynthesis and metabolism of essential nutrients. Finally, I examined the variation in type III secretion system (T3SS) expression in P. aeruginosa isolates obtained from the lungs of CF patients to investigate the prevalence of spontaneous activation of the T3SS. To do this, I compared approximately 1200 isolates from a cohort of nine CF patients collected over a period of six months. P. aeruginosa isolates were cultured in the presence and absence of ion-chelating agent, after which culture supernatants were screened by ELISA for the presence of the T3SS needle tip protein, PcrV. I identified four distinct T3SS expression phenotypes and observed the presence of spontaneous activation of the T3SS in approximately a third of the isolates tested, which has not been observed previously. This work has contributed much needed insight into how changes in genotype may determine changes in virulence during host adaptation in relation to the expression of P. aeruginosa virulence factors and the development of auxotrophic phenotypes, which can potentially serve as a starting point for future studies. In addition, this work has provided indications of possible functions for genes that are currently lacking a confirmed functional annotation which has generated new biological information.","abstract_html":"Pseudomonas aeruginosa is an exceptionally diverse and adaptable Gram-negative bacterial pathogen, with the ability to cause acute infections at multiple sites within human hosts. It is a leading cause of healthcare-associated infections worldwide, and is a leading cause of pulmonary infections in people with cystic fibrosis (CF). Despite the impact of P. aeruginosa infections in the CF population, the underlying mechanisms that govern its pathoadaptiveness to the CF lung, and the full extent to which this contributes to changes in virulence and other biological processes, remains poorly understood. I therefore set out to investigate the effects of gene disruption on P. aeruginosa virulence traits associated with infections of the CF lung, to gain a deeper understanding of genotype-phenotype relationships, uncover potential genes for future investigation, and discover new functions of previously unannotated genes. First, I designed and then conducted high throughput phenotyping assays using an arrayed P. aeruginosa transposon mutant library covering approximately 4000 non-essential genes. Mutants were phenotypically screened using agar-based assays for six virulence factors that are associated with CF lung infections and are frequently observed to be variable in clinical isolates from people with CF (swimming and twitching motility, secretion of gelatinase and caseinase, and production of rhamnolipids and siderophores). Photographic images of phenotyping plates were analysed using machine learning analysis methods. Across all six phenotypes studied, I identified more than 500 hits in 449 genes that resulted in a change in phenotype when disrupted. Of these, 280 were genes that do not currently have functional annotations. Functional enrichment analysis of genes highlighted the importance of amino acid biosynthesis and metabolism, nucleotide biosynthesis and metabolism, and biogenesis of motility related structures. This study has laid the foundations for understanding how gene mutations contribute to changes in P. aeruginosa virulence and describes new methods for high throughput screening of arrayed mutant libraries and automated analysis of image-based data which could be transferred to the study of other pathogens and other phenotypes. Next, I employed the high throughput sequencing based method, TraDIS (Transposon Directed Insertion-site Sequencing) to simultaneously screen pooled P. aeruginosa transposon mutants for genes that are essential for metabolism and biosynthesis of essential nutrients. Pooled mutants were cultured in the presence of overlapping nutrient pools and relative abundance analysis was used to deconvolute the results to the individual substrate level. From this study, I identified 79 genes that contribute to metabolism or biosynthetic pathways of which included 25 with no current functional annotation. Functional enrichment analysis revealed the themes of bacterial motility and biofilm formation to be significant, indicating a possible relationship between changes in the expression of these two virulence behaviours and alterations to pathways involved in biosynthesis and metabolism of essential nutrients. Finally, I examined the variation in type III secretion system (T3SS) expression in P. aeruginosa isolates obtained from the lungs of CF patients to investigate the prevalence of spontaneous activation of the T3SS. To do this, I compared approximately 1200 isolates from a cohort of nine CF patients collected over a period of six months. P. aeruginosa isolates were cultured in the presence and absence of ion-chelating agent, after which culture supernatants were screened by ELISA for the presence of the T3SS needle tip protein, PcrV. I identified four distinct T3SS expression phenotypes and observed the presence of spontaneous activation of the T3SS in approximately a third of the isolates tested, which has not been observed previously. This work has contributed much needed insight into how changes in genotype may determine changes in virulence during host adaptation in relation to the expression of P. aeruginosa virulence factors and the development of auxotrophic phenotypes, which can potentially serve as a starting point for future studies. In addition, this work has provided indications of possible functions for genes that are currently lacking a confirmed functional annotation which has generated new biological information.","abstract_has_math":false,"creators":["Ellison, Louise"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Floto, andres"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-08-04","date_published":"2024-08-04","updated_at":"2026-07-22T22:23:57Z","subjects":["Pseudomonas aeruginosa"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/4444f912-d339-4fbb-8411-df9b28c636c4/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.118097","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Floto, andres"]},{"key":"dc:creator","label":"Author","values":["Ellison, Louise"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-08-04"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/383905"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Pseudomonas aeruginosa"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/4444f912-d339-4fbb-8411-df9b28c636c4/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-05-09"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.118097"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8923e617-e612-4f4b-95df-ace4d75f74f6/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Pseudomonas aeruginosa is an exceptionally diverse and adaptable Gram-negative bacterial pathogen, with the ability to cause acute infections at multiple sites within human hosts. It is a leading cause of healthcare-associated infections worldwide, and is a leading cause of pulmonary infections in people with cystic fibrosis (CF). Despite the impact of P. aeruginosa infections in the CF population, the underlying mechanisms that govern its pathoadaptiveness to the CF lung, and the full extent to which this contributes to changes in virulence and other biological processes, remains poorly understood. I therefore set out to investigate the effects of gene disruption on P. aeruginosa virulence traits associated with infections of the CF lung, to gain a deeper understanding of genotype-phenotype relationships, uncover potential genes for future investigation, and discover new functions of previously unannotated genes. First, I designed and then conducted high throughput phenotyping assays using an arrayed P. aeruginosa transposon mutant library covering approximately 4000 non-essential genes. Mutants were phenotypically screened using agar-based assays for six virulence factors that are associated with CF lung infections and are frequently observed to be variable in clinical isolates from people with CF (swimming and twitching motility, secretion of gelatinase and caseinase, and production of rhamnolipids and siderophores). Photographic images of phenotyping plates were analysed using machine learning analysis methods. Across all six phenotypes studied, I identified more than 500 hits in 449 genes that resulted in a change in phenotype when disrupted. Of these, 280 were genes that do not currently have functional annotations. Functional enrichment analysis of genes highlighted the importance of amino acid biosynthesis and metabolism, nucleotide biosynthesis and metabolism, and biogenesis of motility related structures. This study has laid the foundations for understanding how gene mutations contribute to changes in P. aeruginosa virulence and describes new methods for high throughput screening of arrayed mutant libraries and automated analysis of image-based data which could be transferred to the study of other pathogens and other phenotypes. Next, I employed the high throughput sequencing based method, TraDIS (Transposon Directed Insertion-site Sequencing) to simultaneously screen pooled P. aeruginosa transposon mutants for genes that are essential for metabolism and biosynthesis of essential nutrients. Pooled mutants were cultured in the presence of overlapping nutrient pools and relative abundance analysis was used to deconvolute the results to the individual substrate level. From this study, I identified 79 genes that contribute to metabolism or biosynthetic pathways of which included 25 with no current functional annotation. Functional enrichment analysis revealed the themes of bacterial motility and biofilm formation to be significant, indicating a possible relationship between changes in the expression of these two virulence behaviours and alterations to pathways involved in biosynthesis and metabolism of essential nutrients. Finally, I examined the variation in type III secretion system (T3SS) expression in P. aeruginosa isolates obtained from the lungs of CF patients to investigate the prevalence of spontaneous activation of the T3SS. To do this, I compared approximately 1200 isolates from a cohort of nine CF patients collected over a period of six months. P. aeruginosa isolates were cultured in the presence and absence of ion-chelating agent, after which culture supernatants were screened by ELISA for the presence of the T3SS needle tip protein, PcrV. I identified four distinct T3SS expression phenotypes and observed the presence of spontaneous activation of the T3SS in approximately a third of the isolates tested, which has not been observed previously. This work has contributed much needed insight into how changes in genotype may determine changes in virulence during host adaptation in relation to the expression of P. aeruginosa virulence factors and the development of auxotrophic phenotypes, which can potentially serve as a starting point for future studies. 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Despite the impact of P. aeruginosa infections in the CF population, the underlying mechanisms that govern its pathoadaptiveness to the CF lung, and the full extent to which this contributes to changes in virulence and other biological processes, remains poorly understood. I therefore set out to investigate the effects of gene disruption on P. aeruginosa virulence traits associated with infections of the CF lung, to gain a deeper understanding of genotype-phenotype relationships, uncover potential genes for future investigation, and discover new functions of previously unannotated genes. First, I designed and then conducted high throughput phenotyping assays using an arrayed P. aeruginosa transposon mutant library covering approximately 4000 non-essential genes. Mutants were phenotypically screened using agar-based assays for six virulence factors that are associated with CF lung infections and are frequently observed to be variable in clinical isolates from people with CF (swimming and twitching motility, secretion of gelatinase and caseinase, and production of rhamnolipids and siderophores). Photographic images of phenotyping plates were analysed using machine learning analysis methods. Across all six phenotypes studied, I identified more than 500 hits in 449 genes that resulted in a change in phenotype when disrupted. Of these, 280 were genes that do not currently have functional annotations. Functional enrichment analysis of genes highlighted the importance of amino acid biosynthesis and metabolism, nucleotide biosynthesis and metabolism, and biogenesis of motility related structures. This study has laid the foundations for understanding how gene mutations contribute to changes in P. aeruginosa virulence and describes new methods for high throughput screening of arrayed mutant libraries and automated analysis of image-based data which could be transferred to the study of other pathogens and other phenotypes. Next, I employed the high throughput sequencing based method, TraDIS (Transposon Directed Insertion-site Sequencing) to simultaneously screen pooled P. aeruginosa transposon mutants for genes that are essential for metabolism and biosynthesis of essential nutrients. Pooled mutants were cultured in the presence of overlapping nutrient pools and relative abundance analysis was used to deconvolute the results to the individual substrate level. From this study, I identified 79 genes that contribute to metabolism or biosynthetic pathways of which included 25 with no current functional annotation. Functional enrichment analysis revealed the themes of bacterial motility and biofilm formation to be significant, indicating a possible relationship between changes in the expression of these two virulence behaviours and alterations to pathways involved in biosynthesis and metabolism of essential nutrients. Finally, I examined the variation in type III secretion system (T3SS) expression in P. aeruginosa isolates obtained from the lungs of CF patients to investigate the prevalence of spontaneous activation of the T3SS. To do this, I compared approximately 1200 isolates from a cohort of nine CF patients collected over a period of six months. P. aeruginosa isolates were cultured in the presence and absence of ion-chelating agent, after which culture supernatants were screened by ELISA for the presence of the T3SS needle tip protein, PcrV. I identified four distinct T3SS expression phenotypes and observed the presence of spontaneous activation of the T3SS in approximately a third of the isolates tested, which has not been observed previously. This work has contributed much needed insight into how changes in genotype may determine changes in virulence during host adaptation in relation to the expression of P. aeruginosa virulence factors and the development of auxotrophic phenotypes, which can potentially serve as a starting point for future studies. 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