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Title: Identification and confirmation of essential genes of Actinobacillus pleuropneumoniae in vitro and in vivo
Author: Leanse, Leon
ISNI:       0000 0004 6423 6979
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Actinobacillus pleuropneumoniae (APP) is a bacterial pathogen that infects the porcine respiratory tract, causing the economically significant disease porcine pleuropneumonia. As yet, no highly efficacious or heterologous vaccine has been produced for its prevention. A better understanding of host-pathogen interactions, including the contribution of essential genes is important to elucidate novel strategies for the development of both vaccines and therapeutics. This study looked to identify the essential genes for growth of APP under numerous biologically important conditions using Transposon Directed Insertion Sequencing (TraDIS) and an individual mutant screen. The loss of diversity of an original 43,000 Tn mutant library, meant TraDIS was unsuccessful, however, a total of 16 putative essential genes were identified while screening mutants anaerobically. Using RNA-seq, changes in the APP transcriptome during in vitro and in vivo conditions were also elucidated. Findings illustrated that, compared to published in vivo (acute pig infection) transcriptome data, all conditions (growth in porcine serum, anaerobically in rich medium and in the Waxmoth Galleria mellonella) showed comparable gene expression levels, suggesting their potential as alternative models for the study of APP pathogenesis. Currently, no inducible gene expression system in APP has been published. Therefore, an anhydrotetracycline (aTc) inducible gene expression system was developed to verify predicted essential genes. After initial developmental issues, the aTc-inducible system proved functional and titratable in APP strain MIDG2331 and was used to verify atpB as essential for anaerobic growth. Genes identified as essential for anaerobic growth (atpB) or highly repressed in G. mellonella (lrp, MIDG2331_00346) were evaluated for their contribution to virulence and vaccine potential in G. mellonella. The atpB and lrp mutants were attenuated for virulence in G. mellonella, whereas deletion of a gene (MIDG2331_00346), encoding a hypothetical protein, had no effect on virulence. In addition, testing of the MIDG2331 lrp mutant suggested it may be a potential live- attenuated vaccine (LAV) candidate. Therefore, using these alternative models, coupled with the use of high-throughput methods, can provide suitable alternatives to the pig for the discovery of vaccine targets.
Supervisor: Langford, Paul R. ; Kroll, J. Simon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral