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.

Staphylococcus aureus has developed resistance to several antibiotics including vancomycin, which is often used as a “last resort” treatment. There is an ever-increasing need to develop novel antimicrobial treatments to combat S. aureus and other drug resistant bacteria. Microorganisms are most often found in polymicrobial communities where they either exhibit synergistic or antagonistic relationships. Competition between microorganisms can lead to the discovery of new antimicrobial targets as the specific mechanisms of resistance are elucidated. In addition, synergistic treatments are being evaluated for their combined effect and potential to decrease the concentration of drugs needed, and thus the side effects also. Alcaligenes faecalis is a microorganism that our lab has previously shown to inhibit S. aureus and other various bacterial species. In this study, we found that A. faecalis reduces the planktonic growth of S. aureus by 94.5% and biofilm growth by 76.6%. A. faecalis also has a synergistic effect when paired with bacitracin to reduce the planktonic growth by 99.9% and biofilm growth by 99.7%. Transposon mutagenesis was successfully performed on A. faecalis, and loss of function mutations were attained. Two mutants were no longer able to inhibit the growth of Staphylococcus aureus, Candida albicans, or Bacillus megaterium. Further analysis and genomic sequencing of these mutants is needed to determine the gene(s) that were interrupted and the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus treatments.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173(附属図書館) / 26||人博||722(吉田南総合図書館) / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

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Bovine herpesvirus-1 (BoHV-1) is an important pathogen of cattle associated with respiratory and reproductive disease and is the most common viral agent implicated in the bovine respiratory disease complex (BRDC). BRDC is an economically significant multifactorial disease of feedlot cattle estimated to cost Australian feedlot producers $AU60 million/year in lost production, therapeutics and disease management. Worldwide BRDC is attributed to cost $US2 billion to cattle industries. In an effort to limit the associated economic costs and enhance animal health and welfare of feedlot cattle, the concerted use of vaccination and diseased animal management are practiced. Numerous vaccines are available in North America and Canada however, in Australia, feedlot producers are reliant on three vaccines. These vaccines target either the bacterial or viral agents of the BRDC and encompass antibody, subunit and attenuated live BoHV-1 preparations. Live attenuated vaccines are developed by numerous methods including, deletion or disruption of certain genes. The development of an attenuated live virus vaccine was traditionally a laborious task requiring numerous rounds of in vitro purification. Contrastingly, technological advances introduced this decade, allowing the stable maintenance of the complete herpesvirus genome in bacteria as a bacterial artificial chromosome (BAC), has advanced herpes virology exponentially in that investigation and manipulation of the herpesvirus genome can be conducted independent of a cell culture system. With respect to BRDC and the generation of vaccines to combat the disease, the tools to fully utilise the potential of BoHV-1 as a live vaccine vector are now routine. It is now possible to vii construct BoHV-1 as a delivery vector by inserting appropriate antigens of those bacterial and viral pathogens implicated in the BRDC into a BAC maintained BoHV-1 genome. However, there is a significant lack of genetic information regarding BoHV-1 and inserting several antigenic sequences would expand the genome of BoHV-1 inducing non-viability. Therefore, to further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and nonessential genes required for the in vitro viability of BoHV-1. Identifying the essential and nonessential genes will establish which genes may be preferentially deleted or replaced with exogenous antigenic sequences in a BoHV-1 derived vaccine vector. To define the requirement of genes encoded by BoHV-1, random-insertion mutagenesis utilising a Tn5 transposition system and targeted gene deletion catalysed by GET recombination was employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion was determined by direct sequencing. with the essential or nonessential requirement of either transposed or deleted open reading frames (ORFs) assessed by transfection of respective BoHV- 1 BAC DNA into host cells. Of the 73 recognised ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be nonessential for virus viability in cell culture with the requirement of the two dual copy ORFs inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by Human herpesvirus 1. However, ORFs encoding for glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to Human herpesvirus-1 encoded homologues. Further analysis of clones encompassing restriction digestion profiling, one-step growth and replication kinetic analysis defined the genetic constitution and replicative capacity of the mutant clones. Thirty-three individual ORFs of the 36 defined nonessential ORF were identified as being amenable to deletion without causing significant replicative detriment to a potential BoHV-1 vaccine vector. This study has provided the foundational information required for the future development of BoHV-1 as a multivalent vaccine vector for the protection of feedlot cattle from BRDC. Furthermore, the genetic information generated in this study contributes to the general knowledge of the prototype ruminant herpesvirus, BoHV-1, and contributes to the comparative study of gene function between the large and diverse family that is Herpesviridae.

Bovine herpesvirus-1 (BoHV-1) is an important pathogen of cattle associated with respiratory and reproductive disease and is the most common viral agent implicated in the bovine respiratory disease complex (BRDC). BRDC is an economically significant multifactorial disease of feedlot cattle estimated to cost Australian feedlot producers $AU60 million/year in lost production, therapeutics and disease management. Worldwide BRDC is attributed to cost $US2 billion to cattle industries. In an effort to limit the associated economic costs and enhance animal health and welfare of feedlot cattle, the concerted use of vaccination and diseased animal management are practiced. Numerous vaccines are available in North America and Canada however, in Australia, feedlot producers are reliant on three vaccines. These vaccines target either the bacterial or viral agents of the BRDC and encompass antibody, subunit and attenuated live BoHV-1 preparations. Live attenuated vaccines are developed by numerous methods including, deletion or disruption of certain genes. The development of an attenuated live virus vaccine was traditionally a laborious task requiring numerous rounds of in vitro purification. Contrastingly, technological advances introduced this decade, allowing the stable maintenance of the complete herpesvirus genome in bacteria as a bacterial artificial chromosome (BAC), has advanced herpes virology exponentially in that investigation and manipulation of the herpesvirus genome can be conducted independent of a cell culture system. With respect to BRDC and the generation of vaccines to combat the disease, the tools to fully utilise the potential of BoHV-1 as a live vaccine vector are now routine. It is now possible to vii construct BoHV-1 as a delivery vector by inserting appropriate antigens of those bacterial and viral pathogens implicated in the BRDC into a BAC maintained BoHV-1 genome. However, there is a significant lack of genetic information regarding BoHV-1 and inserting several antigenic sequences would expand the genome of BoHV-1 inducing non-viability. Therefore, to further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and nonessential genes required for the in vitro viability of BoHV-1. Identifying the essential and nonessential genes will establish which genes may be preferentially deleted or replaced with exogenous antigenic sequences in a BoHV-1 derived vaccine vector. To define the requirement of genes encoded by BoHV-1, random-insertion mutagenesis utilising a Tn5 transposition system and targeted gene deletion catalysed by GET recombination was employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion was determined by direct sequencing. with the essential or nonessential requirement of either transposed or deleted open reading frames (ORFs) assessed by transfection of respective BoHV- 1 BAC DNA into host cells. Of the 73 recognised ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be nonessential for virus viability in cell culture with the requirement of the two dual copy ORFs inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by Human herpesvirus 1. However, ORFs encoding for glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to Human herpesvirus-1 encoded homologues. Further analysis of clones encompassing restriction digestion profiling, one-step growth and replication kinetic analysis defined the genetic constitution and replicative capacity of the mutant clones. Thirty-three individual ORFs of the 36 defined nonessential ORF were identified as being amenable to deletion without causing significant replicative detriment to a potential BoHV-1 vaccine vector. This study has provided the foundational information required for the future development of BoHV-1 as a multivalent vaccine vector for the protection of feedlot cattle from BRDC. Furthermore, the genetic information generated in this study contributes to the general knowledge of the prototype ruminant herpesvirus, BoHV-1, and contributes to the comparative study of gene function between the large and diverse family that is Herpesviridae.

Vibrio anguillarum infects many fish species in aquaculture, reducing farm productivity and negatively impacting fish welfare. Deeper understanding of the biology of V. anguillarum, particularly during infections in vivo, will help to improve disease prevention and control. Thus, the aim of this thesis was to provide further insight into the infection biology of V. anguillarum with a view to identifying better ways to reduce the impact of this pathogen in aquaculture. Conventional studies on virulence, particularly those aiming to identify novel virulence factors, often employ transposon mutagenesis where the functions of individual genes in the bacterium are disrupted. These mutant libraries are screened to identify those with attenuated virulence, allowing subsequent identification of the gene responsible. Usually the native fish host would be used but such studies are increasingly difficult to perform due to regulations on vertebrate experiments and ethical concerns. As a result, alternative invertebrate hosts are now an important means to studying microbial infections, but few models have been assessed for bacterial pathogens of fish. In this thesis, larvae of the greater wax moth Galleria mellonella were evaluated as an alternative host to investigate V. anguillarum virulence. Wild-type V. anguillarum isolates killed larvae in a dose-dependent manner, replicated in the haemolymph, and larvae infected with a lethal dose of bacteria could be rescued by antibiotic therapy, thus indicating that V. anguillarum established an infection in G. mellonella. Crucially, virulence of 11 wild-type V. anguillarum isolates correlated significantly between larva and Atlantic salmon infection models, and studies with isogenic mutants knocked out for various virulence determinants revealed conserved roles for some in larva and fish infections, including the pJM1 virulence plasmid and rtxA toxin. Thereafter, 350 strains from a V. anguillarum random transposon insertion library were screened for attenuated virulence in G. mellonella. In total, 12 strains had reduced virulence and in these mutants the transposon had inserted into genes encoding several recognised and putative virulence factors, including a haemolytic toxin (vah1) and proteins involved in iron sequestration (angB/G and angN). Importantly, the transposon in one strain had inserted into an uncharacterised hypothetical protein. Preliminary investigations found this putative novel virulence factor to contain a GlyGly-CTERM sorting domain motif, with sequence similarity to VesB of Vibrio cholerae which is involved in post-translational processing of cholera toxin. Finally, three transposon insertion libraries were mass sequenced on a MiSeq platform to identify V. anguillarum genes lacking transposon insertions. These genes were assumed to be ‘required’ for viability in the conditions under which the mutants were selected, in this case tryptone soya agar. In total, 248 genes lacked a transposon insertion and were the putative ‘required’ genes, and these may be important chemotherapeutic targets for new approaches to combat V. anguillarum infections. This thesis has furthered our understanding of the biology of the important fish pathogen V. anguillarum using an ethically acceptable approach, and the findings may assist with new ways to reduce the burden of this bacterium in aquaculture.