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Understanding the Role of Anaerobic Respiration in Burkholderia Thailandensis and B

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Understanding the Role of Anaerobic Respiration in Burkholderia Thailandensis and B 1 Understanding the role of anaerobic respiration in Burkholderia thailandensis and B. pseudomallei survival and virulence Submitted by Clio Alexandra Martin Andreae to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Science May 2014 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature………………………………………………………………………………… 2 Abstract Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Northern Australia and Southeast Asia. Melioidosis can present with acute, chronic and latent infections and can relapse several months or years after initial presentation. Currently not much is known about the ways in which B. pseudomallei can persist within the host, although it has been speculated that the ability to survive within an anaerobic environment will play some role. B. pseudomallei is able to survive anaerobically for extended periods of time but little is known about the molecular basis of anaerobic respiration in this pathogenic species. Bioinformatic analysis was used to determine the respiratory flexibility of both B. pseudomallei and B. thailandensis, identifying multiple genes required for aerobic and anaerobic respiration, and molybdopterin biosynthesis. Using B. thailandensis as a model organism a transposon mutant library was created in order to identify genes required for anaerobic respiration. From this library one transposon mutant was identified to have disrupted moeA, a gene required for the molybdopterin biosynthetic pathway. This B. thailandensis transposon mutant (CA01) was unable to respire anaerobically on nitrate, exhibiting a significant reduction in nitrate reductase activity, altered motility and biofilm formation, but did not affect virulence in Galleria mellonella. It was hypothesised that the reduction in nitrate reductase activity was contributing to the phenotypes exhibited by the B. thailandensis moeA transposon mutant. To determine whether this was true an in-frame narG deletion mutant was created in B. pseudomallei. Deletion of B. pseudomallei narG (ΔnarG) resulted in a significant reduction in nitrate reductase activity, anaerobic growth, motility and altered persister cell formation, and but did not affect virulence in G. mellonella or intracellular survival within J774A.1 murine macrophages. This study has highlighted the importance of anaerobic respiration in the survival of B. thailandensis and B. pseudomallei. 3 Table of Contents Page Number Title page 1 Abstract 2 List of Contents 3 List of Figures 10 List of Tables 14 List of publications and poster presentations 16 Declaration 17 Acknowledgements 17 Abbreviations 18 Reference list 276 1. Chapter 1 – Introduction 22 1.1 Melioidosis 22 1.1.1 Global distribution and prevalence 22 1.1.2 Transmission and routes of infection 23 1.1.3 Clinical presentations and associated risk factors 25 1.1.4 Recurrent melioidosis 27 1.1.5 Treatment and antibiotic resistance 28 1.1.6 Vaccine development 29 1.2 Burkholderia pseudomallei and B. thailandensis 29 1.2.1 Genome and strain differences 29 1.2.2 Environmental survival 35 1.2.3 Virulence factors 36 1.2.4 Intracellular survival 36 1.2.5 Immune response 42 1.3 Respiratory pathways in prokaryotes 45 1.3.1 Aerobic respiration 45 1.3.2 The nitrogen cycle 45 1.3.3 Nitrate reductase 50 4 1.3.4 Nitrite reductase 50 1.3.5 Nitric oxide reductase 53 1.3.6 Nitrous oxide reductase 54 1.4 Molybdopterin biosynthesis and molybdoenzymes 55 1.4.1 Molybdopterin cofactor biosynthetic pathway 56 1.4.2 Nitrogenase 58 1.4.3 DMSO reductase family 58 1.4.4 Sulfite oxidase family 61 1.4.5 Xanthine oxidase family 61 1.5 Nitrate reductase 62 1.5.1 Membrane-bound nitrate reductase 62 1.5.2 Periplasmic nitrate reductase 66 1.5.3 Assimilatory nitrate reductase 68 1.6 Role of anaerobic respiratory proteins in pathogenesis 69 1.6.1 Wayne’s model for hypoxic shift down 70 1.6.2 A role for the membrane-bound nitrate reductase in 71 virulence 1.6.3 Role of the molybdopterin biosynthetic pathway and 72 molybdopterin in pathogenesis 1.7 Aims of this project 75 2. Chapter 2 – Materials and Methods 76 2.1. Bioinformatics 76 2.1.1. NCBI BLAST and K.E.G.G. analysis 76 2.1.2. Sequence alignments 76 2.1.3. Structure prediction 76 2.2. Burkholderia thailandensis work – transposon mutagenesis, 77 PCR and enzymatic assays 2.2.1. Media, growth conditions and bacterial strains 77 5 2.2.2. Genomic DNA and plasmid extraction 80 2.2.3. Transposon mutagenesis 80 2.2.4. Agarose gel electrophoresis and DNA visualisation 81 2.2.5. Transposon mutagenesis – Polymerase chain reaction 81 (PCR) 2.2.6. PCR confirmation of the transposon mutants 81 2.2.7. Nested PCR using arbitrary and transposon specific 82 primers 2.2.8. Transposon mutant (CA01) complementation 82 2.2.9. Tri-parental mating 85 2.2.10. Competent cell preparation 86 2.2.11. Digestion and ligation 86 2.2.12. Transformation 88 2.2.13. Isolation of chromosomal DNA 88 2.2.14. Southern blot 89 2.2.15. Griess reaction 90 2.2.16. Methyl-viologen assay on cell membrane fractions 90 2.2.17. Reverse Transcriptase PCR 91 2.2.18. Anaerobic viability assay 93 2.3. B. thailandensis in vitro and in vivo virulence assays 93 2.3.1. Swimming motility 93 2.3.2. Biofilm formation 93 2.3.3. Galleria mellonella infection assay 94 2.4. Burkholderia pseudomallei mutagenesis work 94 2.4.1. Growth media and growth conditions used for B. 94 pseudomallei work 2.4.2. pDM4 deletion mutagenesis 95 2.4.2.1 Creation of a knockout cassette 95 2.4.2.2 Ligation of knockout cassettes into pDM4 99 2.4.2.3 Conjugation into B. pseudomallei 99 2.4.2.4 Sucrose counter selection 104 6 2.4.3. Boiled PCR lysates 106 2.4.4. PCR to confirm deletion of target gene 106 2.4.5. Complementation using pBHR-MCS-1 106 2.4.6. Conjugation of pBHR vector constructs into the ΔnarG 108 mutant– Tri-parental mating 2.5. B. pseudomallei in vitro and in vivo experiments 109 2.5.1. Anaerobic growth of B. pseudomallei 109 2.5.2. Determination of NAR activity under aerobic conditions 109 2.5.3. Motility 110 2.5.4. G. mellonella challenge 110 2.5.5. Sensitivity to acidified nitrite (pH 5) 110 2.5.6. Persister cell assay 110 2.5.7. Antibiotic minimal inhibitory concentration determination 111 2.5.8. Hydrogen peroxide sensitivity 112 2.5.9. Murine infection model 112 2.5.10. Macrophage infection 113 2.5.11. Transmission electron microscopy 113 3. Chapter 3 – Bioinformatic analysis of the respiratory flexibility 115 exhibited by B. thailandensis, B. pseudomallei and B. mallei 3.1. Introduction 115 3.2. Identification of genes required for aerobic and anaerobic 117 respiration 3.2.1. Identification of respiratory proteins required for the B. 117 thailandensis E264, B. pseudomallei K96243, and B. mallei ATCC 23344 electron transport chain 3.2.2. Identification of genes required for denitrification 118 3.2.3. Nitric oxide reductase and nitrous oxide reductase in B. 121 thailandensis and B. pseudomallei 7 3.3. Bioinformatic analysis of the NAR and NIR genes encoded by B. 124 pseudomallei and B. thailandensis 3.3.1. Both B. pseudomallei and B. thailandensis encode two 124 membrane-bound nitrate reductase 3.3.2. B. pseudomallei NarG (BPSL2309) structural model 129 3.3.3. B. thailandensis and B. pseudomallei are predicted to 129 encode two putative copper nitrite reductases 3.3.4. Prediction of transmembrane helices in both putative 134 copper nitrite reductases 3.3.5. Structural prediction of both putative copper nitrite 134 reductases in B. pseudomallei 3.4. Molybdopterin biosynthetic pathway in Burkholderia 136 3.5. Discussion 138 3.6. Conclusions 149 4. Role of the molybdopterin biosynthetic pathway in anaerobic 150 growth and survival of B. thailandensis 4.1 Introduction 150 4.2 Results 151 4.2.1 B. thailandensis is an obligate respirer, only growing 151 anaerobically in the presence of an alternative electron acceptor 4.2.2 Construction of a B. thailandensis transposon mutant 156 library and identification of miniTn5Km2 insertion into BTH_I1704 4.2.3 BTH_I1704 is required for anaerobic growth and nitrate 160 reductase activity 8 4.2.4 The B. thailandensis genome encodes two putative moeA 160 genes 4.2.5 B. thailandensis can remain viable for up to one year within 165 an anaerobic environment 4.2.6 BTH_I1704 plays a role in biofilm formation and motility but 168 not virulence in Galleria mellonella 4.2.7 CA01 complementation with pDA-17::BTH_I1704 173 successfully restores anaerobic respiration, NAR activity and biofilm formation 4.3 Discussion 176 4.4 Conclusions 184 5. Chapter 5 – Deletion mutagenesis and characterisation of the 185 role of NarGHI in anaerobic nitrate respiration 5.1. Introduction 185 5.2. Results 186 5.2.1. Anaerobic growth of B. pseudomallei K96243 186 5.2.2. Identification of targets for pDM4 deletion mutagenesis 187 5.2.3. Knockout cassettes for pDM4 mutagenesis were 190 successfully created for targeted mutagenesis for BPSL2309, BPSS2299, BPSL2455 and BPSL1479 deletion 5.2.4. Creation of a BPSL2309 deletion mutant (ΔnarG) 190 5.2.5. Deletion of BPSL2309 (ΔnarG) prevents anaerobic growth 196 on nitrate and significantly reduces NAR activity 5.2.6.
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