Thesis (Ph.D.)--Flinders University, School of Medicine, 2005. / Typescript (photocopy). Includes bibliographical references. Also available online.

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A programme of research was carried out to attempt the molecular characterisation of the human and animal pathogen, Burkholderia pseudomallei, the causative agent of melioidosis and the newly described avirulent species, B.thailandensis for comparative purposes. Melioidosis is still little understood, and so the clinical approach to the prevention and control of melioidosis must ultimately rest upon the basic understanding of the causative organism, particularly the pathogenic properties of B.pseudomallei. A range of B.pseudomallei and B.thailandensis isolates were cultured and the extracellular products were isolated and concentrated and an initial study conducted to identify potential target molecules for cloning. Those isolates tested were shown to have somewhat differing ECP profiles when analysed with SDS-PAGE and antigenic profiles when subject to irnmunoblotting using convalescent human serum although isolates within and between species shared a number of common bands. The ECPs were also tested for a range of activities and it was established that both species had proteolytic and phospholipase activities neither had a haemolytic activity and only isolates of B.pseudomallei had a hexosaminidase activity a putative pathogenicity determinant. Genomic DNA of B.pseudomallei was used to construct genomic libraries in a range of E. coli host vector systems. A λGTII genomic library was screened with antisera for the presence of B.pseudomallei antigens and a number of natural and synthetic substrates for the presence of haemolytic and proteolytic components. Screening yielded one stable immunopositive clone with a novel positive reaction in the form of a "halo" of reaction around the plaque. The 5 kbp cloned fragment was subcloned into a plasmid vector, and the resulting recombinant molecule, pBPGT2 was DNA sequenced and found to contain a putative pilin gene. Attempts were made to determine the size of the recombinant antigen and to further express the pilin gene product all of which were unsuccessful. A southern blot procedure confirmed the fidelity of the cloning procedure proving that the fragment was from the host organism, B.pseudomallei. A further southern blot procedure tested for the presence of the pilin sequence in a range of B.pseudomallei and B.thailandensis isolates proving the presence of the gene in only isolates of B.pseudomallei. PCR primers were designed to amplify the DNA encoding the active site of the ADP-ribosylating toxin (ET A) of Pseudomonas aeruginosa and a PCR reaction was carried out on a number of B.pseudomallei and B.thailandensis isolates. The reaction yielded a 500 bp product in only B.pseudomallei isolates and DNA sequencing of the product revealed no obvious homology to ETA of P.aeruginosa but was used as a probe to isolate a larger fragment of DNA which was found to encode a number of interesting putative genes. These included one with homology to a porin similar to that of the pathogen Neisseria gonorrhoea, with a role in virulence. During attempts to digest the genomic DNA of B.pseudomallei isolate 4845 with the restriction enzyme Sau3A two 12 kbp bands of DNA were resistant to the endonuclease activity. Attempts were made to clone these bands into a range of plasmid vectors with two clones containing deleted products. DNA sequencing proved inadequate with only a small amount of sequence information obtained. However, towards the final stages of the research project sequence information from the B.pseudomallei genome sequencing project facilitated the recognition of a 38 kbp fragment containing the sequence information from one of the clones, which encodes an alkaline protease and a putative haemagglutinin and is postulated to be a Pathogenicity Island encoding secreted virulence factors. The sequencing project also facilitated the isolation of two putative hexosaminidase genes postulated to be responsible for the activities observed when testing the B.pseudomallei isolates concentrated ECPs. Future studies for the putative genes identified and other components of B.pseudomallei are discussed.

Studies were undertaken to clone and characterise a two-component regulatory system from a clinical isolate (204) of the human and animal pathogen Burkholderia pseudomallei. A number of genomic libraries were constructed in E. coli host-vector systems and screened for the presence of a two-component system using oligonucleotide probes based on nucleotide sequence homology. Fragments of genomic DNA were cloned and sequenced and found to possess two open reading frames (ORFs) that overlap with a single nucleotide and are believed to encode a novel two-component regulatory system. A possible promoter region was identified upstream of the two ORFs, mrgR and mrgS, which read in the same direction and may represent an operon. The deduced translation of mrgR reveals a protein, MrgR, which possesses conserved motifs that are consistent with the phosphorylation domains and DNA-binding helix-turn-helix structure of a family of response regulatory proteins. The deduced translation of mrgS reveals that the MrgS protein possesses all the invariant amino acids that characterise other sensor regulatory proteins. Southern hybridisation studies showed that the mrgRS locus was present in 19 isolates of B. pseudomallei from a wide geographical derivation, but not in any closely related bacterial species, including Burkholderia thailandensis. The expression of the two genes was verified using antibodies developed to synthetic peptides based on sequences from the C- and N-terminal regions of MrgR and MrgS, respectively. The specificity of the antibodies was confirmed in Western blotting studies in which almost all of mrgR and the proximal quarter of mrgS were translationally fused with malE (MBP-MrgR and MBP-MrgS) and expressed in E. coli K12. The antibodies were used to probe Western blots of cellular and extracellular extracts of different isolates of B. pseudomallei and identified multiple bands in whole-cell lysates. The sizes of two of these bands were 24 kDa and 115 kDa, which may represent the unprocessed forms of MrgR and MrgS, respectively. It was proposed that the other bands represented either isoforms or degradation products of the full-length proteins. The recognition of all bands was abolished following pre-incubation of the antibodies with the immunising peptide but remained unaffected if an irrelevant peptide, was used for this purpose. Western blot analysis demonstrated that serum antibodies from a patient with acute melioidosis recognised MBP-MrgR but not MBP-MrgS suggesting a possible role for MrgR in the disease process. The expression of mrgR and mrgS was found to be constitutive in B. pseudomallei that had been cultured using different combinations of temperature, pH and NaCl suggesting that the genes perform a number of biological functions. There is some evidence that at 42°C the processing of MrgR and MrgS may be altered and the possible mechanisms for this are discussed. B. pseudomallei grew better at 42°C and pH 5 and less well at 25°C and pH 8 and this was influenced by NaCl concentration partly reflecting the environmental distribution and intracellular nature of the pathogen. Environmental and clinical isolates of B. pseudomallei differed in the pH optimum for growth at 42°C. The DNA flanking the mrgRS locus in isolate 204 was cloned, sequenced, and seven ORFs were identified including a transcriptional regulatory gene similar to bvgR of Bordetella pertussis. Southern blot analysis using three different DNA probes revealed restriction fragment length polymorphisms (RFLPs) in the region downstream of mrgRS. Two distinct RFLP patterns were identified among 16 different isolates of B. pseudomallei. The potential effects of this variation on gene expression and protein function await further investigation.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 132-160).

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Pathology / Master / Master of Philosophy

No description available.

Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy) Bibliography: leaves 189-223.