Molecular characterisation of Burkholderia pseudomallei
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.