Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.
| Identifer | oai:union.ndltd.org:ADTP/256471 |
| Date | January 2007 |
| Creators | Oliveira-Fry, Anna Maria, s9911120@student.rmit.edu.au |
| Publisher | RMIT University. Applied Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.rmit.edu.au/help/disclaimer, Copyright Anna Maria Oliveira-Fry |
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