Signature-tagged mutagenesis (STM) is an in vivo mutation-based genetic screen that allows for the identification of virulence genes. A working model of STM in the temperature-dependent rabbit model (TDRM) of Haemophilus ducreyi has been established. A unique signature-tag (ST) was inserted upstream of the aphA-3 gene in the transposon Tn1545-Delta3. Eleven different ST were used, each consisting of 21 unique nucleotides that were designed for PCR. A library of ST transposon H. ducreyi 35000 mutants was generated. A STM pool consisted of 10 unique ST-mutants that were inoculated intraepithelially into the TDRM. Ulcer development and histopathology of the lesions produced by the STM mutants inoculated at 105 CFU showed the same morphology as those produced by wild-type inoculated at 105 CFU. Ulcers that developed by day 4 were excised and recovered mutants in the output pool were screened by PCR. A previously characterized attenuated hemoglobin (HgbA) mutant was ST to serve as a positive control and was not recovered in the output pool. A total of 176 STM mutants were screened and 26 attenuated H. ducreyi mutants were recovered.
The 26 candidate genes were grouped according to their functional categories: classical virulence factors, cell surface components, stress response, DNA recombination and repair, transport, metabolism, regulation and hypothetical genes. This STM study resulted in the identification of known virulence genes such as the cdtA, hgbA and lspAl which validated the STM approach. The identification of a heat shock protein and genes involved in DNA repair, emphasize the importance of stress response genes in vivo.
Many novel genes were discovered that had not been previously identified as having a role in pathogenesis. Genes involved in metabolism were identified which highlight the fitness contribution these factors play within a hast. Two of these genes (frdA and sucA) may facilitate the ability of H. ducreyi to grow anaerobically. As in other STM studies, genes with hypothetical functions were identified. One gene, hicB, is suggested to encode an RNA toxin-antitoxin cassette. This study provides proof-of-principle for the STM approach for the recovery of genetic determinants in H. ducreyi responsible for the pathogenesis of chancroid.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29822 |
| Date | January 2009 |
| Creators | Yeung, Angela |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 256 p. |
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