Rhodococcus equi is a soil-dwelling actinomycete with the ability to cause pyogranulomatous infections in different animal species and people. Young foals are particularly susceptible and develop a severe pulmonary illness known as rhodococcal pneumonia. The infection is endemic in many horse-breeding farms worldwide and poses a major challenge to the equine industry, as there is no commercial vaccine available. R. equi is a facultative intracellular pathogen. Intracellular survival in macrophages and hence virulence depends on the presence of large plasmids that carry a set of genes encoding virulence-associated proteins (Vaps) of largely unknown functions. Virulence plasmids are of different types and appear to determine host-specific infectivity for horses, pigs and cattle. In this thesis, I explored bacterial chromosomal factors that contribute to the virulence of R. equi. Previous microarray transcription profiling work from the laboratory showed that housekeeping metabolic genes from the R. equi chromosome were co-opted to serve a virulence function via co-regulation with plasmid virulence genes. Here, I identified a further virulence plasmid-co-expressed metabolic chromosomal locus with a key role in R. equi pathogenesis. The identified locus, gltAB1, encodes an NADPH-dependent glutamate synthase required for ammonia assimilation under low nitrogen conditions. Reverse-transcription quantitative rea-ltime PCR confirmed that gltAB1 was co-expressed with the vap genes from the plasmid whereas a homologous chromosomal locus encoding a second NADPH-dependent glutamate synthase, gltAB2, was not. In-frame deletion mutants were constructed and their virulence analysed. gltAB1 but not gltAB2, was found to be involved in virulence and required for intracellular proliferation in J774A.1 macrophages. The ΔgltAB1 mutant showed significant attenuation in vivo in a mouse infection model, in contrast to the ΔgltAB2, which behaved like the wild type. The ability of the ΔgltAB1 mutant strain to act as a live attenuated vaccine was tested in experiments in BALB/c mice. The mutant conferred protection against subsequent challenge of the animals with wild-type virulent bacteria, thus identifying a novel candidate vaccine for the control of R. equi pneumonia in foals. Furthermore, this thesis describes studies of the bovine-type plasmid, previously sequenced in our laboratory. The purpose of this work was to determine if VapN, the bovine-type allelic variant of the VapA protein encoded in the equine-type plasmid, was also essential for R. equi virulence. A plasmid-less derivative strain and a deletion mutant in the vapN gene were examined for their ability to proliferate in two different cell lines and to persist in BALB/c mice. These strains showed the same strong virulence attenuation observed with plasmid-less and ΔvapA strains derived from the equine isolate R.equi 103S, demonstrating that the bovine-type VapN protein also plays a central role in R. equi virulence. Additionally, the thesis includes preliminary work on approaches to explore the role and mechanism of Vap proteins in R. equi virulence. It also describes the construction of GFP-tagged R. equi strains for use in cell biological experiments and live imaging of infected cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:699956 |
| Date | January 2014 |
| Creators | Hapeshi, Alexia |
| Contributors | Vazquez-Boland, Jose ; Haas, Juergen |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17281 |
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