Yersinia enterocolitica is a widespread human gastrointestinal pathogen which causes stomach cramps, nausea, vomiting and diarrhea It possesses an N-acylhomoserine lactone (AHL)-dependent quorum sensing system (QS) composed of a LuxRI homologue pair, YenR and YenI and a second ' orphan' LuxR homologue, YeaR. Its virulence weaponry, which includes the adhesin YadA, a type three secretion system (T3SS) and the Yersinia outer protein (Yap) effectors, is carried by the 70kb plasmid pYVe. When subcultured at 37°C a population of Y enterocolitica will, over time, lose the virulence plasmid and this was more pronounced in the QS mutants. In a recent microarray study the QS regulan of Y. enterocolitica whas shown to regu late a number of virulence, plasmid replication and partition genes. This study aimed to investigate the molecular mechanisms which underlie these observations in order to understand in more detail the relationship between QS. virulence plasmid stability and pathogenesis of Y. enterocoiitica 808 1. To successfully study the QS-associated plasmid loss phenotype it was first necessary to label pYVe to allow easy selection and visualization of plasmid positive ceUs. p YVe was therefore labelled with a combined gentamicin resistance and gfp cassette (GG I). Plasmid loss studies were then conducted on the Y. enleroco/ilica GG I parent and QS mutants and revealed that in the absence of gentamicin the GG 1 insertion actually increased plasmid stabi lity. Further analys is of the region into which GG I had inserted indicated that no apparent open reading frame was disrupted. Because DNA topology plays an important role in virulence plasmid gene regulation in Y enrerocolitica an analysis of the DNA secondary structure around the insertion point of GG I was conducted and revealed that upon insertion the GG 1 cassette had changed the DNA secondary structure of the region from curved to more linear. lux-based transcriptional fusion to the promoter of repA and spyA. two genes which playa central role in plasmid replication and partition, suggested that QS does not regulate their transcription and might rather act at the post-transcriptional level. To further characterize the role of QS on Y. enterocolitica pathogenic lifestyle phenotypical analysis were conducted. Swarming motility of Y. enteroco/itica is known to be QS controlled and this study extended these observations in demonstrating that YenR is directly involved in swarming control, as it activates motility in the absence of AHLs (low cell density) and represses motility the presence of AHLs (high cell density). Adhesion and invasion to Caco-2 cells and Yop production were tested in Y.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594824 |
| Date | January 2012 |
| Creators | Grasso, Marco |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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