Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:765822 |
| Date | January 2016 |
| Creators | Salwa, Taneem |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/23654 |
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