Citrobacter rodentium infection in mice to dissect host pathogen relationship in the gut
Abstract
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.
Authors
Salwa, TaneemCollections
- Theses [4201]