LIVE CELL IMAGING OF BACTERIAL PATHOGENS BREACHING THE BARRIER OF Dissertation

LIVE CELL IMAGING OF BACTERIAL PATHOGENS BREACHING THE BARRIER OF POLARIZED EPITHELIAL CELLS - Dissertation Example2.3 Staining with anti-cortactin and phalloidin152.4 Staining with anti-ZO-1 and HOECHST162.5 Extracting GFP and YFP plasmids from C.jejuni for transformations162.6 Transformation of Cj 11168 wt with GFP and YFP plasmids172.7 Scanning Electron Microscopy 182.7.1 Specimen preparation for SEM (cultured monolayers)183.0 Results19 3.1 Laser Scanning Confocal Microscopy 19Staining with anti-ZO-1 and HOESCHT23 bod 11 (A-D) HOESCHT stains blue to visualise the nuclei and ZO-1 stains green to visualize the tight junctions233.2 Scanning Electron Microscopy 243.3 Campylobacter jejuni invasion induces cytoskeletal rearrangement283.4 Campylobacter jejuni invasion causes tissue layer ruffling283.5 Analysis of wild type and DHtrA mutant C. jejuni by electron microscopy 293.2 Campylobacter jejuni invasion is time dependent293.3 Campylobacter jejuni invasion induces cytoskeletal rearran gement293.4 Campylobacter jejuni invasion causes membrane ruffling303.5 Analysis of wild type and DHtrA mutant C. jejuni by electron microscopy 303.6 Transformation of Cj 11168 wt with GFP and YFP plasmids304.0 Discussion315.0 Conclusion346.0 References35AbstractCampylobacter jejuni is a well known morbific bacteria commonly transmitted through contaminated food (Oyarzabal, et al., 2012). Its presence in the human enteric gut causes disease and is one of the leading causes of gastroenteritis in human beings. By crossing the polarized epithelial barrier in the gut, it leads to increased damage to enteric tissue although the exact mechanism by which it accomplishes this is not yet defined (Boehm, 2011). As such, the research aimed to determine this through a series of infection studies. Results of the modality in which C. jejuni breeches the barrier of polarized epithelial cells was captured through the use of high resolution field emission scanning electron microscopy (FESEM) and c onfocal microscopy, and the results through various strains of the pathogenic bacteria evaluated. The results of the experiments carried out in this study indicated that invasion of an epithelial cell by C. jejuni causes membrane ruffling and cytoskeletal rearrangement. It also shows that invasion by C. jejuni is time dependent and the descend of pathogenic bacteria increases with time. It also shows that C. jejuni strains can also undergo transformation with the GFP and YFP in order to ensure genetic diversity and replication. 1.0 Introduction The human gastro intestinal tract has a mucosal cell layer that forms a strong barrier which protects the body from invasion by pathogenic microbes commonly residing in the intestinal lumen (Boehm et al., 2012). This class of bacteria, commonly referred to as enteric pathogenic bacteria, include Salmonella, Shigella, Heliobacter, and Campylobacter among others possess specific properties that enable them to