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 Example 2.3 Staining with anti-cortactin and phalloidin 15 2.4 Staining with anti-ZO-1 and HOECHST 16 2.5 Extracting GFP and YFP plasmids from C.jejuni for transformations 16 2.6 Transformation of Cj 11168 wt with GFP and YFP plasmids 17 2.7 Scanning Electron Microscopy 18 2.7.1 Specimen preparation for SEM (cultured monolayers) 18 3.0 Results 19 3.1 Laser Scanning Confocal Microscopy 19 Staining with anti-ZO-1 and HOESCHT 23 Figure 11 (A-D) HOESCHT stains blue to visualise the nuclei and ZO-1 stains green to visualize the tight junctions 23 3.2 Scanning Electron Microscopy 24 3.3 Campylobacter jejuni invasion induces cytoskeletal rearrangement 28 3.4 Campylobacter jejuni invasion causes membrane ruffling 28 3.5 Analysis of wild type and DHtrA mutant C. jejuni by electron microscopy 29 3.2 Campylobacter jejuni invasion is time dependent 29 3.3 Campylobacter jejuni invasion induces cytoskeletal rearrangement 29 3.4 Campylobacter jejuni invasion causes membrane ruffling 30 3.5 Analysis of wild type and DHtrA mutant C. jejuni by electron microscopy 30 3.6 Transformation of Cj 11168 wt with GFP and YFP plasmids 30 4.0 Discussion 31 5.0 Conclusion 34 6.0 References 35 Abstract Campylobacter jejuni is a well known pathogenic bacteria commonly transmitted through contaminated food (Oyarzabal, et al., 2012). Its presence in the human intestinal 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 intestinal 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 manner 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 confocal 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 rear rangement. It also shows that invasion by C. jejuni is time dependent and the number 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 gastrointestinal 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