Investigation of the function of Campylobacter jejuni glyceraldehyde-3-phosphate dehydrogenase in iron acquisition from lactoferrin
thesisposted on 09.05.2018, 11:00 by Mohamed-Elamen Mohamed Mahmoud Fadel
Campylobacter jejuni is a major cause of bacterial food-borne disease, but the available knowledge of the biology and molecular basis of its pathogenesis is limited. Iron is an essential co-factor for the effective intestinal colonisation of C. jejuni. However, iron is not readily available due to its being tightly bound to proteins such as haemoglobin, transferrin and lactoferrin (Lf). Studies in different organisms have reported the contribution of GAPDH to iron acquisition from ferric-Tf and ferric-Lf; iron binding proteins. Therefore, this study aimed to determine the role of C.jejuni NCTC11168 GAPDH in iron uptake from host ferric-lactoferrin complex. Due to essentiality of gapA, use of the Campylobacter complementation pC46 plasmids to inactivate this gene was required. Therefore, gapA mutated strains were successfully created in which the wild type gapA allele was knocked out in a gapA merodiploid strains while the expression of the complemented gapA allele is driven by promoters of different strength. In addition, site-directed mutagenesis was used to create C150S-GAPDH protein which lacks glycolytic activity. The results of gene expression studies in this research show that the mutated gapA strains overexpressed GAPDH. Iron growth assays in MEMα medium supplemented with low concentrations of human ferric-Lf as the sole iron source indicated that GAPDH has an important role in iron uptake from Lf. This role was supported by the inhibition of the growth of C. jejuni incubated with anti-GapA antibody in cultures supplemented with ferric-Lf as the sole iron source. Moreover, GAPDH was identified for the first time as an outer membrane associated protein in C. jejuni. Using different binding assays, it was shown that GAPDH could act as an extracellular receptor for Lf. Both purified wild type GAPDH and non-glycolytic functional C150S-cjGAPDH bound different ferric-Lf family iron binding proteins, with high affinity binding of Lf compared to the other iron glycoproteins. Therefore, the binding function of GAPDH is independent from glycolytic function. In conclusion, this research demonstrates that GAPDH of C. jejuni is important agent in iron uptake from human ferric-Lf, and therefore it is probably required for successful colonisation of C. jejuni.