| dc.description.abstract | Worldwide nearly 2.8 million people are infected by STEC with acute illness, among them approximately 0.14% lead to HUS and 0.008% lead to death. Shiga toxins are essential virulence factors of STEC, but also other factors have been reported to be involved in virulence of such bacteria. Adherence to the target intestinal epithelial cell is an important initial step to establish STEC infection, with intimin is the major adhesin. Besides intimin, STEC express fimbrial proteins such as long polar fimbriae (LPF) which also have a role as adhesion factors. For some fimbrial and putative fimbrial proteins the function in virulence is still not clear. The aim of this study was to clone the putative fimbrial operon lpfB1 into a wild type STEC strain and analyse expression of fimbrial operon genes in this strain under different growth conditions.
First PCR primers for the lpfB1 operon and one of the genes within the operon were designed, based on the nucleotide sequence of the operon from a genome sequenced strain. After optimization of the PCR, the operon with up- and downstream adjacent sequence was amplified. The identity of the PCR product was confirmed by gel electrophoresis and Sanger sequencing. The PCR product was then inserted in a cloning vector pCR-4 TOPO, which was first transformed in TOP10 competent cells. This resulted in TOP10 cells with lpfB1 insert-vector. Plasmid was extracted from the transformed cells and plasmid with the lpfB1 insert (named pDA00) and plasmid without insert (named pDA00) were amplified with PCR and performed electrophoresis to confirm the presence of lpfB1 operon. These plasmids (pDA00 and pDA09) were further cloned into a clinical wild type strain STEC O103:H2, which had lost its stx genes. Transformed cells were again tested with PCR and electrophoresis to confirm the lpfB1 operon. Transformed bacterial cells were then cultured in LB broth and SILACE broth. From these cultures RNA was extracted, and reverse transcribed to cDNA using random primers. The amplification efficiency of the real-time PCR for the lpfB1C gene, and the housekeeping gene rpoA was tested, and found to be 99% and 105% respectively. Then cDNA was amplified by real time PCR of the lpfB1C gene, using rpoA as reference, to study the expression of the operon under different growth conditions. The reference gene rpoA was expressed when grown both in LB and SILACE broth, while the lpfB1C gene was not found to be expressed in any of the two growth conditions tested. There could be several reasons why the lpfB1C gene was not expressed in this study, but due to time constrains there was not sufficient time to investigate why the lpfB1C gene was not expressed.
In conclusion, PCR amplification and cloning of the lpfB1 operon were successful, but the operon was not expressed under the two growth conditions tested. Nevertheless, this study has expanded further possibilities to analyze the expression of long polar fimbriae in non-O157 STEC. | |
