Regulation and Role in Pathogenesis of Megaplasmid-encoded Virulence Factors of Shiga Toxin producing Escherichia coli
Final Report Abstract
One part of the research plan was to compare gene expression profiles of the two megaplasmids pO! 13 and p0157 under a variety of different in vitro conditions using a constructed mini-array which represents the combined plasmid genomes. Regrettably, this technique was not longer accessible in the laboratory. Therefore, Reverse transcription (RT) real time PCR analysis and proteomic approaches were used in order to identify differential expression of megaplasmid encoded genes. Microarray analysis is a common tool to screen simultaneously a broad range of genes, whereas RT real-time PCR and whole proteome analysis is limited in the number of analysable target genes/proteins. As a result, the research focus was narrowed. In the first instance expression profiles of a limited number of megaplasmid-encoded genes of strain 98NK2 were analysed. To do so, various conditions such as growth in the presence of several types of tissue culture cells, iron depleted media, bovine serum-containing media and supplementation of growth media with short chain fatty acids to mimic conditions in the colon and ileum were tested. Analysis focused on selected megaplasmid encoded genes, aheady shown or suspected to be involved in the pathogenesis of STEC disease, such as iha, subA, saa, pilS,fliC, epeA, ehxA and a range of genes with unknown functions, including ORF LH0147 later termed sab. Based on these preliminary proteomic (MALDI-TOF-MS) and RT real time PCR data, investigations were focussed on comparative gene expression profiling of the megaplasmid encoded gene iha in LEE-negative strain 98NK2 and in addition in two LEE-positive STEC 0157:H7 strains (Part 1). The identification and characterisation of a novel megaplasmid encoded autotransporter adhesin (Sab) of LEE-negative STEC was the subject of the second project (Part 2). Results of in vitro experiments described in Part 2 illustrate that Sab mediates adherence to two human cell lines and promotes biofilm formation. Due to time constraints I gave preference to investigate the function of Sab in vitro instead of using in vivo methods (streptomycin mouse model), which was originally part of the project. Since LEE-positive STEC are 5a6-negative a comparative study of bacterial interaction with host cells was only carried out using 98NK2.
