Molecular characterization of an environmental bacterial isolate with strong serological cross-reaction with Shigella boydii 15. Implications for live shigellosis vaccine development

Bacillary dysentery caused by Shigella bacteria (shigellosis) is a major health problem in developing countries. Even though contaminated drinking water is expected to play an important role for the dissemination of this infectious disease the survival and distribution of Shigella bacteria in the aquatic environment is not well understood. This project focused on a bacterial isolate, called Iso10, from a lake outside Dhaka in Bangladesh. It strongly cross- reacts serologically with Shigella boydii 15 specific antisera but does not possess the genes associated with Shigella virulence, which makes it a potential candidate for development of a live shigellosis vaccine. The main goals were to identify the relationship between Iso10, Shigella and Escherichia coli using Multi Locus Sequence Analysis and comparison of the proteomic and LPS characteristics of Iso10 with S. boydii 15 trough 2D gel analysis and Western blotting. The results from sequencing of the 16S rRNA gene and 7 housekeeping genes (purA, adk, icd, fumC, recA, mdh and gyrB) from Iso10 showed that it shares significant sequence identity with the species Esherichia fergusonii. A phylogenetic tree based on concatemers of the 7 genes showed that Iso10 is placed together with E. fergusonii outside the main clusters of E. coli and Shigella spp. Membrane proteins were successfully extracted from Iso10 and separated on a 2D gel. Ten different proteins were identified by deNovo sequencing (MALDI TOF-TOF MS) and Peptide Mass Fingerprinting with MALDI-TOF MS. One protein, showing a very strong cross- reaction with Shigella- specific antisera, was identified as Outer membrane protein A (OmpA). A comparative study with OmpA extracted from Iso10, E. coli K12, S. boydii 15 and E. fergusonii showed that all the proteins reacted with Shigella- specific antisera, indicating that OmpA is a highly conserved porin in the Escherichia genus. OprF, a membrane protein previously only detected in Pseudomonas spp., was identified by deNovo sequencing which indicates an exchange of antigens between species which has not earlier been observed. LPS from Iso10, E. fergusonii and S. boydii was extracted and probed with S. boydii 15 type specific antisera. The results showed that Iso10 and S. boydii cross- reacted but no cross-reaction could be detected between E. fergusonii and Shigella- anitsera, which indicates a previous horizontal gene transfer of the O- antigen gene cluster (rfb) between S. boydii 15 and Iso10. Iso10 may induce an immune response capable of protecting against S. boydii 15 infections. However, since the virulence of E. fergusonii still is unclear further characterization has to be carried out before it can be evaluated as a possible live vaccine