Activities of enteropathogenic/enterohemorrhagic E. coli effector proteins
Final Report Abstract
The project focused on the study of hitherto uncharacterized EHEC non-LEE effector proteins. A total of 15 bacterial effector genes were cloned into both, plasmids for overexpression as affinity-tagged fusion proteins in E. coli and plasmids for cell culture studies. Subsequently, these constructs were used to screen expression in E. coli lab strains and to study subcellular localization and the effect of heterologous expression in HeLa cells. Conditions for overexpression in E. coli lab strains and the respective purification protocols could be established for four effector proteins, namely espR1, espY1, espY2, and espY4. The purified proteins were used for in vitro studies aiming at identifying potential host target proteins. Unexpectedly, it was not possible to detect any protein-protein interactions via pulldown experiments with porcine brain extract under the conditions tested. In cell culture experiments, one of the effectors – espW – displayed an intriguing localization pattern when expressed as CFP-fusion protein in mammalian cells hinting at an association with the actin cytoskeleton, which is known as a prominent target of many EHEC effectors. Therefore, this effector was chosen for a more detailed examination. Expression of CFP- espW did not result in any noticeable cellular phenotype in HeLa or HT1080 cells, nor did it seem to affect the infection of HeLa cells by EHEC. Similarly, the generated espW knockout strain did not behave differently from the parental wildtype EHEC in HeLa infection studies giving no hint to a putative target and function of espW. Despite numerous efforts including the construction of truncated versions and fusions to a variety of different affinity tags as well as co-expression experiments it was not possible to obtain purified espW for crystallization and in vitro studies. Instead, a yeast two hybrid system was used to screen a human brain library for espW interaction partners. Thus, an interaction of epW with the cytoplasmatic domain (CPD) of Brain-specific angiogenesis inhibitor 3 (BAI3), a seven-transmembrane protein belonging to the adhesion-GPCR family, was detected. CPD-BAI3 was overexpressed in E. coli and could be purified in sufficient amount for pull-down experiments confirming its interaction with espW. The production and purification of larger amounts of CPD-BAI3 for crystallization studies is feasible, but has not yet been realized. Unfortunately, to date it is still unclear in which cellular pathways BAI3 is involved exactly. Recently, an interaction with the Rac1 regulator ELMO1 was reported. However, no difference in Rac1 activation levels could be detected in HeLa cells infected with WT EHEC or ΔespW EHEC, respectively. Other recent findings by Mauldin et al. have also linked Elmo1 with the regulation of expression of cytokine genes, so the effect of espW on an array of selected human cytokines and chemokines was monitored. Interestingly, in Caco2 and even more pronounced in macrophage-like THP1 cells, there was a marked increase in the level of cytokine macrophage migration inhibitory factor (MIF) in cells infected with ΔespW EHEC compared to cells infected with WT EHEC. As MIF is known to be an important mediator of host responses against gram-negative bacteria, it seems likely that a) BAI3 is involved in mediating an immune response to bacterial infection and that b) espW dampens down the production of pro-inflammatory mediators during EHEC infections. However, since this function of espW is only a hypothesis based on preliminary data, further studies are warranted to confirm the proposed role of espW in modulating the host immune response and to elucidate the exact nature of its mode of action.
