Work on this project was subdivided into 4 work packages (WP): 1) Characterization of SteD-provoked effects on T cell activation and attenuation of STm ΔsteD strains in vivo; 2) Analysis of SteD-MARCH8 interaction; 3) Identification of new SteD targets and characterization of their role in pathogenesis; 4) Assessment of human-adapted SteD functionality and its use in biomedicine application.
To characterize the effects of SteD on T cell activation in vivo (WP1), T cells obtained from spleens and mesenteric lymph nodes (MLN) of orally or intraperitoneally infected permissive (Nramp1- C57BL/6) or resistant (Nramp1+ 129/SvjxB6) mice were examined. Oral infection of C57BL/6 mice with WT STm led to significantly increased amounts of regulatory T cells (Tregs) in mesenteric lymph nodes of infected animals when compared to animals infected with STm ΔsteD. The obtained results supported the original hypothesis that translocation of SteD by Salmonella leads to increased proportion of Tregs. The observed difference in levels of Tregs between WT or ΔsteD Salmonella infected mice was, however, too small to allow further exploitation of this phenomenon.
Next, the molecular mechanism of SteD action was investigated (WP2). In depth mutational analysis of SteD was done to identify regions of SteD involved in its function and interactions with its partners. This led to identification of 7 amino acid residues crucial for targeting MHCII and formation of stable complex between SteD and its functional partners. Moreover, examination of interaction of SteD with its partners helped to describe a physiological function of a formerly uncharacterised human protein.
The mechanism identified during work on WP2 has been proven valid for a newly identified SteD target (WP3). This mammalian protein was found to be important for establishing a productive interaction between DCs and T lymphocytes and its targeting by SteD helps explain influence of SteD on development of specific T cell response (as studied in WP1). Moreover, this function appears to be an important general mechanism in DC-T cell interactions, beyond Salmonella pathogenesis.
Finally, a potential exploitation of SteD in clinical practice (WP4) did not appear feasible due to the weak phenotype observed and described in WP1.
The results obtained throughout the project were published in two peer-reviewed articles in open access format (four more articles are in preparation) and were presented to both scientific audiences (EIMID 2018, Salmonella Biology and Pathogenesis Conference GRC 2019 meetings) and at Institute of Microbiology of the Czech Academy of Sciences (invited talk) as well as to broader public during Imperial festivals (2018, 2019) and festival Pint of Science (2018). Moreover these results are of general interest beyond the field of bacterial pathogenesis and will be interesting for a broad readership (eg. in fields of cancer and general immunology).
