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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SupaSteD (Suppresion of adaptive immunity by the Salmonella effector SteD)

Teaser

Summary

Efficient immune response towards intracellular bacterial pathogens (eg. Salmonella) depends on proper mode of activation of CD4+ T lymphocytes. This is mediated by antigen presenting cells such as dendritic cells (DCs). DCs trigger CD4+ T lymphocyte-dependent specific immune response by presenting foreign antigens on major histocompatibility complex class II (MHCII). Salmonella evolved a mechanism to specifically block antigen presentation by MHCII thus inhibiting onset of appropriate immune response.
The aim of this project was to reach a deep characterization of the functions of the Salmonella effector SteD on host MHCII-dependent specific immunity in an attempt to prepare a new tool for wider use in clinical practice. To reach this main goal, the work was divided into the following objectives: i) characterization of the effects of SteD on T cell activation in context of host organism; ii) obtaining a detailed understanding of SteD activity on molecular level; iii) identification of proteins targeted by SteD to modulate functions of T lymphocytes; iv) development of SteD as a tool usable for controlled modulation of adaptive immunity.
Results obtained throughout the project helped to uncover a function of a formerly uncharacterised human protein which is crucial for SteD activity. Interaction between SteD and this protein is a key step for inhibition of antigen presentation by MHCII and for preventing establishment of productive interaction between DCs and T lymphocytes. Therefore, T lymphocytes developing under the influence of SteD-manipulated DCs show features of regulatory T cells rather than immunostimulatory CD4+ helper T lymphocytes.

Work performed

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 Î”steD 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 Î”steD. 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 Î”steD 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).

Final results

It is known that Salmonella infection leads to increased amounts of Tregs in the host. The exact mechanism of this is, however, unknown. The results obtained throughout the project showed that Salmonella infection increases amounts of Tregs at least partially in an SteD-dependent manner. The potential clinical use of SteD in this respect still remains to be explored.
The results of this project also showed the involvement of a formerly uncharacterised protein in SteD-mediated inhibition of proliferation of T lymphocytes. This protein is involved in SteD-dependent decrease in DC-T lymphocyte interaction and early activation of T lymphocytes. This appears to be an important general mechanism in DC-T cell interaction beyond Salmonella pathogenesis. Thus, these findings have a potential to bring a substantial progress to fields of cell biology and immunology.