Discussion
Introduction:
The observation that some helminth infections are negatively correlated with the prevalence of allergic inflammatory disease has driven research into parasite immunomodulation. Type 2 immune responses are commonly associated with host defence against helminth infections and the development of allergic inflammation. Helminths (or their secreted molecules) can ameliorate allergic airway inflammation in mouse models of asthma by modulating host type 2 immune responses. Recently, a series of immunomodulatory molecules secreted by the murine intestinal parasite
Heligmosomoides polygyrus have been identified: HpARI binds to IL-33 and DNA in host necrotic cells; HpBARI binds and blocks the IL-33 receptor ST2; and HpTGM induces regulatory T cells through engagement of the host TGF-β receptor. Interestingly, HpARI, HpBARI and HpTGM all consist of a string of consecutive atypical Complement Control Protein (CCP) domains. CCP domain-containing proteins are over-represented in the
H. polygyrus genome. This led to the hypothesis that CCP domain-containing proteins may represent a family of immunomodulatory proteins in
H.polygyrus.
Method:
Taking a bioinformatic approach, an atypical CCP motif was developed by using sequence alignment of HpARI, HpBARI, HpTGM and all
H.polygyrus genes containing conventional CCP domains defined by InterPro IPR000436. This motif was used to search against an in-house
H.polygyrus transcriptome and genomic data on WormBase ParaSite to predict and identify new atypical CCP domain-containing proteins. Selected candidates were produced as recombinant proteins using an Expi293 expression system and were assessed for interaction with host immune proteins using the Avidity-based Extracellular Interaction Screening (AVEXIS) assay.
Results:
After screening approximately 200,000 genome predicted proteins, and selecting candidates for features of CCP domains, we identified 70 potential CCP domain-containing proteins, from which 15 candidates were selected for expression as “baits” in the AVEXIS assay. Furthermore, the known immunomodulators HpARI, HpBARI and HpTGM were cloned for expression as baits. To assess immune targets of these candidate immunomodulators, 58 immune genes (chemokines, cytokines and their receptors) were selected for expression as “preys” in the AVEXIS assay. This system is sensitive and has now been optimised to show robust interactions between HpARI and IL-33, HpBARI and ST2, and HpTGM and the TGF-b receptor. We are now screening for further novel protein-protein interactions between parasite molecules and immune targets.
Conclusion:
The methods and analyses described here may assist the development of a larger pipeline in identifying, producing, and characterising new helminth immunomodulatory molecules. Thus, accelerating the progress in finding new treatments or preventions against allergic asthma.
