Fasciola hepatica Glutathione S‐Transferase (FhGST) conformational studies and its effect on the suppression of toll‐like receptors stimulation

Abstract

Fasciola hepatica is a parasite well known as a master of immunomodulation because of its ability to suppress the Th1 immune responses and elicit a strong Th2/Treg response. This occurs when the parasite releases excretory‐secretory products (ESPs), which are comprised of a myriad of glycoproteins that are believed to play an important role in the immune response. F. hepatica Glutathione S‐Transferase (FhGST) is one of the ESP components recognized for its anti‐oxidant properties and its capacity to provide part of the parasite's defense by detoxifying the secondary products of lipid peroxidation that are produced due to the immune free‐radical attack on the host or the parasite membrane. Our preliminary studies demonstrate that FhGST exerts a suppressive effect on the activation of TLR4 in macrophages which is evidenced by significant suppression of NF‐kB activation and down‐regulation of TNFα and IL1‐β in response to LPS‐stimuli. We speculated that the integrity of the tertiary structure, as well as the glycans that decorate the protein moiety of FhGST, could be essential for its capacity to suppress the NF‐kB activation. To test our hypothesis, we first subjected FhGST to a thermal denaturing process by boiling it at 90°C for 10 min followed by a treatment with PNGase F. Circular dichroism analysis (CD) comparing the FhGST spectra before and after heat denaturing served to confirm that FhGST was full and irreversibly denatured. Next, denatured or native FhGST were added alone or in the presence of the whole extract of 1 x 108 cells Klebsiella pneumonia (Kp) to a culture of human monocytes (THP1‐CD14) that express most TLRs as well as an NF‐kb inducible secreted embryonic alkaline phosphatase reporter gene. Cells stimulated with an attenuated whole extract of 1 × 108 cells Klebsiella pneumonia (Kp) were used as activation control. Our results demonstrated that native FhGST suppresses NF‐kB activation by 83%, whereas the heat‐denatured protein suppresses NF‐kB activation by 84%. These suppressions suggest that in contrast to expected, the capacity of FhGST to suppress the NF‐κB activation in THP1‐CD14 cells is independent of the integrity of its secondary/tertiary structure and is not associated to the glycans. Docking analysis in which the FhGST‐protein moiety was docked with the CD14 and MD2 co‐receptor structures suggested that FhGST could target these co‐receptors as part of its mechanisms of action.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, 5R25GM061151‐16 and NIH grants G12MD007600 and 2P40OD012217This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.