Living under stress: Role of a bacterial-like mechanosensitive channel in Trypanosoma cruzi osmoregulation

WONG B; BARRERA P; DAVE N; JIMENEZ ORTIZ V

Garden Grove

Simposio; 28th CSU Annual Biotechnology Symposium; 2016

California State University Program for Education and Research in Biotechnology

In order to complete its life cycle, Trypanosoma cruzi-the protozoan parasite that causes Chagas disease- faces various environmental changes as it propagates from an insect vector to a mammalian host. Previous studies have shown that T. cruzi has a robust osmoregulatory response, however the osmosensors involved in the detection and compensation pathways have not been identified. Mechanosensitive channels, which are activated by a stretch of the plasma membrane, have been associated with sensing of environmental changes in other organisms, but the function of these channels in T. cruzi is still unknown. In silico analysis of T. cruzi genome reveals the presence of mechanosensitive channels similar to the ones described in bacteria. The protein has very low homology with E. coli channels, but shows conserved structural features, including a hinge-like structure in the pore-forming domain.We hypothesize that a bacterial-like mechanosensitive channel, TcMcS, is involved in osmoregulatory processes in T. cruzi. Overexpressing mutants using the pTcIndex inducible system were developed to investigate the role of TcMcS in T. cruzi osmoregulation. Knockout mutants mediated by CRISPR-Cas9 were generated to test the essentiality of the protein. The localization and expression pattern of TcMcS varied in the three main life stages of T. cruzi. TcMcS seems to be localized in the contractile vacuole of epimastigote and trypomastigote forms, and in the plasma membrane of amastigote forms. Under hyposmotic stress, cells overexpressing TcMcS swell significantly less than wild- type parasites. Under EGTA treatment, this advantage was eliminated, suggesting that calcium plays a role in the osmoregulatory response. Known mechanosensitive channel blockers, including gadolinium and streptomycin, were found to elicit significant differences in the parasite?s ability to detect and compensate for the osmotic stress. Overall, our results support the idea that TcMcS is involved in sensing and compensation of osmotic stress in T. cruzi. Funding: NIH Grant R00AI101167, CSUF MHIRT Program, CSUF HHMI