Abstract 2103 Environmental and chemical factors impacting osmoregulation in Crithidia fasciculata parasites

Osmoregulation is important for most living cells, and involves regulatory volume decrease (RVD) to offset swelling caused by hypotonic stress. Some parasites, including the non-pathogenic trypanosome parasite model, Crithidia fasciculata, use a contractile vacuole (CV) complex to quickly pump water out of the cell during RVD. C. fasciculata is related to the human-infective parasites causing Chagas' disease and Leishmaniasis. A series of experiments were conducted to assess the effects of environmental and chemical conditions on C. fasciculata's RVD. Firstly, the extracellular ionic milieu (presumably impacting the parasite plasma membrane potential) was modulated. Using light scatter visible spectroscopy to follow RVD, we found that the removal of sodium (Na+) and (Ca2+) ions extracellularly did not impact RVD kinetics. Secondly, we studied the impact of temperature on osmotic response. In five-minute kinetics experiments, RVD was observed over a large temperature range (5-35˚C) and occurred faster at higher temperatures. Over longer 90-minute periods, C. fasciculata cells were remarkably resistant to hypotonic stress, with the majority of cells 25 °C or cooler surviving 90% hypotonic dilution without noticeable swelling after 90minutes. However, when hypotonic stress was combined with high temperatures, cells became irreversibly swollen and eventually died as followed via microscopy. Lastly, potential inhibitors and activators of cAMP signaling were tested for their impact on RVD kinetics. The adenylate cyclase inhibitor and antifungal agent miconazole dramatically inhibited RVD, leading to irreversibly rounded cells after 15min of treatment (in the low μM range). These findings could be utilized to understand osmoregulation in human-infective trypanosome parasites and design contractile-vacuole targeted treatments.