An X-Domain Phosphoinositide Phospholipase C (PI-PLC-like) of Trypanosoma brucei Has a Surface Localization and Is Essential for Proliferation.

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

Development of Gamified, Interactive, Low-Cost, Flexible Virtual Microbiology Labs That Promote Higher-Order Thinking during Pandemic Instruction.

The COVID-19 pandemic radically and without warning changed the laboratory learning environment for students and instructors. Students were faced with having to be receptive to new learning methods; instructors scrambled to devise innovative ways of providing a realistic lab experience for students. The demand for creative online teaching strategies and the expansion of gamified training platforms created an opportunity for the development of new and interactive lab experiences. Current online labs offer some elements of a "real" lab experience, but a system that incorporates all the tools needed to create a realistic, immersive lab environment has yet to be developed. This study examines using different gamification elements implemented in a PowerPoint-based platform. There was no cost associated with the virtual lab and it could be easily downloaded, increasing accessibility. In true gaming style, a student could "play" without restriction, without the limitations that accompany wet labs. Students were challenged with various scenarios throughout the lab, making choices and receiving feedback through the process. These features positively impacted student outcomes and improved engagement, as expressed in end-of-course evaluations. The implementation also stressed the need for further development of embedded assessments, competitive and interactive opportunities for students, and access to detailed learning analytics for instructors.

Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C.

We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca(2+) concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca(2+) sensitivity and substrate preference, demonstrating the role of this motif in Ca(2+) regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP2. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP2 and a contribution to the inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway.

The role of acidocalcisomes in the stress response of Trypanosoma cruzi.

Acidocalcisomes of Trypanosoma cruzi are acidic calcium-containing organelles rich in phosphorus in the form of pyrophosphate (PP(i)) and polyphosphate (poly P). Acidification of the organelles is driven by vacuolar proton pumps, one of which, the vacuolar-type proton pyrophosphatase, is absent in mammalian cells. A calcium ATPase is involved in calcium uptake, and an aquaporin is important for water transport. Enzymes involved in the synthesis and degradation of PPi and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, participate in PP(i) and poly P metabolism and volume regulation and are essential for virulence. A signalling pathway involving cyclic AMP generation is important for fusion of acidocalcisomes to the contractile vacuole complex, transference of aquaporin and volume regulation. This pathway is an excellent target for chemotherapy as shown by the effects of phosphodiesterase C inhibitors on parasite survival.

Chemical validation of phosphodiesterase C as a chemotherapeutic target in Trypanosoma cruzi, the etiological agent of Chagas' disease.

Trypanosoma cruzi phosphodiesterase (PDE) C (TcrPDEC), a novel and rather unusual PDE in which, unlike all other class I PDEs, the catalytic domain is localized in the middle of the polypeptide chain, is able to hydrolyze cyclic GMP (cGMP), although it prefers cyclic AMP (cAMP), and has a FYVE-type domain in its N-terminal region (S. Kunz et al., FEBS J. 272:6412-6422, 2005). TcrPDEC shows homology to the mammalian PDE4 family members. PDE4 inhibitors are currently under development for the treatment of inflammatory diseases, such as asthma, chronic pulmonary diseases, and psoriasis, and for treating depression and serving as cognitive enhancers. We therefore tested a number of compounds originally synthesized as potential PDE4 inhibitors on T. cruzi amastigote growth, and we obtained several useful hits. We then conducted homology modeling of T. cruzi PDEC and identified other compounds as potential inhibitors through virtual screening. Testing of these compounds against amastigote growth and recombinant TcrPDEC activity resulted in several potent inhibitors. The most-potent inhibitors were found to increase the cellular concentration of cAMP. Preincubation of cells in the presence of one of these compounds stimulated volume recovery after hyposmotic stress, in agreement with their TcrPDEC inhibitory activity in vitro, providing chemical validation of this target. The compounds found could be useful tools in the study of osmoregulation in T. cruzi. In addition, their further optimization could result in the development of new drugs against Chagas' disease and other trypanosomiases.