Background

Trypanosoma cruzi, the parasite causative of Chagas disease, has a digenetic life cycle, whereby it exists in insect vectors from the Reduviidae family, and is transmitted to mammalian hosts, and vice versa. The T. cruzi life cycle involves four main morphogenetic stages, namely epimastigotes (which replicate in the midgut of the insect host), metacyclic trypomastigotes (non-replicative forms capable of penetrating the body of the mammalian host and invading cells within such host), amastigotes (replicative forms into which the metacyclic trypomastigotes develop) and bloodstream trypomastigotes (forms released into the circulatory system, infecting new cells).

For the full completion of its life cycle to be successful, the parasite must be prepared to cope with abrupt changes in environmental conditions between hosts and even within different compartments within the hosts. Signal transduction pathways are essential for parasites to recognize environmental fluctuations and respond through cellular changes. Various second messenger pathways are involved in the regulation of cell proliferation, and passage from epimastigotes to metacyclic trypomastigotes. This process is known as metacyclogenesis. To date, several mechanisms and events involved in the onset of metacyclogenesis remain poorly understood. Among players in key pathways, the AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses that result in a reduction of ATP and an increase in AMP levels. Thus, AMPK is thought to function both as a nutrient and as an energy sensor that maintains energy homeostasis and protects cells from death by nutrient starvation. In their work, Sternlieb et al (1) characterize the AMPK complexes in T. cruzi (TcAMPK) for the first time, and describe its function as a regulator of nutritional stress in epimastigote forms.

Key findings and developments

The authors begin their work by identifying in silico, TcAMPK subunits, based on the orthologues of the previously identified 2 isoforms of the alpha AMPK subunit, and the beta and gamma subunits of T. brucei. Key amino acids for the subunit interactions remained conserved, however, the kinase catalytic domain was the only conserved region predicted in the alpha subunit candidates. Specific to the alpha subunit were two observations namely a) that the C-terminal portion of AMPKα usually has an autoinhibitory sequence or domain, a linker, and a βγ-interaction domain, and b) the activation loop, which contains a conserved threonine residue (Thr172), which must be phosphorylated to reach maximum levels of kinase activity. Converse to TcAMPKα1, TcAMPKα2 has a serine residue replacing the threonine residue at the activation loop. The authors mention this phosphorylatable residue in TcAMPKα2 is the only coding sequence presenting this divergence.

The authors proceeded to evaluate the functional capability of each of the putative TcAMPK subunits by performing complementation assays in S. cerevisiae conditional mutant strains, deficient either for the alpha subunit, the 3 beta subunits, or the gamma subunit. The mutants were unable to grow in media containing any carbon source other than glucose. The authors went on to generate HA-tagged TcAMPK subunits for each of the corresponding mutants. Upon complementation, all mutants restored their capability to use raffinose as a carbon source.

The authors then went on to study the modulation of TcAMPK catalytic activity in vivo, as well as a biochemical characterization. The authors used various assays, including antibody-based reactions and treatment with AMPK activity modulators, to visualize the activation status of the AMPK catalytic subunits. Moreover, they performed mass spectrometry analysis and detected the TcAMPKα2 and the phosphorylation in the serine replacing the threonine in the activation loop.

Given that in many organisms AMPK is a metabolic regulator activated when energy metabolites and nutrients are limited, the authors went on to explore the sensing and metabolic role of TcAMPK in epimastigotes as they passage through the insect gut. For this, T. cruzi epimastigotes were exposed to nutritional stress for 17h, which resulted in significantly increased AMPK catalytic activity. The authors conclude that TcAMPK can be activated in the absence of a carbon source, and may play a role in initiating metabolic responses to face prolonged nutritional stress in T. cruzi epimastigotes.

Then, TcAMPKα1-HA and TcAMPKα2-HA were then transfected into T. cruzi (leading to the generation of over-expressors), and the intracellular localization of both proteins was investigated. Both proteins were found to distribute in the cytosol in a granulated pattern, suggesting a partial association with glycosomes or acidocalcisomes. Overexpression of each isoform of the catalytic subunit had an opposite effects on T. cruzi epimastigote proliferation. While overexpression of TcAMPKα1-HA resulted in increased duplication time followed by arrest and death, overexpression of TcAMPKα2-HA led to a decrease in duplication time, without any other phenotypic effect. Altogether this suggests that each of the isoforms plays a different role in the parasite’s life cycle.

Finally, the authors investigated the relationship between TcAMPK and autophagy in epimastigote cells, and whether TcAMPK could modulate autophagosome formation. For this, the authors compared the WT line with the TcAMPKα2-overexpressor, and found that the overexpressor had higher autophagic capacity.

What I like about this preprint

This is one of the studies we included in a preList related to the Molecular Parasitology Meeting in Woods Hole in 2020 (virtual). I like that the authors focused on a relatively neglected parasite, and seem to have bridged a gap in knowledge to better understand the metabolic processes the parasite undergoes when transitioning between phases. I think the findings are very interesting and relevant to T. cruzi but also other parasitology areas.

References

1. Sternlieb T, et al, AMP-activated protein kinase: a key enzyme to manage nutritional stress responses in parasites with complex life cycles, bioRxiv, 2020.

 

Posted on: 11 December 2020

doi: https://doi.org/10.1242/prelights.26369

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