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AMP-activated protein kinase: A key enzyme to manage nutritional stress responses in parasites with complex life cycles

Tamara Sternlieb, Alejandra C. Schoijet, Patricio D. Genta, Guillermo D. Alonso

Preprint posted on April 09, 2020 https://www.biorxiv.org/content/10.1101/2020.04.08.032284v1

Insights into nutritional stress responses in parasites with complex life cycles.

Selected by Mariana De Niz

Categories: cell biology, microbiology

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.

Figure 1. Schematic showing AMP-activated protein kinase: a key enzyme to manage nutritional stress responses in T. cruzi. (Credit: Tamara Sternlieb).

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: 11th December 2020

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

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Author's response

Tamara Sternlieb shared

Open questions 

1.You focused from a certain point on, on the alpha subunit of TcAMPK. Can you expand further on your findings regarding the role of the beta and gamma subunits of TcAMPK?

Besides the complementation of S. cerevisiae, which already supports the role of these subunits in an AMPK complex, we tried overexpressing the HA tagged versions in epimastigotes. For the gamma subunit we succeeded and were able to analyse cellular localization and protein expression. Its localization coincided with the one we observed for the alpha subunit, in granules in the cytosol, which could point to glycosomes or acidocalcisomes. When we performed a western blot revealed with anti-HA antibody, we saw two bands close together just below 70 kDa. This was close to the expected molecular weight, and we are still trying to identify the post-translational modification that produces the appearance of a second band. No phenotypic effect resulted from this overexpression.
On the other hand, we could never obtain an epimastigote culture that overexpresses the beta subunit. We transfected several times and confirmed integration of the tagged sequence and mRNA expression, but the protein didn’t show in a western blot. It is still a mystery for us.

2.Further, you discuss along your work, the epimastigote stage. In the context of your work, what do you expect in terms of nutritional stress responses in other forms, such as the metacyclic trypomastigotes, the amastigotes, and the bloodstream trypomastigotes?

That is a very interesting question. T. cruzi modifies its metabolism quite a lot through its life cycle and faces varying nutritional “menus” at each stage. The epimastigote stage is robust and can endure through lack of glucose and most aminoacids, and survive with very little. But in those conditions, the nutritional stress is a signal for it to prepare for the next stage. So along differentiation, a metabolic adaptation begins. Nutritional stress in the other replicative stage, the amastigote, can lead to growth arrest and a kind of quiescent state, where the intracellular parasites persist but don’t replicate as much. This phenomenon is being studied with great effort to find the regulatory mechanisms involved, since it is supposed to be related to drug resistance and inability to eliminate the parasite in chronic infections. The absence of certain aminoacids during infection with metacyclic trypomastigotes in vitro can lead to reduced amastigote count, while the absence of other aminoacids has no effect whatsoever. So nutrient availability and sensing is essential for T. cruzi to progress through its life cycle successfully.

3.While the idea of tissue tropism for T. cruzi has been a matter of controversy, again in the context of your work, how do you expect that tropism to certain organs affects nutrient availability, and therefore parasite development, or fate choices within different organs once in the mammalian host?

It is interesting to think if nutrient availability in different tissues could be one of the reasons the parasites may persist longer in them. It has been observed that parasites can modulate the host cells’ metabolism and metabolic enzymes expression. Maybe the tissues for which tropism exist present some nutritional advantages or are more susceptible to metabolic manipulation. However, we haven’t studied nutritional stress in relation with tropism in our lab. Everything is possible.

4.You have touched upon the topic of autophagy. Various groups, particularly in the context of Plasmodium liver stages, have suggested that autophagy is a double edged sword when it comes to benefits for parasite nutrition and survival, as autophagy is also a cellular response to eliminate intracellular pathogens. You have opened an interesting door in the context of T. cruzi. What would be key hypotheses you consider in the context of your work, in relation to autophagy and T. cruzi development?

This is quite an important thing to keep in mind if TcAMPK is evaluated as a potential drug target in the future. AMPK is a well conserved enzyme, and it has been widely studied in humans to modulate metabolic responses to fight cancer, modulate weight loss, among others. On the other hand, autophagy is essential for the parasite to remodel its metabolic pathways and enzyme repertoires when differentiation occurs or under stress. AMPK is involved in autophagy in both organisms, as far as we know. Hence, to consider TcAMPK as a potential drug target, it will be of essence to evaluate how the tested drugs affect the host cells.

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