Biology subjects

Simek, P.

Publications and source records attributed to Simek, P..

4 recordsLinked to original sources

bioRxiv2023

Metabolism of glucose and trehalose by cyclic pentose phosphate pathway is essential for effective immune response in Drosophila

Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. Neutralization of the parasitoid egg involves the differentiation of lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg; meanwhile the capsule created also protects the host from the toxic reaction. We combine transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters, and accordingly uptake of sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH production. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose, within the cell, to fuel cyclic PPP. Intracellular trehalose metabolism is not required for resistance, but may be associated with host protection from its own toxic response. Thus, our results suggest that sugar metabolism within immune cells, and specifically in cyclic PPP, may be important for not only fighting infection, but also for protecting the host from its own immune response and ensuring sufficient fitness of the survivor.

immunology↗

bioRxiv2023

Trypanosoma brucei bloodstream form mitochondrion is capable of ATP production by substrate phoshorylation

The bloodstream form Trypanosoma brucei maintains essential mitochondrial membrane potential ({Delta}{Psi}m) through the reverse activity of FoF1-ATP synthase. The ATP that drives this activity is thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). We have shown that this carrier is the only carrier that can import ATP into the mitochondrial matrix to power the FoF1-ATPase. Contrary to expectations, its deletion has no effect on parasite growth, virulence and levels of {Delta}{Psi}m, suggesting that ATP is produced intramitochondrially by substrate phosphorylation pathways. Therefore, we knocked out the succinyl-CoA synthetase (SCoAS) gene, a key enzyme that produces ATP through substrate phosphorylation. Its absence resulted in changes in the metabolic landscape of the parasite, lower virulence, and reduced mitochondrial ATP content. This minimal mitochondrial ATP pool was maintained by AAC activity as evidenced by the 25- fold increase in sensitivity of the mutant parasites to AAC inhibitor carboxyatractyloside. Under nutrient-limited conditions, suppression of SCoAS expression by RNA interference negatively affected cell growth and levels of {Delta}{Psi}m. We concluded that the bloodstream mitochondrion is capable of generating ATP via substrate phosphorylation pathways, the importance of which depends on environmental conditions.

microbiology↗

bioRxiv2022

Integrative assessment of the transcriptome and virome of the poultry red mite Dermanyssus gallinae.

Dermanyssus gallinae is a blood-feeding mite that parasitises on wild birds and farmed poultry. The D. gallinae mite has a short life cycle of fewer than two weeks from the egg to an egg-laying female. The remarkably swift processing of blood, together with the capacity to blood-feed in most developmental stages, makes this mite a highly debilitating pest. We have constructed developmental stage-specific transcriptomes, through Illumina RNA-seq, to mine the repertoire of protein-encoding mRNA transcripts, products of which participate in key processes that ensure the success of blood digestion, rapid ontogeny, and immunity. As a result of high reproductive capacity, the prevalence of D. gallinae in egg-producing poultry farms globally causes significant economic losses. Acaricides that are used to limit the reproduction of D. gallinae mites target cys-loop ion channels, which are widely shared across the phylogeny of invertebrates. To catalogue a comprehensive list of potential invertebrate-specific ion channels, we have constructed and analysed an additional RNA-seq library of D. gallinae micro-dissected midguts, a tissue with direct exposure to host blood and potential anti-parasitics. We phylogenetically defined groups of cys-loop proteins and probed their sensitivity to selected acaricides. Ultimately, we have catalogued all assembled transcripts and their expression values in a hyper-linked excel sheet with available sequences of individual contigs. The transcriptomic data were complemented by mass-spectrometry (MS)-based metabolite identification and by viability assays using selected inhibitors applied either by microinjection or through artificial membrane feeding. Additionally, we have described the RNA-virome of D. gallinae and identified a novel virus dubbed Red Mite Quaranjavirus 1.

microbiology↗

bioRxiv2021

A mixture of innate cryoprotectants is key for cryopreservation of a drosophilid fly larva.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on mechanisms of how the individual CPs interact, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata that survives long-term cryopreservation in liquid nitrogen. The CPs (proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine, and sarcosine) accumulate in hemolymph in a ratio of 313:108:55:26:6:4:3:0.5 mmol.L-1. Using calorimetry, we show that the artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using MALDI-MSI, we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it likely forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen. Summary statementThe composition of natural cryoprotectant mixture of the extremely freeze-tolerant insect is revealed. Components of the mixture work in synergy and behave differently during organismal freezing and cryopreservation.

physiology↗