Vaccine Strategies for Cryptococcus neoformans.

Cryptococcus neoformans infections are a major worldwide concern as current treatment strategies are becoming less effective in alleviating the infection. The most extreme and fatal cases are those of immunocompromised individuals. Clinical treatments for cryptococcosis are limited to a few classes of approved drugs, and due to a rise in drug resistance, these drugs are becoming less effective. Therefore, it is essential to develop innovative ways to control this infection. Vaccinations have emerged as a safe, viable, and cost-effective solution to treat a number of diseases over the years. Currently, there are no clinically available vaccines to treat cryptococcal infections, but a number of studies have shown promising results in animal models. Here, we present step-by-step experimental protocols using live-attenuated or heat-killed C. neoformans cells as a vaccination strategy in a preventive or in a therapeutic murine model of cryptococcosis.

Lipidome remodeling in response to nutrient replenishment requires the tRNA modifier Deg1/Pus3 in yeast.

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation-like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mutant increases specific misreading errors. This could lead to altered expression of the main regulators of neutral lipid synthesis which are the acetyl-CoA carboxylase (Acc1), an enzyme that catalyzes a key step in fatty acid synthesis, and its regulator, the Snf1/AMPK kinase. We demonstrate that upregulation of the neutral lipid content of LD in the deg1 mutant is achieved by a mechanism operating in parallel to the known Snf1/AMPK kinase-dependent phosphoregulation of Acc1. While in wild-type cells removal of the regulatory phosphorylation site (Ser-1157) in Acc1 results in strong upregulation of triacylglycerol (TG), but not steryl esters (SE), the deg1 mutation more specifically upregulates SE levels. In order to elucidate if other lipid species are affected, we compared the lipidomes of wild type and deg1 mutants, revealing multiple altered lipid species. In particular, in the exponential phase of growth, the deg1 mutant shows a reduction in the pool of phospholipids, indicating a compromised capacity to mobilize acyl-CoA from storage lipids. We conclude that Deg1 plays a key role in the coordination of lipid storage and mobilization, which in turn influences lipid homeostasis. The lipidomic effects in the deg1 mutant may be indirect outcomes of the activation of various stress responses resulting from protein aggregation.

Role of sphingolipids in the host-pathogen interaction.

Fungal pathogens have been under the spotlight as their expanding geographic range combined with their potential harm to vulnerable populations turns them into increasingly threats to public health. Therefore, it is ultimately important to unveil the mechanisms associated with their infection process for further new treatment discovery. With this purpose, sphingolipid-based research has gained attention over the last years as these molecules have key properties that can regulate fungal pathogenicity. Here we discuss some of these properties as well as their role in fungal diseases, focusing on the subgroup of glycosphingolipids, as they represent promising molecules for drug discovery and for the development of fungal vaccines.

TORC1 inhibition induces lipid droplet replenishment in yeast.

Lipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In Saccharomyces cerevisiae, LD dynamics are modulated by the growth phase; however, the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LD replenishment and growth inhibition. The discovery that osmotic stress (1 M sorbitol) also induced LD synthesis but not growth inhibition suggested that the induction of LDs in yeast is not a secondary response to reduced growth. The induction of LDs by rapamycin was due to increased triacylglycerol but not sterol ester synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p, but not Msn2p and Msn4p, were required for full induction of LDs by rapamycin. Furthermore, we show that the deletion of Gln3p and Gat1p transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast.