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Trypanosomatids are a diverse group of uniflagellate protozoa that include globally important pathogens such asTrypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase (FAS)-I system typically used forde novosynthesis of long chain fatty acids (LCFA) in other eukaryotes. Instead, these microbes have evolved a modular fatty acid elongase (ELO) system comprised of individual ELO enzymes that operate processively. The role of the ELO system in maintaining lipid homeostasis in trypanosomes has not been determined. Here we demonstrate that ELO2 and ELO3 are required for global lipidome maintenance in the insect stage ofT. cruziwhereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that merits examination of the trypanosomatid ELO pathway as critical for central metabolism.

SARS-CoV-2 extensively N-glycosylates its surface spike (S) proteins. This post-translational modification is essential to modulate protein conformation and host cell invasion. Each S monomer can be modified with up to 22 N-glycans. To meet the high demand of protein glycosylation during virus replication, SARS-CoV-2 upregulates the expression of host N-glycosylation genes. Although a substantial amount of detail is known about the structure of S protein N-glycans, the role of N-glycosylation in SARS-CoV-2 infection remains largely undetermined. Here, we investigated the essentiality of the host N-glycosylation pathway and viral N-glycans for SARS-CoV-2 infection. When either monkey or human cells were preincubated with glycosylation inhibitors, including FDA-approved iminosugars, virus infection was significantly reduced. This infection phenotype was confirmed after RNAi knockdown of several glycosylation genes. In addition, enzymatic deglycosylation of whole viral particles confirmed that accessible oligosaccharides on the SARS-CoV-2 surface are essential for host cell infection. Altogether, we show evidence that the normal functioning of the host N- glycosylation machinery is essential not only for SARS-CoV-2 to infect, but also to produce new functional virions. These findings open the door for developing new approaches targeting N-glycosylation against COVID-19.

The ability to undergo morphological changes during adaptation to distinct environments is exploited byCandida albicansand has a direct impact on virulence. In this study, we investigated the influence of fungal extracellular vesicles (EVs) during yeast growth, biofilm formation, and morphogenesis inC. albicans. Addition ofC. albicansEVs (CaEVs) to the culture medium positively affected yeast growth. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated thatCaEVs inhibited biofilm formation byC. albicans in vitro. By time-lapse microscopy and SEM, we showed thatCaEV-treatment stops filamentation promoting pseudohyphae formation with multiple sites for yeast budding. The ability ofCaEVs to regulate dimorphism was further compared to EVs isolated from differentC. albicansstrains,Saccharomyces cerevisiae, andHistoplasma capsulatum.CaEVs from distinct strains robustly inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 hours. A minor inhibitory effect was promoted by EVs fromS. cerevisiaeandH. capsulatumonly after 24 hours of incubation. The inhibitory effect ofCaEVs was promoted by a combination of lipid compounds identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably,CaEVs were also able to reverse filamentation, transforming hyphal growth to yeast forms. Transcriptomic analysis demonstrated that treatment withCaEVs modified the expression of more than 300 genes. The most effectively upregulated pathways were related to DNA metabolism. The downregulated genes were mostly associated with extracellular and adhesion proteins. Finally, yeast cells treated withCaEVs for 24 hours lost their agar invasive ability and were avirulent when inoculated inGalleria mellonellalarvae. In summary, our results indicate that fungal EVs can profoundly modifyC. albicansgrowth and regulate yeast-to-hypha differentiation inhibiting biofilm formation and virulence.

Excessive progesterone (PRG) may increase breast cancer risk under hormone therapy during postmenopause or hormonal contraception. As a sex steroid hormone, PRG exerts its cellular responses through signaling cascades involving classic (genomic), non-classic (non-genomic), or both combined responses by binding to either classic nuclear PRG receptors or non-classic membrane PRG receptors. Currently, the intricate balance and switch mechanisms between these two signaling cascades remain elusive. Three genes, KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), have been demonstrated to form a CCM signaling complex (CSC). In this report, we discover that the CSC plays an essential role in coupling both classic and non-classic PRG signaling pathways by mediating crosstalk between them. The coupled signaling pathways were detailed through high throughput omics.One Sentence SummaryWe discover a novel signaling network among the CCM signaling complex (CSC), classic and non-classic progesterone receptors, and their ligands-progesterone/mifepristone, is dynamically modulated and fine-tuned with a series of feedback regulations; perturbation of this intricate balance, such as hormone therapy in the postmenopause or hormonal contraception regimen, or perturbed CSC signaling could result in increased risks in breast cancer or compromising tumor therapy.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50–300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction.