Biology subjects

Cestari, I.

Publications and source records attributed to Cestari, I..

8 recordsLinked to original sources

bioRxiv2026

A genome wide antigen-antibody screen identifies a yeast-based therapeutic vaccine candidate for Chagas disease

Chagas disease is caused by Trypanosoma cruzi infection and results in decades-long, chronic, debilitating, and lethal disease. There is no vaccine available, largely due to the lack of vaccine targets. We generated a T. cruzi genome-wide library for yeast surface display and screened it with antibodies from Chagas disease patients to identify antigen-antibody interactions. We identified hundreds of T. cruzi antigens that are immunogenic in humans and mapped their antibody-binding sites at nucleotide resolution. Hundreds of immunogens were conserved across strains, divergent from human proteins, and expressed across all T. cruzi infectious forms, revealing potential vaccine targets. We vaccinated mice with a 63-amino-acid-long immunogenic region using recombinant protein (rIR1) or yeast surface expression (yIR1). yIR1 surpassed rIR1, reducing parasitemia in blood and muscle tissues in a prophylactic, acute-stage vaccination model, validating IR1 as an immunogen and yeast as a vaccine vehicle for Chagas disease. Notably, therapeutic vaccination of chronically infected mice with yIR1 cleared parasites from muscle tissues and was accompanied by the production of -IR1 antibodies and an increased proportion of CD8+ T cells, indicating that a vaccine for treating chronic Chagas disease may be feasible. The antigen-antibody screen revealed hundreds of immunogens for vaccine, diagnostic, and biomarker discovery, and uncovered yIR1 as a therapeutic vaccine candidate for Chagas disease. One Sentence SummaryA yeast-based therapeutic vaccine candidate for treating chronic Chagas disease

microbiology↗

bioRxiv2025

Stochastic variation in surface protein expression diversifies Trypanosoma cruzi infection

Trypanosoma cruzi possesses hundreds of genes associated with its pathogenesis. The extent and organization of this diverse gene repertoire, expression, and role in infection remain unclear. Using accurate long-read sequencing and chromatin conformation capture, T. cruzi chromosomes were assembled from telomere-to-telomere. The genome revealed multigene families of virulence genes accounting for [~]70% of some chromosomes, organized in clusters or scattered through housekeeping genes. Quantitative proteomics identified stage-specific proteins and numerous trans-sialidases upregulated in trypomastigotes. Notably, the expression of virulence gene families changed stochastically in trypomastigotes, conferring T. cruzi fitness to cardiomyocyte infection while diversifying the invasion to multiple tissues. A T. cruzi genome-wide yeast surface display screen against Chagas disease patients antibodies revealed virulence genes expressed during human infections. However, limited conservation in their antibody-binding sites suggests their sequence diversity and variation help parasites avert antibody recognition. The data point to a role for virulence multigene families in infection persistence.

microbiology↗

bioRxiv2025

Chromosome compartment assembly is essential for subtelomeric gene silencing in trypanosomes

Genome three-dimensional organization is essential for the coordination of eukaryote gene expression. The chromosomes of the pathogen Trypanosoma brucei contain hundreds of silent variant surface glycoprotein (VSGs) genes in subtelomeric regions. However, T. brucei transcribes a single VSG gene and periodically changes the VSG expressed by transcriptional or recombination mechanisms, altering its surface coat to escape host antibodies by antigenic variation. We show that VSG-rich silent subtelomeric regions form distinct chromosome compartments from transcribed regions, with subtelomeric compartments of different chromosomes co-interacting. We uncovered chromatin-associating factors at the boundaries of transcribed and silent compartments. Among these, repressor activator-protein 1 (RAP1) marks the compartment boundaries and spreads over silent regions. Inactivation of phosphatidylinositol phosphate 5-phosphatase removed RAP1 from compartment boundaries and subtelomeric regions, disrupting compartment assembly and derepressing all VSG genes. The data show spatial segregation of repressed from transcribed chromatin and phosphoinositides regulation of silent compartment assembly and genome organization.

microbiology↗

bioRxiv2023

High-efficiency transformation and expression of genomic libraries in yeast

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield about 106 cfu/{micro}g of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/{micro}g plasmid DNA. The protocol generates sufficient transformants for 10-100x coverage of diverse genome libraries with small amounts of genomic libraries (0.1{micro}g of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the methods efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

molecular biology↗

bioRxiv2023

A PI(3,4,5)P3-dependent allosteric switch controls antigenic variation in trypanosomes

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

microbiology↗

bioRxiv2023

Helminth-derived metabolites induce tolerogenic functional, metabolic, and transcriptional signatures in dendritic cells that attenuate experimental colitis

Helminth infections can restore immune tolerance and suppress intestinal inflammation, yet the molecular mediators underlying these effects are poorly defined. Here, we show that nonpolar metabolites derived from the helminth Heligmosomoides polygyrus bakeri (HnpM) program dendritic cells (DCs) toward a tolerogenic DC (tolDC) phenotype. Adoptive transfer of HnpM-treated DCs significantly ameliorated DSS-induced intestinal inflammation in mice. Transcriptomic and metabolomic profiling revealed that HnpM induces early metabolic reprogramming followed by gene expression signatures characteristic of tolerogenic DCs that are shared by established tolDC-inducing agents. Together, these findings identify that helminth-derived metabolites are a novel source of small molecule therapeutics to restore immune tolerance in immune-mediated disorders. Summary SentenceMetabolites produced by Heligmosomoides polygyrus induce metabolic and transcriptional changes in DCs consistent with tolDCs, and adoptive transfer of these DCs attenuate DSS-induced intestinal inflammation.

immunology↗

bioRxiv2022

Genome-wide libraries for protozoan pathogens drug target screening using yeast surface display

The lack of genetic tools to manipulate protozoan pathogens has limited the use of genome-wide approaches to identify drug or vaccine targets and understand these organisms biology. We have developed an efficient method to construct genome-wide libraries for yeast surface display (YSD) and developed a YSD fitness screen (YSD-FS) to identify drug targets. We show the robustness of our method by generating genome-wide libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of [~]105 to 106 clones, representing [~]6 to 30-fold of the parasites genome. Nanopore sequencing confirmed the libraries genome coverage with multiple clones for each parasite gene. Western blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS assay, we identified bonafide interactors of metronidazole, a drug used to treat protozoan and bacterial infections. We also found enrichment in nucleotide-binding domain sequences associated with yeast increased fitness to metronidazole, indicating that this drug might target multiple enzymes containing nucleotide-binding domains. The libraries are valuable biological resources for discovering drug or vaccine targets, ligand receptors, protein-protein interactions, and pathogen-host interactions. The library assembly approach can be applied to other organisms or expression systems, and the YSD-FS assay might help identify new drug targets in protozoan pathogens.

microbiology↗

bioRxiv2019

Plasmodium secretion induces hepatocyte lysosome exocytosis and promotes parasite entry

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosomal-associated membrane protein (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of the SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates new parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen, Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens.

cell biology↗