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 approximately 10⁶ cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 10⁸ cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10–100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg 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 method’s 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.

Trypanosoma cruzi possesses hundreds of genes associated with 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, we assembled T. cruzi Sylvio X10 strain chromosomes from telomere-to-telomere. The genome provides accurate organization of multigene family genes, confirming their distribution in expanded clusters or scattered throughout the chromosomes. Quantitative proteomics shows stage-specific proteins and numerous trans-sialidases upregulated in trypomastigotes. The expression of virulence gene families varied in trypomastigotes after each round of cell infection, resulting in heterogeneous parasite populations with variable cell invasion capacity. A T. cruzi genome-wide yeast surface display screen against Chagas disease patients’ antibodies reveals genes expressed during human infections. However, limited conservation in their antibody-binding sites suggests their sequence diversity and variation might help parasites avert antibody recognition. The data point to a role for some multigene families in infection persistence.

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.

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 α-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.