Understanding and identifying protein-ligand interactions is essential for elucidating fundamental biological processes and developing translational applications such as vaccine and drug targets. Yeast surface display (YSD) systems expressing genome-wide or combinatorial libraries in Saccharomyces cerevisiae are powerful tools to identify protein-ligand interactions in an unbiased fashion. In the following protocol, we couple the YSD system expressing a genome-wide library of the protozoan pathogen Trypanosoma cruzi with magnetic-activated cell sorting to identify antigen-antibody interactions. We describe the enrichment of pathogen antigens targeted by antibodies of infected patients using the YSD library. We also detailed a DNA sequencing methodology and a data analysis computational pipeline. The approach can be easily adapted to identify protein-protein or protein-drug interactions.

Abstract 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. One Sentence Summary A yeast-based therapeutic vaccine candidate for treating chronic Chagas disease

Trypanosoma brucei is a single-celled, flagellated, extracellular protozoan parasite and the causative agent of African trypanosomiasis in humans and animals. T. brucei evades the host antibody responses by periodically switching its Variant Surface Glycoprotein (VSGs) coat. VSGs are highly antigenic proteins expressed in a monogenic manner from a repertoire of ~2,500 genes and pseudogenes, at subtelomeric expression sites (ES). However, the mechanisms that control VSG switching remain unclear. Previously, immunoprecipitation coupled with cross‑linking and mass spectrometry revealed that, as part of the phosphatidylinositol signaling pathway, phosphatidylinositol 5‑phosphatase (PIP5Pase) associates with the VSG silencer repressor‑activator protein 1 (RAP1) to regulate VSG expression and switching in T. brucei. Additionally, PIP5Pase and RAP1 strongly interact with several additional proteins, including eight candidate regulators which we hypothesize are essential genes for parasite survival and VSG expression control. Our objective is to investigate the role of these candidate regulators in antigenic variation in T. brucei. We constructed tetracycline‑inducible RNA‑interference (RNAi) cell lines in bloodstream form T. brucei SM427 to induce gene-specific knockdowns, followed by gene expression analysis. Five genes were cloned into the MC177VSG221RNAi vector, and two constructs were successfully transfected to generate tetracycline-inducible RNAi cell lines. RNAi-mediated knockdown of the two candidate regulators did not significantly affect parasite growth or morphology. Therefore, these two genes are unlikely to be essential for parasite viability. Gene expression analysis and VSG switching assays are in progress to determine the consequence of the knockdowns on VSG expression and better understand the host evasion mechanisms used by T. brucei.

In vitro enzyme assays provide sensitive and quantitative approaches to studying activity, identifying substrates or performing inhibitor analysis. These approaches can help to study enzymes that regulate different cellular processes in an organism. In addition, enzyme assays can be used to screen protein inhibitors to identify novel drug candidates. In eukaryotes, phosphatidylinositol kinase and phosphatase are enzymes that regulate the level of phosphorylated phosphatidylinositol, which controls several cellular processes. They also regulate several processes in many pathogens, such as kinetoplastid and apicomplexan parasites, and are validated drug targets. Here, we describe protocols to determine the activity of purified phosphoinositide kinases and phosphatases and screens to identify potential inhibitors. The assays are luminescence- and absorbance-based to measure the activity of phosphatidylinositol kinase and phosphatase enzymes, respectively. These assays can be adapted to other protein kinases and phosphatases.

Genome three-dimensional organization is essential for eukaryotic gene expression. The chromosomes of the pathogen Trypanosoma brucei contain hundreds of silent variant surface glycoprotein (VSG) genes in subtelomeric regions. T. brucei transcribes a single VSG gene and periodically changes the VSG expressed, altering its surface coat to escape host antibodies by antigenic variation. We show that T. brucei core and subtelomeric chromosome compartments are separated by distinct boundaries and display topologically associating domains and loops. Chromosomes co-interact through compartment boundaries, which insulate silent subtelomeric from transcribed core compartments. We uncover chromatin-associating factors at the boundaries, including repressor-activator protein 1 (RAP1), which spreads over silent compartments. Inactivation of the RAP1 regulator, phosphatidylinositol phosphate 5-phosphatase, removes RAP1 from boundaries and subtelomeric compartments, disrupting chromatin compartment contacts and activating all VSG genes. The data show spatial segregation of repressed from transcribed chromatin and phosphoinositide regulation of compartment assembly and genome organization.

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. ### Competing Interest Statement The authors have declared no competing interest.

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.

Chromatin compartmentalization is essential for the monogenic expression of variant surface glycoprotein (VSG) genes in African trypanosomes. Trypanosomes express one out of 2,500 VSG genes at a time from telomeric expression sites (ESs). VSGs form a homogenous surface coat essential for parasite evasion of host antibodies by antigenic variation. Trypanosomes periodically switch the VSG expressed by transcriptional switching among telomeric ESs or by VSG gene recombination. Most silent VSG genes are subtelomeric and silenced by mechanisms still not understood. We found that subtelomeric chromatin is organized within repressive compartments essential for silencing VSG genes. Chromatin capture (Pore-C) experiments showed significant enrichment in interactions of silent subtelomeres, whereas the actively transcribed subtelomeric VSG gene interacts with highly transcribed chromosome loci. Fluorescence in situ hybridization and microscopy analysis confirmed telomeric and subtelomeric chromatin compartmentalization. In vivo crosslinking and mass spectrometry interaction analysis of the phosphatidylinositol phosphate 5-phosphatase (PIP5Pase) protein, which regulates VSG gene silencing, revealed a network of interactions involved in compartment assembly and gene repression. Genetic mutations that affect PIP5Pase activity resulted in the reorganization of subtelomeric chromatin, followed by transcription of silent subtelomeric genes and the switching in VSG gene expression. The data indicate that subtelomeric chromatin assembles into repressive compartments that coordinates the repression of hundreds of VSG genes. The compartments are essential for coordinated expression of surface antigens during antigenic variation. This work was funded by Canadian Institutes of Health Research (IC); The Natural Sciences and Engineering Research Council of Canada (IC); Fonds de Recherche du Québec - Nature et Technologie (IC); Canada Foundation for Innovation (IC); and FRQNT-Ukraine postdoctoral fellowship (OK).

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases in which abdominal pain, bloody diarrhea, weight loss, and fatigue collectively result in diminished quality of patient life. The disappearance of intestinal helminth infections in Western societies is associated with an increased prevalence of IBD and other immune-mediated inflammatory diseases. Evidence indicates that helminths induce tolerogenic dendritic cells (tolDCs), which promote intestinal tolerance and attenuate intestinal inflammation characteristic of IBD, but the exact mechanism is unclear. Helminth-derived excretory-secretory (HES) products including macromolecules, proteins, and polysaccharides have been shown to modulate the antigen presenting function of DCs with down-stream effects on effector CD4 + T cells. Previous studies indicate that DCs in helminth-infected animals induce tolerance to unrelated antigens and DCs exposed to HES display phenotypic and functional features of tolDCs. Here, we identify that nonpolar metabolites (HnpM) produced by a helminth, the murine gastrointestinal nematode Heligmosomoides polygyrus bakeri (Hpb), induce tolDCs as evidenced by decreased LPS-induced TNF and increased IL-10 secretion and reduced expression of MHC-II, CD86, and CD40. Furthermore, these DCs inhibited OVA-specific CD4 + T cell proliferation and induced CD4 + Foxp3 + regulatory T cells. Adoptive transfer of HnpM-induced tolDCs attenuated DSS-induced intestinal inflammation characteristic of IBD. Mechanistically, HnpM induced metabolic and transcriptional signatures in BMDCs consistent with tolDCs. Collectively, our findings provide groundwork for further investigation into novel mechanisms regulating DC tolerance and the role of helminth secreted metabolites in attenuating intestinal inflammation associated with IBD. Summary Sentence: Metabolites produced by Heligmosomoides polygyrus induce metabolic and transcriptional changes in DCs consistent with tolDCs, and adoptive transfer of these DCs attenuated DSS-induced intestinal inflammation.

EDITORIAL article Front. Immunol., 12 January 2024Sec. Parasite Immunology Volume 15 - 2024 | https://doi.org/10.3389/fimmu.2024.1362719

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.

Abstract 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.

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.

Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single -celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of similar to 2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug -selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell -specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes.

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 ∼10 5 to 10 6 clones, representing ∼6 to 30-fold of the parasite’s 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.

Human trypanosomiasis and leishmaniasis are vector-borne neglected tropical diseases caused by infection with the protozoan parasitesTrypanosomaspp. andLeishmaniaspp., respectively. Once restricted to endemic areas, these diseases are now distributed worldwide due to human migration, climate change, and anthropogenic disturbance, causing significant health and economic burden globally. The current chemotherapy used to treat these diseases has limited efficacy, and drug resistance is spreading. Hence, new drugs are urgently needed. Phenotypic compound screenings have prevailed as the leading method to discover new drug candidates against these diseases. However, the publication of the complete genome sequences of multiple strains, advances in the application of CRISPR/Cas9 technology, and in vivo bioluminescence-based imaging have set the stage for advancing target-based drug discovery. This review analyses the limitations of the narrow pool of available drugs presently used for treating these diseases. It describes the current drug-based clinical trials highlighting the most promising leads. Furthermore, the review presents a focused discussion on the most important biological and pharmacological challenges that target-based drug discovery programs must overcome to advance drug candidates. Finally, it examines the advantages and limitations of modern research tools designed to identify and validate essential genes as drug targets, including genomic editing applications and in vivo imaging.

Abstract Background Inflammatory bowel diseases (IBD) are chronic inflammatory diseases characterized by abdominal pain, bloody diarrhea, fatigue, weight loss, and diminished quality of life. The morbidity associated with IBD is a result of loss of tolerance towards the gastrointestinal commensal microbiota. The high incidence of IBD in Western societies is inversely correlated with the low incidence of intestinal helminth parasite infections, potentially due to the ability of the helminth parasite to induce tolerance by inducing tolerogenic dendritic cells (tolDC) polarization. Although macromolecules from helminth such as proteins and polysaccharides have been shown to polarize tolDCs, to characterize a novel pathway, in this study we focused on small molecules such as metabolites in the helminth secretome. We hypothesized that helminth-derived metabolites (HDMs) polarize DCs towards a tolerogenic phenotype, which alleviates colitis. Aims To evaluate and characterize the tolerogenic response induced by HDMs in DCs, and its ability to alleviate colitis. Methods Heligmosomoides polygyrus worms were culture for 24 h and HDMs were isolated from conditioned media by chromatography. Bone marrow dendritic cells (BMDCs) were differentiated with GM-CSF for 8 days and then incubated with HDM for 4 h before LPS stimulation for 20 h. Cytokine secretion was measured by ELISA. The transcriptome of DCs treated with HDMs was assessed by RNAseq. Colitis was induced by giving 3% DSS in drinking water for 5 days followed by 3 days of tap water. The anti-colitic effect of HDMs was assessed by daily treatment with HDM or DCs treated with HDM in the 3 days of tap water. Results Pre-treatment with HDM decreased LPS-induced TNF and increased IL-10 release by BMDCs, compared to control BMDCs. Colitic mice treated with HDM presented lower disease activity scores, less colon shortening, decreased weight loss, and healthier histopathology compared to vehicle-treated colitic mice. Importantly, there was an increased frequency of CD11c+ CD103+ DCs in the colon of HDM-treated mice, suggesting that HDM alleviates colitis by increasing the abundance of tolDCs in the colon. Adoptive transfer of HDM-treated DCs also reduced the severity of colitis compared with vehicle-treated mice or mice that received naïve DCs. These results indicate that HDM induced tolerogenic DCs, which in turn ameliorates DSS colitis. RNAseq showed that HDM upregulated 183 and downregulated 76 genes. These differentially expressed genes may indicate a novel mechanism by which helminths induce a tolerogenic profile in DCs. Conclusions HDMs induce tolerogenic DCs and alleviate DSS-induced colitis. Funding Agencies NSERC and FRQNT

Genome-wide screens using yeast or phage displays are powerful tools for identifying protein-ligand interactions, including drug or vaccine targets, ligand receptors, or protein-protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 10 5 -10 7 DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system.