Conspicuous chloroplast with light harvesting-photosystem I/II megacomplex in marine Prorocentrum cordatum.

Marine photosynthetic (micro)organisms drive multiple biogeochemical cycles and display a large diversity. Among them, the bloom-forming, free-living dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) stands out with its distinct cell biological features. Here, we obtained insights into the structural properties of the chloroplast and the photosynthetic machinery of P. cordatum using microscopic and proteogenomic approaches. High-resolution FIB/SEM analysis revealed a single large chloroplast (∼40% of total cell volume) with a continuous barrel-like structure, completely lining the inner face of the cell envelope and enclosing a single reticular mitochondrium, the Golgi apparatus, as well as diverse storage inclusions. Enriched thylakoid membrane fractions of P. cordatum were comparatively analyzed with those of the well-studied model-species Arabidopsis (Arabidopsis thaliana) using 2D BN DIGE. Strikingly, P. cordatum possessed a large photosystem-light harvesting megacomplex (>1.5 MDa), which is dominated by photosystems I and II (PSI, PSII), chloroplast complex I, and chlorophyll a-b binding light harvesting complex proteins. This finding parallels the absence of grana in its chloroplast and distinguishes from the predominant separation of PSI and PSII complexes in A. thaliana, indicating a different mode of flux balancing. Except for the core elements of the ATP synthase and the cytb6f-complex, the composition of the other complexes (PSI, PSII, and pigment-binding proteins, PBPs) of P. cordatum differed markedly from those of A. thaliana. Furthermore, a high number of PBPs was detected, accounting for a large share of the total proteomic data (∼65%) and potentially providing P. cordatum with flexible adaptation to changing light regimes.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Comparative analyses of parasites with a comprehensive database of genome-scale metabolic models.

Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings and discovered that phylogeny is not the sole predictor of metabolic similarity. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.

The non-LTR retrotransposons of Entamoeba histolytica: genomic organization and biology.

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.

Protocol for design, construction, and selection of genome phage (gPhage) display libraries.

This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage.

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)-mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1-dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome's homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole-induced damage, indicating that the prodrug-induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1-activated 2-nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription-coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention.

Single-cell genome sequencing of protozoan parasites.

Despite considerable genetic variation within hosts, most parasite genome sequencing studies focus on bulk samples composed of millions of cells. Analysis of bulk samples is biased toward the dominant genotype, concealing cell-to-cell variation and rare variants. To tackle this, single-cell sequencing approaches have been developed and tailored to specific host-parasite systems. These are allowing the genetic diversity and kinship in complex parasite populations to be deciphered and for de novo genetic variation to be captured. Here, we outline the methodologies being used for single-cell sequencing of parasitic protozoans, such as Plasmodium and Leishmania spp., and how these tools are being applied to understand parasite biology.

Genomic and Genetic Approaches to Studying Antimalarial Drug Resistance and Plasmodium Biology.

Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum, the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses.

Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives.

The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies.

Essential Genes of the Parasitic Apicomplexa.

Genome-scale mutagenesis screens for genes essential for apicomplexan parasite survival have been completed in three species: Plasmodium falciparum, the major human malaria parasite, Plasmodium berghei, a model rodent malaria parasite, and the more distantly related Toxoplasma gondii, the causative agent of toxoplasmosis. These three species share 2606 single-copy orthologs, 1500 of which have essentiality data in all three screens. In this review, we explore the overlap between these datasets to define the core essential genes of the phylum Apicomplexa. We further discuss the implications of these groundbreaking studies for understanding apicomplexan parasite biology, and we identify promising areas of focus for developing new pan-apicomplexan parasite interventions.

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum.

Establishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen's biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes.

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes-parasites that cause lethal diseases in humans and livestock-employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes2. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space3-5. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation6,7. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.

Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease.

The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.

Molecular Epidemiology of Giardia Infections in the Genomic Era.

Giardia duodenalis is a major gastrointestinal parasite of humans and animals across the globe. It is also of interest from an evolutionary perspective as it possesses many features that are unique among the eukaryotes, including its distinctive binucleate cell structure. While genomic analysis of a small number of isolates has provided valuable insights, efforts to understand the epidemiology of the disease and the population biology of the parasite have been limited by the molecular tools currently available. We review these tools and assess the impact of affordable and rapid genome sequencing systems increasingly being deployed in diagnostic settings. While these technologies have direct implications for public and veterinary health, they will also improve our understanding of the unique biology of this fascinating parasite.

Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives

The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies.

Structure of the Plasmodium-interspersed repeat proteins of the malaria parasite.

The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.

Identity-by-descent with uncertainty characterises connectivity of Plasmodium falciparum populations on the Colombian-Pacific coast.

Characterising connectivity between geographically separated biological populations is a common goal in many fields. Recent approaches to understanding connectivity between malaria parasite populations, with implications for disease control efforts, have used estimates of relatedness based on identity-by-descent (IBD). However, uncertainty around estimated relatedness has not been accounted for. IBD-based relatedness estimates with uncertainty were computed for pairs of monoclonal Plasmodium falciparum samples collected from five cities on the Colombian-Pacific coast where long-term clonal propagation of P. falciparum is frequent. The cities include two official ports, Buenaventura and Tumaco, that are separated geographically but connected by frequent marine traffic. Fractions of highly-related sample pairs (whose classification using a threshold accounts for uncertainty) were greater within cities versus between. However, based on both highly-related fractions and on a threshold-free approach (Wasserstein distances between parasite populations) connectivity between Buenaventura and Tumaco was disproportionally high. Buenaventura-Tumaco connectivity was consistent with transmission events involving parasites from five clonal components (groups of statistically indistinguishable parasites identified under a graph theoretic framework). To conclude, P. falciparum population connectivity on the Colombian-Pacific coast abides by accessibility not isolation-by-distance, potentially implicating marine traffic in malaria transmission with opportunities for targeted intervention. Further investigations are required to test this hypothesis. For the first time in malaria epidemiology (and to our knowledge in ecological and epidemiological studies more generally), we account for uncertainty around estimated relatedness (an important consideration for studies that plan to use genotype versus whole genome sequence data to estimate IBD-based relatedness); we also use threshold-free methods to compare parasite populations and identify clonal components. Threshold-free methods are especially important in analyses of malaria parasites and other recombining organisms with mixed mating systems where thresholds do not have clear interpretation (e.g. due to clonal propagation) and thus undermine the cross-comparison of studies.

Exploring Eimeria Genomes to Understand Population Biology: Recent Progress and Future Opportunities.

Eimeria, protozoan parasites from the phylum Apicomplexa, can cause the enteric disease coccidiosis in all farmed animals. Coccidiosis is commonly considered to be most significant in poultry; due in part to the vast number of chickens produced in the World each year, their short generation time, and the narrow profit margins associated with their production. Control of Eimeria has long been dominated by routine chemoprophylaxis, but has been supplemented or replaced by live parasite vaccination in a minority of production sectors. However, public and legislative demands for reduced drug use in food production is now driving dramatic change, replacing reliance on relatively indiscriminate anticoccidial drugs with vaccines that are Eimeria species-, and in some examples, strain-specific. Unfortunately, the consequences of deleterious selection on Eimeria population structure and genome evolution incurred by exposure to anticoccidial drugs or vaccines are unclear. Genome sequence assemblies were published in 2014 for all seven Eimeria species that infect chickens, stimulating the first population genetics studies for these economically important parasites. Here, we review current knowledge of eimerian genomes and highlight challenges posed by the discovery of new, genetically cryptic Eimeria operational taxonomic units (OTUs) circulating in chicken populations. As sequencing technologies evolve understanding of eimerian genomes will improve, with notable utility for studies of Eimeria biology, diversity and opportunities for control.