Biology subjects

Robello, C.

Publications and source records attributed to Robello, C..

13 recordsLinked to original sources

bioRxiv2025

Tracking the Spread of a Naturally Occurring Leishmania infantumMutant: A qPCR-Based Investigation in Strains and Clinical Samples

The circulation of Leishmania infantum strains carrying a 12 Kb genomic deletion (DEL) alongside wild-type (NonDEL) parasites in the Americas raises critical epidemiological questions. To support molecular surveillance efforts, we developed and validated a qPCR-based genotyping tool capable of detecting and quantifying DEL, NonDEL, and coinfection (MIX) profiles. The strategy targets a constitutive region present in all strains and a specific region exclusive to NonDEL parasites. It was validated using DNA from cultured parasites and applied to a panel of diverse clinical samples from dogs, humans, and other hosts across South and Central America. DEL genotypes were found to be more frequent and widespread, both in clinical samples and cultured parasites, suggesting their significant role in the transmission cycle. The tools application revealed coinfections in paired samples and enabled relative quantification of genotypes. It also showed good correlation with parasite load estimations and high discriminatory power when integrated with the Genotype function of real-time PCR software. This protocol provides a robust, scalable approach for monitoring L. infantum genotypes, with direct implications for understanding infection dynamics, treatment outcomes and public health surveillance. Author SummaryLeishmania infantum is the parasite responsible for visceral leishmaniasis in the Americas. Recent studies have shown that two distinct genotypes--those carrying a specific gene deletion (DEL) and those without it (NonDEL)--coexist and circulate in the region. Understanding how these genotypes are distributed and whether they affect disease transmission or treatment is critical, especially because Miltefosine, a drug used to treat dogs, may not work equally well against both genotypes. In this study, we developed a simple and accurate molecular test to detect and quantify these genotypes in both cultured parasites and clinical samples from different animal hosts, including humans. We found that DEL genotypes are common and widely distributed, and that some animals carry both types of parasites simultaneously. Our method also distinguishes L. infantum from other related species, making it useful for diagnosis and surveillance. This tool can help researchers and health authorities monitor parasite populations more effectively and better understand how different genotypes might impact disease outcomes and control strategies.

microbiology↗

bioRxiv2025

Transcriptomic response to different heme sources in Trypanosoma cruzi

Heme is an essential molecule for most organisms, yet some parasites, like Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize it. These parasites must acquire heme from their hosts, making this process critical for their survival. In the midgut of the insect vector, T. cruzi epimastigotes are exposed to both hemoglobin (Hb) and free heme resulting from its degradation. Despite the importance of this nutrient, how different heme sources influence parasite gene expression remains poorly understood. Here, we showed that heme restitution either as hemin or Hb to heme-starved parasites induces an early and distinct transcriptional response in T. cruzi epimastigotes. Using RNA sequencing at 4- and 24-hours post-supplementation, we identified gene subsets commonly or uniquely regulated by each heme source, including genes putatively linked to heme acquisition and metabolism. We also presented here the first studies focused on CRAL/TRIO domain-containing protein (TcCRAL/TRIO), a novel heme responsive hemoprotein identified from this study. Our results provide a more detailed picture of T. cruzi biology and highlights heme acquisition as a promising point of vulnerability. These findings may ultimately contribute to the identification of potential molecular targets for the development of new therapeutic strategies against Chagas disease.

biochemistry↗

bioRxiv2025

Trypanosoma cruzi Has 32 Chromosomes: A Telomere-to-Telomere Assembly Defines Its Karyotype.

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits remarkable genomic variability and possesses an expanded genome rich in multigene families. However, its precise chromosomal composition has remained elusive due to the challenges posed by extensive repetitive regions. Using PacBio HiFi long-read sequencing, we assembled the complete nuclear genome of the Dm28c strain into 32 telomere-to-telomere chromosomes. This assembly revealed conserved chromosomal structures and synteny patterns when compared to the independently sequenced Dm25 strain, indicating that the 32-chromosome karyotype is a stable and conserved feature of T. cruzi. The species is diploid for all chromosomes, except for chromosome 16, which is consistently tetraploid in all strains analyzed, and homologous to chromosome 31 of Leishmania, also tetraploid. Through a comprehensive annotation pipeline, we refined gene content and resolved haplotypes, resulting in an approximately 25% reduction in core gene redundancy. All chromosomes display a conserved, compartmentalized architecture comprising core, disruptive, and newly defined subtelomeric regions. Subtelomeres, enriched in RHS, TS, and DGF-1 genes and depleted in MASP and mucins, constitute a distinct third genomic compartment, that is transcriptionally active, and represents the main source of genome variability among strains. Altogether, this work defines the complete chromosomal complement of T. cruzi, establishing a robust framework for comparative genomics and enabling detailed studies into genome organization, antigenic variability, and evolutionary dynamics across strains and clades. This assembly redefines the genomic reference landscape and opens new avenues for exploring T. cruzi biology, pathogenic diversity, and adaptive mechanisms.

genomics↗

bioRxiv2024

Modulation of Host Cell Membrane Biophysics Dynamics by Neospora caninum: A Study Using LAURDAN Fluorescence with Hyperspectral Imaging and Phasor Analysis

Neospora caninum is known to manipulate host cell organelles and recruit lipids for its survival. However, the impact of this lipid redistribution on host cell membranes remains poorly understood. This study used LAURDAN fluorescence, hyperspectral imaging, and phasor plot analysis to investigate how N. caninum modifies membrane order in Vero cells. The results revealed a significant decrease in host cell plasma and internal membrane order upon infection, suggesting that cholesterol is redistributed from the host plasma membrane to the parasitophorous vacuoles. To mimic cholesterol depletion, uninfected cells were treated with methyl-{beta}-cyclodextrin (MBCD), which increased membrane fluidity. Conversely, replenishing infected cells with cholesterol-loaded MBCD restored membrane fluidity to levels lower than control cells, indicating cholesterol enrichment. These findings provide novel insights into how N. caninum modulates host cell membrane dynamics through lipid manipulation, potentially aiding its intracellular survival.

biophysics↗

bioRxiv2023

Genomic organization of Trypanosoma cruzi tRNA genes

An often neglected class of genes in large-scale genome analyses is the non-protein-coding genes. In particular, due to the incompleteness of the genome assembly, it had not been possible to determine the genomic organization of the tRNA genes in Trypanosoma cruzi --a eukaryotic unicellular pathogen causative of disabling human Chagas disease--. Here, we analyzed the genic content and distribution of tRNA genes in the nuclear genome of different strains compared with the genome organization of other related trypanosomatids. We found synteny in most of the tDNAs clusters between T. cruzi and Trypanosoma brucei, demonstrating the importance of the genome location of these Pol III transcribed genes. The vast majority of the isoacceptor species are encoded by two genes, except for tDNASeC, which is present as a tandem of 11 copies in the core compartment, associated with well-positioned nucleosomes. Finally, we describe a group of tRNA genes located at chromatin folding domain boundaries, possibly acting as chromatin insulators in T. cruzi.

genomics↗

bioRxiv2023

3D genome organization drives gene expression in trypanosomes

In trypanosomes --eukaryotic unicellular pathogens that cause disabling human and animal diseases-- very few transcriptional regulatory elements have been described and it is largely accepted that they regulate gene expression mainly post-transcriptionally. In this regard, the role of the spatial organization of the genome on gene expression and vice versa remains practically unexplored. The genome of these parasites is partitioned into core (highly conserved syntenic) and species-specific disruptive regions (synteny disruption), containing multigene families encoding for surface glycoproteins. By mapping genome-wide chromatin interactions we demonstrate that these regions constitute 3D compartments (C and D). These chromatin compartments present significant differences in DNA methylation, nucleosome positioning and chromatin interactions, affecting genome expression dynamics. We show that the genome is organized into chromatin folding domains and transcription is dramatically determined by the local chromatin structure. Our results support a model in which epigenetic mechanisms dramatically impact gene expression in these eukaryotic pathogens.

microbiology↗

bioRxiv2022

Trypanosoma cruzi isolates naturally adapted to congenital transmission display a unique strategy of transplacental passage

Chagas disease is mainly transmitted by vertical transmission (VT) in non-endemic areas and in endemic areas where vector control programs have been successful. For the present study, we isolated natural strains vertically transmitted through three generations and proceeded to study their molecular mechanism of VT. No parasitemia was detected in immunocompetent mice, but they were able to induce an immune response and colonize different organs. VT experiments revealed that infection with the different strains did not affect mating, pregnancy or resorptions, but despiting low parasitemia, VT strains reached the placenta and resulted in higher vertical transmission rates than strains of either moderate or high virulence. While the virulent strain modulated more than 2500 placental genes, VT strains modulated 150, and none of the modulated genes is shared between them. VT strains downregulate genes associated with cell division and replication and upregulate immunomodulatory genes leading to anti-inflammatory responses and tolerance. The virulent strain stimulates a strong pro-inflammatory immune response, and this molecular footprint correlated with histopathological analyses. We herein describe a unique placental response regarding the passage of T cruzi VT isolates across the maternal-fetal interphase, challenging the current knowledge derived mainly from studies of laboratory-adapted or highly virulent strains.

microbiology↗

bioRxiv2021

Indigenous ancestry and admixture in the Uruguayan population

The Amerindian group known as the Charruas inhabited Uruguay at the timing of European colonial contact. Even though they were extinguished as an ethnic group as a result of a genocide, Charruan heritage is part of the Uruguayan identity both culturally and genetically. While mitochondrial DNA studies have shown evidence of Amerindian ancestry in living Uruguayans, here we undertake whole-genome sequencing of 10 Uruguayan individuals with Charruan heritage. We detect chromosomal segments of Amerindian ancestry supporting the presence of indigenous genetic ancestry in living descendants. Specific haplotypes were found to be enriched in Charruas and rare in the rest of the Amerindian groups studied. Some of these we interpret as the result of positive selection, as we identified selection signatures and they were located mostly within genes related to the infectivity of specific viruses. Historical records describe contacts of the Charruas with other Amerindians, such as Guarani, and patterns of genomic similarity observed here concur with genomic similarity between these groups. Less expected, we found a high genomic similarity of the Charruas to Diaguita from Argentinian and Chile, which could be explained by geographically proximity. Finally, by fitting admixture models of Amerindian and European ancestry for the Uruguayan population, we were able to estimate the timing of the first pulse of admixture between European and Uruguayan indigenous peoples in 1658 and the second migration pulse in 1683. Both dates roughly concurring with the Franciscan missions in 1662 and the foundation of the city of Colonia in 1680 by the Spanish.

genomics↗

bioRxiv2021

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1023 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A, B and D, and group 2 containing clade C. No significant differences were found in hybrid strains that justify the existence of TcV and Tc VI as separate clades: our results indicate that a unique event of hybridization between TcII and TcIII occurred. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

genomics↗

bioRxiv2020

Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements

Neospora caninum primarily infects cattle causing abortions with an estimated impact of a billion dollars on worldwide economy, annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen, Toxoplasma gondii. By revisiting the genome sequence, assembly and annotation using third generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii. We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally annotated as ChrVIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing over 500 new genes. We sequence and annotate the non-photosynthetic plastid and mitochondrial genomes, and show that while apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exists between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii, but more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.

genomics↗

bioRxiv2018

Nanopore sequencing significantly improves genome assembly of the eukaryotic protozoan parasite Trypanosoma cruzi

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a two-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ~16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource since it now represents the best-quality assembly available for TcII, a highly prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of T. cruzi and facilitates more comprehensive evolutionary inferences. Our work represents the first report of Nanopore technology used to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.

genomics↗

bioRxiv2018

The Tritryps comparative repeatome: insights on repetitive element evolution in Trypanosomatid pathogens

The major human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are collectively known as the Tritryps. The initial comparative analysis of their genomes has uncovered that Tritryps share a great number of genes, but repetitive DNA seems to be extremely variable between them. However, the in-depth characterization of repetitive DNA in these pathogens has been in part neglected, mainly due to the well-known technical challenges of studying repetitive sequences from de novo assemblies using short reads. Here, we compared the repetitive DNA repertories between the Tritryps genomes using genome-wide, low-coverage Illumina sequencing coupled to RepeatExplorer analysis. Our work demonstrates that this extensively implemented approach for studying higher eukaryote repeatomes is also useful for protozoan parasites like trypanosomatids, as we recovered previously observed differences in the presence and amount of repetitive DNA families. Additionally, our estimations of repetitive DNA abundance were comparable to those obtained from enhanced-quality assemblies using longer reads. Importantly, our methodology allowed us to describe a previously unknown transposable element in Leishmania major (TATE family), highlighting its potential to accurately recover distinctive features from poorly characterized repeatomes. Together, our results support the application of this low-cost, low-coverage sequencing approach for the extensive characterization of repetitive DNA evolutionary dynamics in trypanosomatid and other protozooan genomes.

genomics↗

bioRxiv2018

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degree of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi{acute}s genome since they permit directly determining the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, allows not only accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (DTU TcVI) and the non-hybrid Dm28c (DTU TcI), determined by PacBio SMRT technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a \"core compartment\" and a \"disruptive compartment\" which exhibit opposite gene and GC content composition. New tandem and disperse repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families MUC and trans-sialidases allows now a better overview of these complex groups of genes.

genomics↗