Biology subjects

Machado, H.

Publications and source records attributed to Machado, H..

10 recordsLinked to original sources

bioRxiv2024

m6A landscape is more pervasive when Trypanosoma brucei exits the cell cycle

N6-methyladenosine (m6A) is a mRNA modification with important roles in gene expression. In African trypanosomes, this post-transcriptional modification is detected in hundreds of transcripts and it affects the stability of the variant surface glycoprotein (VSG) transcript in the proliferating blood stream form. However, how m6A landscape varies across the life cycle remains poorly defined. Using full-length, non-fragmented RNA, we immunoprecipitated and sequenced m6A-modified transcripts across three life cycle stages of Trypanosoma brucei - slender (proliferative), stumpy (quiescent), and procyclic forms (proliferative). We found that 1037 transcripts are methylated in at least one of these three life cycle stages. While 21% of methylated transcripts are common in the three stages of the life cycle, globally each stage has a distinct methylome. Interestingly, 47% of methylated transcripts are detected in the quiescent stumpy form only, suggesting a critical role for m6A when parasites exit the cell cycle and prepare for transmission by the Tsetse fly. In this stage, we found that a significant proportion of methylated transcripts encodes for proteins involved in RNA metabolism, which is consistent with their reduced transcription and translation. Moreover, we found that not all major surface proteins are regulated by m6A, as procyclins are not methylated, and that, within the VSG repertoire, not all VSG transcripts are demethylated upon parasite differentiation to procyclic form. This study reveals that the m6A regulatory landscape is specific to each life cycle stage, becoming more pervasive as T. brucei exits the cell cycle. SummaryAfrican trypanosome parasites adapt to mammalian and insect hosts by adjusting gene expression, morphology, and metabolism. In this study, we focus on how N6-methyladenosine (m6A), a post-transcriptional modification, affects the parasites transcriptome throughout its differentiation from the mammalian host to the fly. We found that methylation is differentially regulated as the life cycle progresses, being particularly prevalent in the non-proliferative stumpy form, as more methylated transcripts are found at this insect-infective stage than in slender and procyclic forms. We further show that the not all parasite surface proteins are regulated by m6A and that the previously identified link between m6A methylation and the expression level of the major surface protein of bloodstream forms applies to the active variant surface glycoprotein, but not always to silent genes, suggesting two distinct regulatory mechanisms of (de)methylation.

molecular biology↗

bioRxiv2023

The lymphatic system favours survival of a unique T. brucei population, and its invasion results in major host pathology.

Trypanosoma brucei colonize and multiply in the blood vasculature, as well as in various organs of the hosts body. Lymph nodes have been previously shown to harbour large numbers of parasites, and the lymphatic system has been proposed as a key site that allows T. brucei distribution through, and colonization of the mammalian body. However, visualization of host-pathogen interactions in the lymphatic system has never captured dynamic events with high spatial and temporal resolution throughout infection. In our work, we used a mixture of tools including intravital microscopy and ex vivo imaging to study T. brucei distribution in 20 sets of lymph nodes. We demonstrate that lymph node colonization by T. brucei is different across lymph node sets, with the most heavily colonized being the draining lymph nodes of main tissue reservoirs: the gonadal adipose tissue and pancreas. Moreover, we show that the lymphatic vasculature is a pivotal site for parasite dispersal, and altering this colonization by blocking LYVE-1 (i.e. surface receptor of lymphatic vessel endothelial cells) is detrimental for parasite survival. Additionally, parasites within the lymphatic vasculature have unique morphological and behavioural characteristics, different to those found in the blood, demonstrating a physical separation of environments across both types of vasculature. Finally, we demonstrate that the lymph nodes and the lymphatic vasculature undergoes significant alterations during T. brucei infection, resulting in oedema throughout the hosts body.

microbiology↗

bioRxiv2022

Adipocyte lipolysis protects the host against Trypanosoma brucei infection

Trypanosoma brucei, an etiological agent of African trypanosomiasis, colonizes the interstitial spaces of the adipose tissue in disproportionately high numbers, inducing a robust local immune response. Loss of body weight, including loss of adipose mass, is a hallmark symptom of African trypanosomiasis. Nevertheless, it is unclear which molecular mechanisms drive this loss of adipose mass and in turn whether it contributes to pathology. Here we show that lipolysis is activated early in infection in adipose tissue of T. brucei-infected mice. This activation is dependent on immune activation, as mice deficient for both B and T lymphocytes failed to upregulated adipocyte lipolysis upon infection and retained higher fat mass. Genetic ablation of the rate limiting adipose triglyceride lipase specifically from adipocytes in mice (AdipoqCre/+-Atglfl/fl) prevented the upregulation of adipocyte lipolysis during infection, leading to reduced loss of fat mass and adipocyte volume. Surprisingly infected AdipoqCre/+-Atglfl/fl mice succumbed earlier to infection and presented a higher parasite burden in the gonadal adipose tissue, indicating that lipolysis limits the growth of the parasite population. Collectively, this work provides molecular mechanistic insight into the loss of fat mass in African trypanosomiasis and identifies adipocyte lipolysis as a host-protective mechanism during a T. brucei infection.

microbiology↗

bioRxiv2021

Clonal dynamics of haematopoiesis across the human lifespan

Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer. The cellular alterations that underpin the abruptness of this functional decline after the age of 70 years remain elusive. We sequenced 3579 genomes from single-cell-derived colonies of haematopoietic stem cell/multipotent progenitors (HSC/MPPs) across 10 haematologically normal subjects aged 0-81 years. HSC/MPPs accumulated 17 mutations/year after birth and lost 30bp/year of telomere length. Haematopoiesis in adults aged <65 was massively polyclonal, with high indices of clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. In contrast, haematopoiesis in individuals aged >75 showed profoundly decreased clonal diversity. In each elderly subject, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before age 40, but only 22% had known driver mutations. Genome-wide selection analysis estimated that 1/34 to 1/12 non-synonymous mutations were drivers, occurring at a constant rate throughout life, affecting a wider pool of genes than identified in blood cancers. Loss of Y chromosome conferred selective benefits on HSC/MPPs in males. Simulations from a simple model of haematopoiesis, with constant HSC population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.

genomics↗

bioRxiv2021

Genome-wide mutational signatures of immunological diversification in normal lymphocytes

A lymphocyte suffers many threats to its genome, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. After developing protocols for single-cell lymphocyte expansions, we sequenced whole genomes from 717 normal naive and memory B and T lymphocytes and hematopoietic stem cells. Lymphocytes carried more point mutations and structural variation than stem cells, accruing at higher rates in T than B cells, attributable to both exogenous and endogenous mutational processes. Ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every one on-target IGV mutation during the germinal center reaction. Structural variation was 16-fold higher in lymphocytes than stem cells, with ~15% of deletions being attributable to off-target RAG activity. One Sentence SummaryThe mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

genomics↗

bioRxiv2021

Note: Updating the metadata of four misidentified samples in the DrosRTEC dataset

This note details the consortiums rationale behind its decision to modify the metadata for putatively misidentified European samples in the DrosRTEC dataset. In brief, we use PCA on published datasets from North America and Europe to generate phylogeographic clusters reflective of worldwide D. melanogaster demography. We used this PCA to train a DAPC model in order the predict the group membership. Our results indicate that 4 out of 73 samples were misclassified and the metadata was updated accordingly. These samples are a spring-fall pair from Spain and Austria.

genomics↗

bioRxiv2020

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses. One-sentence summaryHeterologous expression of the Castanea crenata allene oxide synthase gene in Arabidopsis thaliana improves the defense response to the pathogen Phytophthora cinnamomi.

plant biology↗

bioRxiv2020

Environmental conditions dictate differential evolution of vancomycin resistance in Staphylococcus aureus

While microbiological resistance to vancomycin in Staphylococcus aureus is rare, clinical vancomycin treatment failures are common, and methicillin-resistant S. aureus (MRSA) strains isolated from patients after prolonged vancomycin treatment failure remain susceptible. Adaptive laboratory evolution was utilized to uncover mutational mechanisms associated with MRSA vancomycin resistance in a bacteriological medium used in clinical susceptibility testing and a physiological medium. Sequencing of resistant clones revealed shared and media-specific mutational outcomes, with an overlap in cell wall regulons (walKRyycHI, vraSRT). Evolved strains displayed similar genetic and phenotypic traits to resistant clinical isolates. Importantly, resistant phenotypes that developed in physiological media did not translate into resistance in bacteriological media. Further, a bacteriological media-specific mechanism for vancomycin resistance enabled by a mutated mprF was confirmed. This study bridges the gap of understanding between clinical and microbiological vancomycin resistance in S. aureus and expands the number of allelic variants that result in vancomycin resistance phenotypes.

microbiology↗

bioRxiv2020

Revealing 29 sets of independently modulated genes in Staphylococcus aureus, their regulators and role in key physiological responses

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its Transcriptional Regulatory Network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying Independent Component Analysis (ICA) to a compendium of 108 RNAseq expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed (1) high confidence associations between 21 i-modulons and known regulators; (2) an association between an i-modulon and {sigma}S, whose regulatory role was previously undefined; (3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR and Vim-3, (4) the roles of three key transcription factors (codY, Fur and ccpA) in coordinating the metabolic and regulatory networks; and (5) a low dimensional representation, involving the function of few transcription factors, of changes in gene expression between two laboratory media (RPMI, CAMHB) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation. Significance StatementStaphylococcus aureus infections impose an immense burden on the healthcare system. To establish a successful infection in a hostile host environment, S. aureus must coordinate its gene expression to respond to a wide array of challenges. This balancing act is largely orchestrated by the Transcriptional Regulatory Network (TRN). Here, we present a model of 29 independently modulated sets of genes that form the basis for a segment of the TRN in clinical USA300 strains of S. aureus. Using this model, we demonstrate the concerted role of various cellular systems (e.g. metabolism, virulence and stress response) underlying key physiological responses, including response during blood infection.

microbiology↗

bioRxiv2018

Broad geographic sampling reveals predictable and pervasive seasonal adaptation in Drosophila

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel among Drosophila melanogaster populations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation in Drosophila.

evolutionary biology↗