Biology subjects

Olmo, F.

Publications and source records attributed to Olmo, F..

12 recordsLinked to original sources

bioRxiv2026

Shared and divergent features of the immune response to acute and chronic Trypanosoma cruzi infection in mice revealed by cardiac transcriptomics

Chagas disease is caused by infection with Trypanosoma cruzi and can result in chronic cardiomyopathy. However, the molecular programmes that define infected cardiac tissue, and how they change as the host-parasite relationship moves from acute pathogenic infection to long-term persistence, remain incompletely understood. Here, we used RNA sequencing to characterise host transcriptional responses in cardiac tissue during both acute and chronic infection in a mouse model. Cardiac parasite distribution was defined using bioluminescence imaging and histology, and RNA sequencing was performed on anatomically standardised cardiac tissue samples collected at both stages. Acute infection was associated with a broad interferon-driven immune activation, whereas during chronic infection this network contracted, with preservation of a tightly connected antigen presentation core centred on major histocompatibility complex genes and immunoproteasome components. Genes associated with extracellular matrix remodelling and fibrosis were not broadly activated. These findings indicate that reduced, but sustained immune activation, rather than widespread fibrotic transcriptional remodelling, represents a prominent feature of cardiac tissue in chronically infected mice. They identify a transition from an overtly inflammatory, pathogen-driven acute response to a more restricted chronic host-parasite equilibrium in which immune surveillance remains transcriptionally active despite very low and spatially intermittent cardiac infection. We identified a conserved 20-gene signature shared between acute and chronic stages. This overlap is consistent with continued or episodic parasite-derived stimulation and/or intrinsic host mechanisms that maintain cardiac immune surveillance. Thus, chronic T. cruzi infection can be viewed as a persistent host-parasite association in which biological coexistence does not imply benignity: immune containment and parasite persistence can occur alongside cumulative cardiac injury.

genomics↗

bioRxiv2026

Experimental Trypanosoma cruzi infection cured with a mechanistically distinct drug combination

Infections with the protozoan parasite Trypanosoma cruzi are widespread in the Americas and can lead to severe cardiac and/or gastrointestinal pathology. Current treatments are limited to monotherapies characterised by prolonged dosing regimens, disputed efficacy and toxic side-effects. Here, we demonstrate that short duration co-administration of well-tolerated sub-efficacious oral doses of the parasite-selective proteasome inhibitor GNF6702 and the pro-drug benznidazole produce parasitological cure in an experimental model of chronic Chagas disease.

microbiology↗

bioRxiv2025

Transcriptomic and proteomic analysis of quiescent epimastigotes as a resource for investigating Trypanosoma cruzi persistence.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi. Despite triggering a strong immune response, infections are typically life-long and can result in severe cardiac and/or digestive tract pathology. Current drugs have limited efficacy, and treatment failure is a common outcome. Eliminating a non-replicating T. cruzi sub-population that can persist after therapy has been a key challenge for the drug-development community. Here, we describe the transcriptome and proteome profiles of quiescent epimastigote forms of the parasite isolated from exponentially growing cultures on the basis of reduced turnover of transiently-induced red fluorescent protein. This quiescent sub-population was characterised by down-regulation of genes/proteins involved in translation, metabolism, mitochondrial function and DNA replication, and by up-regulation of proteins that promote exit from the cell-cycle in other organisms. These data represent a resource that can be exploited to dissect the mechanistic basis of quiescence and to refine the drug-development screening cascade.

microbiology↗

bioRxiv2025

Statins do not reduce the parasite burden during experimental Trypanosoma cruzi infection

Cardiomyopathy is the most common pathology associated with Trypanosoma cruzi infection. Reports that statins have both cardioprotective and trypanocidal activity have generated interest in their potential as a therapeutic treatment. Using a highly-sensitive bioluminescent mouse model, we show that 5 days treatment with statins has no significant impact on parasite load. The free systemic concentrations fail to reach the level required for potency. Hence, clinical trials to investigate trypanocidal activity of statins lack experimental justification.

microbiology↗

bioRxiv2024

Intermittent suppressive posaconazole therapy is ineffective at mitigating cardiac and digestive tract pathologies in an experimental model of chronic Chagas disease

Infections with Trypanosoma cruzi cause Chagas disease, a chronic condition that can give rise to debilitating cardiac and/or gastrointestinal damage. However, it is unclear why only [~]30% of individuals progress to symptomatic pathology, why this can take decades to become apparent, and why there is such a wide range of disease outcomes. Disease pathology is a long-term cumulative process resulting from collateral damage caused by inflammatory immune responses that continually eliminate transient parasite infections in the heart and/or gastrointestinal tract. The guiding principle behind anti-parasitic drug development is that sterile cure is required to prevent progression to symptomatic pathology. Evidence suggests that the cumulative damage required to reach the symptomatic threshold is determined by a number of factors, including host and parasite genetics, which govern the intensity and location(s) of infection. Therefore, an alternative therapeutic strategy could involve long-term intermittent treatment, which may not confer sterile cure, but is able to suppress the parasite burden to a level where the disease does not become symptomatic within the life-time of the infected individual. To test this hypothesis, we used an experimental murine model that displays both cardiac and digestive tract pathology. Mice were given intermittent treatment with posaconazole, under conditions that reduced the parasite burden by >99%, but did not confer sterile clearance. Our results show that this did not provide long-term protection against the key cardiac or gastrointestinal manifestations of Chagas disease, and that sterile cure should remain the single goal of the drug development community.

microbiology↗

bioRxiv2024

A panel of phenotypically and genotypically diverse bioluminescent: fluorescent Trypanosoma cruzi strains as a resource for Chagas disease research

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that displays considerable genetic diversity. Infections result in a range of pathological outcomes, and different strains can exhibit a wide spectrum of anti-parasitic drug tolerance. The genetic determinants of infectivity, virulence and therapeutic susceptibility remain largely unknown. As experimental tools to address these issues, we have generated a panel of bioluminescent:fluorescent parasite strains that cover the diversity of the T. cruzi species. These reporters allow spatio-temporal infection dynamics in murine models to be monitored in a non-invasive manner by in vivo imaging, provide a capability to detect rare infection foci at single-cell resolution, and represent a valuable resource for investigating virulence and host:parasite interactions at a mechanistic level. Importantly, these parasite reporter strains can also contribute to the Chagas disease drug screening cascade by ensuring that candidate compounds have pan-species in vivo activity prior to being advanced into clinical testing. The parasite strains described in this paper are available on request. AUTHOR SUMMARYChagas disease results from infection with the protozoan parasite Trypanosoma cruzi and is a major public health problem throughout Latin America. T. cruzi is a genetically diverse species and infection can result in a wide range of pathological outcomes, mainly associated with the heart and/or digestive tract. Research on Chagas disease, ranging from fundamental biology to drug development, has been greatly aided by the availability of genetically modified parasite reporter strains that express bioluminescent:fluorescent fusion proteins. In combination with mouse models and imaging technology, these strains allow infections to be monitored in real-time, with high sensitivity, and infection foci to be visualised at single-cell resolution. Here, we describe an extensive panel of bioluminescent and fluorescent strains that cover the diversity of the T. cruzi species. These reporter strains, that are available on request, should have wide utility in many areas of Chagas disease research. In particular, as part of the drug development screening programme, they can be used to ensure that candidate compounds have in vivo activity across the species prior to being advanced into clinical testing.

microbiology↗

bioRxiv2023

Trypanosoma cruzi persisters that survive benznidazole treatment in vitro and in vivo are in a transient non-proliferative state.

Benznidazole is the front-line drug used to treat infections with Trypanosoma cruzi, the causative agent of Chagas disease. However, for reasons that are unknown, treatment failures are common. To assess the nature of parasites that persist after treatment, we first exposed infected mammalian cell monolayers to a benznidazole regimen that reduces the intracellular amastigote population to <1% of the pre-treatment level. Of host cells that remained infected, the vast majority contained only one or two surviving intracellular amastigotes. Analysis, using incorporation of the thymidine analogue EdU, revealed these surviving parasites to be in a transient non-replicative state. Furthermore, treatment with benznidazole led to widespread damage to parasite DNA. When parasites that survived treatment in mice were examined using in vivo and ex vivo bioluminescence imaging, we found that recrudescence is not due to persistence of parasites in a specific organ or tissue that preferentially protects them from drug activity. Surviving parasites were widely distributed and located in host cells where the vast majority contained only one or two amastigotes. Therefore, infection relapse does not arise from a small number of intact large nests. Rather, persisters are either survivors of intracellular populations where co-located parasites have been killed, or amastigotes in single/low-level infected cells exist in a state where they are less susceptible to benznidazole. Assessment by EdU incorporation revealed that the small number of parasites which persist in mice after treatment are initially non-replicative. A possible explanation could be that triggering of the T. cruzi DNA damage response pathway by the activity of benznidazole metabolites results in exit from the cell cycle as parasites attempt DNA repair, and that metabolic changes associated with non-proliferation act to reduce drug susceptibility. Alternatively, a small percentage of the parasite population may pre-exist in this non-replicative state prior to treatment. Author SummaryTrypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. For reasons that are not established, the front-line drug benznidazole often fails to achieve sterile cure. Here, we used highly sensitive imaging technology to investigate the impact of benznidazole on T. cruzi infected mice. Following non-curative treatment, we found that persistence is not restricted to a specific organ or tissue that preferentially protects the parasite from drug activity. Rather, surviving parasites are widely distributed, although overall tissue levels are extremely low. These persisters are located in host cells that typically contain only one or two non-replicating intracellular amastigotes. However, these parasites re-initiate DNA replication within several days of treatment cessation and begin to proliferate. Therefore, being in a non-replicative state seems to confer protection against drug-mediated trypanocidal activity. Benznidazole treatment results in widespread damage to parasite DNA. One possibility therefore, is that this triggers the T. cruzi DNA damage response pathway, resulting in exit from the cell cycle as parasites attempt DNA repair. Alternatively, persisters may be derived from a small parasite sub-population that pre-exists in a non-replicative state prior to treatment.

microbiology↗

bioRxiv2021

Local association of Trypanosoma cruzi chronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale

Gastrointestinal (GI) disease affects a substantial subset of chronic Chagas disease (CD) patients, but the mechanism of pathogenesis is poorly understood. The lack of a robust, predictive animal model of chronic T. cruzi infection that exhibits functional digestive disease has held back research. To address this, we combined GI tracer assays and bioluminescence in vivo infection imaging systems for diverse parasite strains to discover models exhibiting chronic digestive transit dysfunction. We identified the colon as a specific site of both tissue parasite persistence and delayed transit. Digestive CD mice exhibited significant retention of faeces in both sated and fasted conditions. Histological and immunofluorescence analysis of the enteric nervous system (ENS) revealed a dramatic reduction in the number of neurons and a loss of immunoreactivity of the enteric neural network in the colon. This model therefore recapitulates key clinical manifestations of human digestive CD. We also exploited dual bioluminescent-fluorescent parasites to analyse rare chronic infection foci in the colon at the single cell level, revealing co-localisation with ENS lesions. This indicates that long-term T. cruzi-host interactions in the colon drive pathogenesis and thus chronic disease may be preventable using anti-parasitic chemotherapy.

microbiology↗

bioRxiv2020

Trypanosoma cruzi amastigotes have a reduced replication rate during chronic stage infections

Chronic Trypanosoma cruzi infections are typically life-long, with small numbers of parasites surviving in restricted tissue sites, which include the gastro-intestinal tract. There is considerable debate about the replicative status of these persistent parasites. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2deoxyuridine incorporation into DNA to provide "snapshots" of parasite replication. Highly sensitive imaging of infection foci at single cell resolution revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections are associated with a reduced rate of parasite replication. Despite this, very few host cells survive infection for >14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. Therefore, long-term persistence in the colon is more likely to be associated with reduced proliferation than with dormancy.

microbiology↗

bioRxiv2020

Non-invasive monitoring of drug action: a new live in vitro assay design for Chagas' disease drug discovery

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the point immediately before the fold change becomes negative, independent of the drug concentration and exposure time. This time-to-kill over drug concentration revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole. Author SummaryChagas disease, caused by Trypanosoma cruzi, is a chronic debilitating infection occurring mostly in Latin America. There is an urgent need for new, well tolerated drugs. However, the latest therapeutic candidates have yielded disappointing outcomes in clinical trials, despite promising preclinical results. This demands new and more predictive in vitro assays. To address this, we have developed an assay design that enables the growth of T. cruzi intracellular forms to be monitored in real time, under drug pressure, for 6 days post-infection. This allowed us to establish the tipping point of drug action, when the parasites stop multiplying and start to die. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole.

microbiology↗

bioRxiv2019

Intracellular DNA replication and differentiation of Trypanosoma cruzi is asynchronous within individual host cells in vivo at all stages of infection

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge. AUTHOR SUMMARYChagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains of T. cruzi carrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication in vivo during both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.

microbiology↗

bioRxiv2019

Immunity conferred by drug-cured experimental Trypanosoma cruzi infections is long-lasting and cross-strain protective

BackgroundThe long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi.\n\nMethodology/Principal FindingsMice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Mice infected for 14 days or more, prior to the initiation of treatment, were highly protected from challenge with both homologous and heterologous strains (>99% reduction in parasite burden). Sterile protection against homologous challenge was frequently observed. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-{gamma}+ T cell response.\n\nConclusions/SignificanceOur findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.\n\nAUTHOR SUMMARYChagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines, with many instances of sterile protection. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where sterile protection was not achieved. This suggested that to confer sterile protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination.

immunology↗