Biology subjects

Ward, A. I.

Publications and source records attributed to Ward, A. I..

7 recordsLinked to original sources

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

Abundance of Nef and p-Tau217 in brains of individuals diagnosed with HIV-associated neurocognitive disorders correlate with disease severance

HIV-associated neurological disorders (HAND) is a term used to describe a variety of neurological impairments observed in HIV-infected individuals. The pathogenic mechanisms of HAND and its connection to HIV infection remain unknown. The brain samples from HIV-infected individuals, both with and without HAND, were characterized by increased abundance of p-Tau217 peptide, which correlated with the abundance of flotillin 1, a marker of lipid rafts. HIV-1 Nef was detected in some, but not all, samples from HAND-affected individuals. Samples positive for Nef had lower abundance of cholesterol transporter ABCA1, higher abundance of flotillin 1 and p-Tau217, and were obtained from individuals with higher severity of HAND relative to Nef-negative samples. These results highlight the contribution of Nef and Nef-dependent effects on cholesterol metabolism and lipid rafts to the pathogenesis of HAND and support a connection between pathogenesis of HAND and Alzheimers disease.

neuroscience↗

bioRxiv2021

Incomplete recruitment of protective T cells facilitates Trypanosoma cruzi persistence in the mouse colon

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain, which allows experimental infections to be imaged at single-cell resolution, we have characterised the hyper-local immunological microenvironment of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.

immunology↗

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

Developing methods for measuring national distributions and densities of wild mammals using camera traps: A Kosovo study

Understanding the distributions and density of wild mammals is integral to the implementation of wildlife management strategies, particularly for controlling diseases and conservation management. Recent advances in camera trap technology together with the development of the Random Encounter Model have provided a non-invasive method for estimating mammal densities. In addition, the development of citizen science initiatives have advanced ecological data collection. This study describes a national camera trap survey delivered by local stakeholders in eleven forest sites in Kosovo from 2014 to 2015 to measure the distributions and abundance of medium to large wild mammals as part of the Control and/or eradication of animal diseases project. The Random Encounter Model was used to calculate density data for each species, which appear realistic when compared to densities found in other European countries. The study particularly focussed on the red fox (Vulpes vulpes) and the grey wolf (Canis lupus) as potential vectors of rabies and wild boar (Sus scrofa) as a vector of classical swine fever. These species were found to be three of the most widely distributed species in Kosovo and were present at the majority of sites at high densities. The camera survey also provided information on species of conservation concern such as the Eurasian brown bear (Ursus arctos) and provided the first physical evidence of a live Eurasian golden jackal (Canis aureus) in Kosovo. Although sources of bias were identified, these estimates are likely to be more accurate than those devised from methods such as hunting bags and the findings of this study suggest that, with a moderate amount of development, camera trapping implemented by local stakeholders can be used as an effective and practicable method to estimate national distributions and population sizes of medium to large sized wild mammals.

ecology↗

bioRxiv2020

In vivo analysis of Trypanosoma cruzi persistence foci at single cell resolution

Infections with Trypanosoma cruzi are usually life-long despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understand how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which has allowed us to visualise infected host cells in murine tissue, with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we show that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells containing >200 parasites, that we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites found in large mega-nests within the muscle fibres. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission, and have implications for drug development. IMPORTANCETrypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence in experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon, and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a life-long infection, despite a vigorous immune response. It also informs drug-development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

microbiology↗