Biology subjects

Tarleton, R. L.

Publications and source records attributed to Tarleton, R. L..

8 recordsLinked to original sources

bioRxiv2024

Glycosomal phosphoenolpyruvate carboxykinase CRISPR/Cas9-deletion and its role in Trypanosoma cruzi metacyclogenesis and infectivity in mammalian host

Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes - unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD+ regeneration within the organelle. Using CRISPR/Cas9 editing, PEPCK gene was disrupted in T. cruzi, producing single- allele knockout epimastigotes (TcPEPCK-sKO) with reduced enzyme activity. This disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed in in vivo infection, where TcPEPCK-sKO infection in IFN{gamma}-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the PEPCK critical role in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that PEPCK is crucial for T. cruzi energy metabolism, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.

microbiology↗

bioRxiv2024

Serial 'deep-sampling PCR of fragmented DNA reveals the wide range of Trypanosoma cruzi burden among chronically infected hosts and allows accurate monitoring of parasite load following treatment

Infection with the protozoan parasite Trypanosoma cruzi is generally well-controlled by host immune responses, but appears to be rarely eliminated. The resulting persistent, low-level infection results in cumulative tissue damage with the greatest impact generally in the heart in the form of chagasic cardiomyopathy. The relative success in immune control of T. cruzi infection usually averts acute phase death but has the negative consequence that the low-level presence of T. cruzi in hosts is challenging to detect unequivocally. Thus, it is difficult to identify those who are actively infected and, as well, problematic to gauge the impact of treatment, particularly in the evaluation of the relative efficacy of new drugs. In this study we employ DNA fragmentation and high numbers of replicate PCR reaction ( deep-sampling) to extend the quantitative range of detecting T. cruzi in blood by at least 3 orders of magnitude relative to current protocols. When combined with sampling blood at multiple time points, deep sampling of fragmented DNA allowed for detection of T. cruzi in all infected hosts in multiple host species. In addition, we provide evidence for a number of characteristics not previously rigorously quantified in the population of hosts with naturally acquired T. cruzi infection, including, a > 6-log variation between chronically infected individuals in the stable parasite levels, a continuing decline in parasite load during the second and third years of infection in some hosts, and the potential for parasite load to change dramatically when health conditions change. Although requiring strict adherence to contamination-prevention protocols and significant resources, deep-sampling PCR provides an important new tool for assessing new therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease. Author SummaryInfection by the protozoan Trypanosoma cruzi normally results in a life-long, but low-level parasitization of muscle tissues, often leading to chagasic heart disease. A major challenge in the Chagas disease field has been the difficulty in detecting and quantifying parasite load in infected hosts. In this study we show that collection of serial blood samples and performance of sometimes high numbers of replicate PCR reactions on fragmented blood DNA, allows detection and quantification of relative parasite load in non-human primates, dogs, and humans with naturally acquired T. cruzi infection. This deep-sampling approach reveals a mostly stable, 100,000-fold or greater difference in parasite load among chronically infected hosts and can detect alterations in parasite levels due to changes in health status or following therapeutic treatment in individual hosts, thus providing a powerful tool for assessing treatment outcomes in T. cruzi infection, including for evaluation of new therapeutics. Additionally, the ability to accurately and sensitively monitor parasite load in hosts provides the means to address highly contentious issues in the Chagas field, including the relative role of parasites and hosts in establishing the persistent parasite burden and the relationship between parasite burden and the presence and severity of clinical disease.

microbiology↗

bioRxiv2023

Validation of a multiplex microsphere immunoassay for detection of Trypanosoma cruzi antibodies in dogs

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern US where triatomine insect vectors occur, and there are limited veterinary diagnostic options; currently, only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. This study evaluated a multiplex microsphere immunoassay (MIA) for detecting antibodies against T. cruzi in dogs and compared with existing serological methods to establish cut-off values and relative sensitivity/specificity. Dog sera (n=135) which were previously characterized using the IFA and off-label use of two commercial rapid assays were tested on the multiplex MIA against 12 different antigens: nine T. cruzi antigens, a negative control recombinant protein (green fluorescent protein), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvovirus vaccination). For each sample, the ratio of median fluorescence intensity (MFI) for each T. cruzi antigen to that of GFP was calculated. Samples with an antigen/GFP ratio greater than 4 standard deviations above the mean of 25 known negative sera were considered positive on that antigen. Samples testing positive on 2 or more antigens were considered positive for T. cruzi antibodies. Compared to the IFA, the multiplex MIA demonstrated a relative sensitivity of 100% and specificity of 96.97%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, the multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

microbiology↗

bioRxiv2023

Frequency variation and dose modification of benznidazole administration for the treatment of Trypanosoma cruzi infection in mice, dogs and non-human primates

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and non-human primates (NHP). Collectively these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are [~]2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

microbiology↗

bioRxiv2022

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections. Author SummaryTrypanosoma cruzi, the parasite that causes Chagas disease, circulates extensively in the southern U.S. and working dog populations in south-central Texas are at very high risk of infection, and morbidity and early mortality due to this infection. In this study, we used low level administration of an FDA-approved drug during the transmission season to attempt to prevent new infections in these dogs. Although that effort failed, the study revealed new information about the transmission dynamics and generation of antibody responses and immune control of infection in this high-transmission setting.

microbiology↗

bioRxiv2021

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern U.S. provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTUs) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established. Author SummaryWe evaluated naturally occurring infections of Trypanosoma cruzi, the causative agent of human Chagas disease, in an indoor/outdoor primate colony at a breeding facility in Texas, USA. Using serial quantitative PCR and hemoculture, we confirmed infection in 92% of the 64 seropositive animals, but neither of these two methods confirmed more than 80% of the cases. Parasites by hemoculture fell into two genetic groups (discrete typing units I and IV), and displayed large variation in growth characteristics, elicited cellular and humoral immune responses as well as virulence and drug susceptibility when tested in mice. EKG abnormalities were found in 13 out of 51 qPCR-positive macaques. Our results demonstrate the complexity of these infection parameters in this colony in spite of the uniform and geographically constrained housing conditions of the macaques.

microbiology↗

bioRxiv2020

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 gene family members 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. These processes also impact genes not required for or involved in immune evasion, creating unique challenges with respect to preserving core genome function while maximizing genetic diversity.

microbiology↗

bioRxiv2017

Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy pathway in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

microbiology↗