Biology subjects

White, B. E.

Publications and source records attributed to White, B. E..

4 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

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

Identification of regulatory elements in primary sensory neurons involved in neuropathic pain

Chronic pain is a significant public health issue that is often refractory to existing therapies. Here we use a multiomic approach to identify cis-regulatory elements that show differential chromatin accessibility, and reveal transcription factor (TF) binding motifs with functional regulation in the dorsal root ganglion (DRG), which contain cell bodies of primary sensory neurons, after nerve injury. We integrated RNA-seq to understand how differential chromatin accessibility after nerve injury may influence gene expression. Using TF protein arrays and chromatin immunoprecipitation-qPCR, we confirmed C/EBP{gamma} binding to a differentially accessible sequence and used RNA-seq to identify processes in which C/EBP{gamma} plays an important role. Our findings offer insights into TF motifs that are associated with chronic pain. These data show how interactions between chromatin landscapes and TF expression patterns may work together to determine gene expression programs in DRG neurons after nerve injury.

neuroscience↗