Biology subjects

Dews, E. A.

Publications and source records attributed to Dews, E. A..

3 recordsLinked to original sources

bioRxiv2025

A Humanized IFN-γ Mouse Model Reveals Skin Eschar Formation, Enhanced Susceptibility and Scrub Typhus Pathogenesis

Scrub typhus, caused by Orientia tsutsugamushi (Ot) bacteria, is a serious acute febrile illness associated with significant mortality. An estimated one million cases occur annually, with more than one billion people at risk. No effective vaccine is currently available, largely due to the complex Ot strain diversity and an incomplete understanding of protective immune mechanisms. To overcome these challenges, there is a critical need for a suitable animal model that mimics human disease through the natural route of infection via mites. Here, we report for the first time that a genetically engineered humanized mouse strain (with triple knockout/knock-in of IFN-{gamma} and its receptors), exhibits increased susceptibility to intradermal Ot infection compared to wild-type (WT) mice. This is evidenced by greater body weight loss, elevated bacterial burden, and reduced expression of interferon-stimulated genes (ISGs). Humanized mice exhibit pronounced biochemical abnormalities and tissue pathology accompanied by dysregulated T cell and neutrophil responses following infection. Notably, these immunocompetent mice develop skin eschar-like lesions resembling those observed in human patients. Overall, our study introduces a promising humanized mouse model to dissect the immunopathogenesis of scrub typhus and evaluate future vaccine candidates. Author SummaryScrub typhus is a serious disease caused by the obligately intracellular bacterium Ot that spreads to humans through the bite of larval mites called chiggers. It affects over a million people each year, primarily in Asia, and can lead to life-threatening complications. Unfortunately, we still lack a clear understanding of how this infection causes disease, partly because there is not a good laboratory model that closely reflects how humans respond to infection. Our recent reports have suggested an important role of IFN-{gamma} in host protection against Ot infection. In this study, we used a new genetically modified mouse strain that carries human IFN-{gamma} signaling in place of its mouse counterpart. We found that these humanized mice are more vulnerable to infection, develop skin eschar lesions like those in patients, and show signs of systemic inflammation and organ damage. Their immune response also resembled what has been observed in human patients. This new mouse model can help scientists better understand the mechanisms as to how this bacterial species causes severe disease outcomes in patients. Once those mechanisms are understood, this mouse model will serve a further purpose as a tool for testing new vaccines and treatments to aid humans at-risk for scrub typhus.

immunology↗

bioRxiv2025

A Propidium Iodide-based In Vitro Screen of the "Bug Box" Against Babesia duncani Reveals Potent Inhibitors

Incidence and endemic range of human babesiosis are expanding. Standard therapy for human babesiosis consists of antimicrobials developed for other indications. While these treatments are adequate in immunocompetent hosts, infections in the immunocompromised can be severe, relapsing, and drug-resistant despite use of multi-drug regimens. Existing drugs are ineffective because they cannot safely achieve and maintain adequate serum concentrations to inhibit Babesia. Discovery of improved agents against Babesia spp. is of growing importance and efficient techniques for high throughput compound screening can assist in this effort. We developed a high throughput in vitro drug screening assay for Babesia duncani that is conducted in 384 well plates and makes use of the fluorescent DNA stain propidium iodide (PI) with relative fluorescence measured by a microplate reader. A Z factor of >0.81 was calculated which suggests an excellent ability to detect inhibitory compounds. A screen of the 41-compound library Structural Genomics Consortium Bug Box was conducted yielding five hits: trimethoprim, atovaquone, SDDC M7, diphenyleneiodonium chloride, and panobinostat. Panobinostat, a histone deacetylase complex (HDAC) inhibitor, was selected for further evaluation given that its target had not been previously explored in B. duncani. Follow up dose-response testing of structurally related compounds revealed multiple potential leads including nanatinostat and quisinostat, both of which were potent at the nanomolar level and showed favorable selectivity index in cytotoxicity studies. High throughput screening using PI and 384 well plates is an advance in drug discovery for babesiosis and HDAC inhibitors show promise as lead compounds worthy of further investigation.

microbiology↗

bioRxiv2023

Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D

Many advances have been made recently in our understanding of Cryptosporidiums asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation of Cryptosporidium. To facilitate exploration of sexual reproduction in C. parvum we developed an organoid-based culture system that supports Cryptosporidiums full life cycle and a novel fertilization reporter. Organoid derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM derived oocysts were infectious in vivo. Fertilization was confirmed by successfully mating two strains of C. parvum and with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry positive parasites in the first generation and offspring that express mNeonGreen. In vivo validation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate the Cryptosporidium lifecycle post sexual differentiation in a physiologically relevant in vitro system. ImportanceOrganoid derived monolayers provide an opportunity to elucidate previously inaccessible aspects of Cryptosporidiums biology. This system overcomes the disadvantages of previous organoid-based methods for Cryptosporidium culture. It is faster and simpler than previously described systems, uses defined media to increase reproducibility and consistency, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate studies of Cryptosporidium cell biology. The ODM system could facilitate the study of host-pathogen interactions, Cryptosporidium-host specificity, or innate or cellular immune responses to Cryptosporidium infection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere with Cryptosporidiums sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in Cryptosporidium, leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.

microbiology↗