Biology subjects

Knoll, L.

Publications and source records attributed to Knoll, L..

4 recordsLinked to original sources

bioRxiv2026

Proteotyping reactivant toxoplasmic encephalitis reveals virulence-associated dense granule protein GRA5 polymorphisms

A fatal case of toxoplasmic encephalitis, and others like it, has caused microbiologists and clinicians to question whether different strains of T. gondii have more pathogenic potential than others. This raises significant concern, as T. gondii is a widely spread parasitic organism that is presumed to lie dormant in a third of the worlds general population. In this study, we expand on a previously published proteomicanalysis reactivated toxoplasmic encephalitis and have been able to identify T. gondii-specific peptides in the cerebrospinal fluid (CSF) of this patient and two additional cases of toxoplasmic reactivation. Multilocus PCR-restriction fragment-length polymorphism (PCR-RFLP) was used to genetically identify the T. gondii strain that resulted in this fatal case, which belonged to ToxoDB PCR-RFLP genotype #7. Using other T. gondii strains of the same genotype, we performed bioassays to compare the pathogenicity of this genotype with that of a clinically relevant strain, ME49. Both of the tested genotype #7 strains appear to have a greater pathogenic potential, although through likely different mechanisms. Of the most abundant T. gondii-specific CSF peptides across multiple patients, we identified a polymorphic region of the dense granule protein GRA5 that appears to have strain specificity. This approach could represent a "proteotype" that allows for T. gondii strain risk stratification within clinical samples. Ultimately spinal fluid could be a valuable tool in distinguishing between T. gondii exposed individuals with no cyst forms in the brain versus those exposed individuals that harbor "clinically silent" but viable brain cysts. SIGNIFICANCEA third of the worlds population is exposed to the parasite Toxoplasma gondii, residing in a dormant, encysted stage within neurons. T. gondii is a diverse microorganism with some strains having greater reactivation potential. There is no means of identifying which individuals are at risk of reactivation. Proteomic analysis of cerebrospinal fluid from patients with reactivated toxoplasmosis demonstrated a consistent pattern of T. gondii peptides, including GRA5, a secreted virulence factor. Postmortem analysis identified a ToxoDB PCR-RFLP genotype #7 strain associated with a fatality. Cerebrospinal fluid could provide clues to persistent brain infection and may identify strains able to reactivate.

microbiology↗

bioRxiv2024

Fibroblasts enhance the growth and survival of adult feline small intestinal organoids

AbstractIntestinal organoids are important cell culture models that complement live animal studies of intestinal biology. Adult feline small intestinal organoids are needed for infectious disease research but are difficult to work with due to slow growth and rapid senescence. We introduce a method of co-culturing adult feline small intestinal organoids with growth inhibited human foreskin fibroblast feeder cells to enhance organoid proliferation and survival. With feeder cells, feline jejunal and ileal organoids survived at least nine months in culture until cryopreservation. Fibroblast supplementation also increased the maximum size of cat and mouse intestinal organoids, making them easier for microinjection. The increased proliferation, longevity, and size make fibroblast-supplementation of feline small intestinal organoids a significant improvement on current methods. These methods have high potential to reduce the number of cats used for research and may be applicable for intestinal cells from other animals that are difficult to culture.

cell biology↗

bioRxiv2023

Metabolic changes to host cells with Toxoplasma gondii infection

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90%. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. Here, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring metabolic response over time using non-invasive autofluorescence lifetime imaging of single cells, seahorse metabolic flux analysis, reactive oxygen species (ROS) production, and metabolomics. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H with infection. These findings are consistent with changes in mitochondrial and glycolytic function, decrease of intracellular glucose, fluctuations in lactate and ROS production in infected cells over time. We also examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which similarly showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours. Glucose metabolic flux analysis indicated that these changes are driven by NADH and NADP+ in T. gondii infection. In sum, metabolic changes in host cells with T. gondii infection were similar during full infection, and kiss and spit. Autofluorescence lifetime imaging can non-invasively monitor metabolic changes in host cells over a microbial infection time-course.

microbiology↗

bioRxiv2022

Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system

Protozoan parasites that infect humans are widespread, and lead to varied clinical manifestations, including life-threatening illnesses in immunocompromised individuals. Animal models have provided insight into innate immunity against parasitic infections, however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for novel in vitro systems that can elucidate mechanisms of immune control and parasite dissemination. We have developed a human microphysiological system of intestinal tissue to evaluate parasite-immune-specific interactions during infection, which integrates primary intestinal epithelial cells and immune cells to investigate the role of innate immune cells during epithelial infection by the protozoan parasite, Toxoplasma gondii, which affects billions of people worldwide. Our data indicate that epithelial-infection by parasites stimulates a broad range of effector functions in neutrophils and NK cell-mediated cytokine production that play immunomodulatory roles, demonstrating the potential of our system for advancing the study of human-parasite interactions. TeaserNovel engineered model of human intestinal tissue for study of dissemination and immune control of parasitic infections.

bioengineering↗