Laura J. Knoll

Intestinal protists represent an underappreciated yet functionally significant component of the human gut microbiome. Historically dismissed as parasites or transient contaminants, many of these microbial eukaryotes, particularly Entamoeba spp., are now recognized as integral to gut ecosystem function and host immune homeostasis. This review examines the complex roles of Entamoeba spp. in the mammalian gut, positioning them as dynamic microbiome “landscapers” that influence host-pathogen interactions, immune tone, and microbial diversity. We explore the evolutionary adaptation of Entamoeba to the gut’s anaerobic and immunologically active environment, highlighting both pathogenic ( E. histolytica ) and non-pathogenic species ( E. dispar , E. coli ) and their distinct immunomodulatory strategies. Special attention is given to the host immune responses shaped by E. histolytica , including inflammasome activation, macrophage polarization, and suppression of protective type-2 responses. The review also details Entamoeba ’s interactions with the gut microbiota, emphasizing their capacity for selective bacterial predation, disruption or enhancement of microbial community structure, and synergistic or antagonistic relationships with commensals and pathogens alike. Methodological challenges in protist detection, genome annotation, and cultivation are discussed, alongside promising advances in sequencing, host DNA depletion, and animal modeling. Taken together, current evidence reframes Entamoeba spp. not as mere pathogens but as key ecological players whose presence can signal resilience or susceptibility within the gut ecosystem. Understanding the context-dependent functions of intestinal protists may offer new insights into microbial therapeutics, immune modulation, and disease prevention strategies.

Intracellular bacteria and protists rely on the host cell to supply many metabolites, but the mechanisms through which pathogens manipulate host metabolism to their benefit are not understood. Here, we demonstrate that when the obligate intracellular parasite Toxoplasma gondii secretes its rhoptry organelle contents into the host cytoplasm before invasion—a process called “kiss and spit”—host cell metabolite abundance is altered in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and amino acid synthesis. U-13C6-labeling metabolomics confirmed that kiss and spit increased the flow of carbon through the pentose phosphate pathway and nucleotide synthesis. An increase in 2,3-bisphosphoglycerate abundance led us to investigate the activation of host cytosolic nucleosidase II (cN-II) to provide purines for the parasite. We found that T. gondii manipulates the host cN-II enzyme to dephosphorylate GMP and IMP that it needs for replication. Furthermore, we found that the approved anti-cancer drug fludarabine, which inhibits cN-II, also inhibits Toxoplasma replication. These results reveal Toxoplasma host cell manipulation and highlight potential therapies for toxoplasmosis. IMPORTANCE: A fundamental challenge in parasitology is understanding how intracellular parasites rapidly reprogram host metabolism to support replication. This study reveals that Toxoplasma gondii initiates profound metabolic reprogramming through a “kiss-and-spit” mechanism, secreting effector molecules without invasion. We demonstrate that T. gondii specifically hijacks host cytosolic 5′-nucleotidase II (cN-II) by elevating 2,3-bisphosphoglycerate levels, which allosterically activates this enzyme to generate purines essential for parasite survival. Genetic deletion of host cN-II significantly impairs parasite replication, establishing cN-II as a critical host dependency factor. These findings have important implications for antiparasitic drug development while advancing our understanding of purine metabolism in apicomplexan parasites. More broadly, elucidating the molecular mechanism linking parasite effector secretion to specific host enzyme activation provides a framework for understanding metabolic manipulation across other intracellular pathogens.

Intestinal protists are emerging as key modulators of host immunity and microbial ecology, yet their impact on mammalian hosts remains poorly defined. Here, we investigated the role of two distinct protists, the amoeba Entamoeba muris , and the parabasalid, Tritrichomonas , to determine how they shape gut immunity in vivo individually and together. Unlike the well-characterized inducer of type 2 immunity, Tritrichomonas , which activates the tuft cell–IL-25–ILC2 circuit in the small intestine, E. muris failed to elicit robust immune responses in the intestine or colon. However, the introduction of E. muris into mice naturally colonized by Tritrichomonas spp., or co-infection with E. muris and Tritrichomonas spp. reduced Tritrichomonas -induced type-2 response in the small intestine and Tritrichomonas- dependent immune activation in the colon. Our data suggest that E. muris may limit the abundance of Tritrichomonas spp., with reduced protist loads in the cecum specifically correlating with diminished tuft cell activation. We also identified sex-specific differences in the intestinal response to Tritrichomonas spp., which have not previously been reported. Taken together, these findings reveal that the presence of E. muris reduces Tritrichomonas -dependent activation of type 2 immunity in the small intestine, even when both protists can be detected in the cecum, without triggering overt inflammation. This work provides a framework for understanding how protists interact within the gut ecosystem and shape mucosal immunity in the absence of pathogenicity. IMPORTANCE: Single-cell parasites called protists are common in mammalian intestinal tracts, yet their modulation of the host immune response and interactions with each other remain poorly defined. Here, we investigated the role of two protists, Entamoeba and Tritrichomonas , to determine how they shape gut immunity individually and together. Unlike the well-characterized inducer of type 2 immunity, Tritrichomonas , which activates the tuft cell circuit, Entamoeba failed to elicit a robust immune response. The introduction of Entamoeba into mice naturally colonized by Tritrichomonas , or co-infection with Entamoeba and Tritrichomonas, reduced the Tritrichomonas -induced immune response. Our data suggest that Entamoeba limits the abundance of Tritrichomonas , correlating with diminished tuft cell activation. We also identified sex-specific differences in the intestinal response to Tritrichomonas . These findings show that Entamoeba reduces Tritrichomonas -dependent activation of type 2 immunity without triggering much inflammation. It helps our understanding of how protists interact within the gut and shape immunity without disease.

Primary fibroblasts are essential cell culture models for studying cellular homeostasis, disease mechanisms, and host–pathogen interactions. Primary cells offer physiological relevance that immortalized cell lines cannot recapitulate. However, existing protocols for isolating and culturing primary fibroblasts lack standardization for long-term applications. Contamination by non-fibroblast cell types, variable media formulations, and poorly monitored passage numbers remain critical challenges for reproducibility. Here, we present a streamlined, cost-optimized protocol for isolating and culturing mouse ear fibroblasts (MEarFs) suitable for extended passages. We demonstrate that enriched media (Advanced DMEM/F-12+15% FBS) consistently promoted adipocyte-like cell contamination in long-term MEarF cultures. In contrast, minimal media (DMEM+10% FBS) proved sufficient for both isolation and long-term cultures, with fibroblast gene expression confirmed through passage 9. Cells displayed characteristic fibroblast morphology, expressed the mesenchymal marker vimentin, and maintained fibroblast-associated transcripts with minimal epithelial and adipocyte marker expression throughout the culture. Bulk RNA sequencing revealed that 17% of mapped genes exhibited differential abundance between passage 3 and passage 9, with enrichment in functional categories related to cell cycle regulation and paracrine signaling. This protocol provides an accessible primary fibroblast cell culture model with broad research applications, emphasizing reproducibility and resource-conscious practices.

Toxoplasma gondii is a widespread parasite that impacts both human and animal health. Increasing use of organoid model systems has made previously challenging aspects of the T. gondii lifecycle more accessible. The media for these organoid systems are highly complex with many growth factors and pathway inhibitors that promote stem cell retention or differentiation of the cells. We noticed changes in T. gondii growth and development in our intestinal organoid system and wanted to determine if this was driven by cell type or media components. We found that low concentrations of SB202190 (a p38 MAPK inhibitor) and A83-01 (an ALK 4/5/7 receptor inhibitor) are each sufficient to alter T. gondii growth even in fibroblast cells. Further investigation with our qPCR panel of T. gondii stage markers revealed that these compounds promote bradyzoite cyst development and prime parasites for pre-sexual and sexual stage gene expression. As these complex organoid systems become more common in microbiology research, this study highlights the role of organoid media components in controlling pathogen growth and development. IMPORTANCE: The use of complex cell culture model systems is becoming more common across many fields. Maintenance of these systems often includes growth factors and inhibitors not found in standard media. These added components have the potential to impact pathogen growth and development and can influence how experimental results may be interpreted. This study improves our understanding of the Toxoplasma gondii lifecycle in one of those systems. It also serves as a template for other pathogen researchers to consider influences within their own systems.

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 world’s 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. SIGNIFICANCE: A third of the world’s 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.

Toxoplasma gondii is an obligate intracellular parasite capable of subverting host defenses to establish infection. Necroptosis, a lytic pro-inflammatory form of programed cell death, has emerged as a host defense mechanism against intracellular pathogens. However, its relevance in controlling T. gondii replication remains unclear. Here, we investigated the role of necroptosis in limiting T. gondii replication using bone marrow-derived macrophages (BMDMs) deficient in key necroptotic mediators, RIPK3 and MLKL. We demonstrate that under naïve conditions, T. gondii replication proceeds unimpeded in RIPK3 ^−/− and MLKL ^−/− BMDMs. However, co-treatment with TNF-α and the pan-caspase inhibitor Z-VAD-FMK, conditions that promote necroptosis, significantly reduced parasite replication in wild-type BMDMs but not in those lacking RIPK3 or MLKL. This suppression was dependent on RIPK1 activity, as pharmacological inhibition with Necrostatin-1 abrogated the effect. We further confirmed that TNF-α and Z-VAD-FMK treatment induced necroptotic cell death characterized by loss of plasma membrane integrity, both of which were absent in RIPK3 ^−/− and MLKL ^−/− cells. These findings establish that the activation of necroptosis can effectively limit T. gondii replication in BMDMs and underscore the importance of RIPK1-RIPK3-MLKL signaling in mounting a cell-intrinsic immune defense. Our study provides new insight into the functional capacity of necroptosis in restricting intracellular parasites and highlights its potential as a therapeutic target in toxoplasmosis.

Intestinal organoids are important cell culture models that complement live animal studies of many intestinal pathogens. Adult feline small intestinal organoids are needed for infectious disease research but are difficult to work with due to slow growth and premature 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 9 months in culture until cryopreservation. Fibroblast supplementation increased the maximum size of cat and mouse intestinal organoids as well. The increased longevity and size of these organoids are a significant improvement on current methods. These organoids also supported pre-sexual development of the medically important parasite Toxoplasma gondii , as evidenced by expression of the merozoite-specific marker GRA11b. This GRA11b positivity was higher in mature cat organoid-derived monolayers grown for 21 days prior to infection, compared with monolayers grown for 10 days. These methods have high potential to reduce the number of cats used for infectious disease research and may be applicable for intestinal cells from other animals that are difficult to culture. IMPORTANCE: Many microbial pathogens are acquired orally through contaminated food or water. Being able to model these infections in cell culture has been greatly enhanced by the development of intestinal organoid technology. One of the species that hosts several infections is cats, but cat intestinal organoids have been notoriously difficult to grow. Here, we describe a co-culture method with fibroblast cells that dramatically improves the longevity of adult cat intestinal organoids. These cat organoid cells can support the pre-sexual development stages of the intestinal pathogen Toxoplasma gondii , a parasite whose sexual cycle is restricted to cats and is the reason that pregnant women are told not to change the litter box. These culture conditions will be a resource to study other cat intestinal pathogens and intestinal organoids from other animals that are difficult to culture.

During an immune response, macrophages specifically reprogramme their metabolism to support functional changes. Here, we revealed that nucleotide metabolism is one of the most significantly reprogrammed pathways upon classical activation. Specifically, de novo synthesis of pyrimidines is maintained up to uridine monophosphate, but blocked at cytidine triphosphate and deoxythymidine monophosphate synthesis; de novo synthesis of purines is shut off at the last step (catalysed by AICAR transformylase/IMP cyclohydrolase, ATIC), and cells switch to increased purine salvage. Nucleotide degradation to nitrogenous bases is upregulated but complete oxidation of purine bases (catalysed by xanthine oxidoreductase, XOR) is inhibited, diverting flux into salvage. Mechanistically, nitric oxide was identified as a major regulator of nucleotide metabolism, simultaneously driving multiple key changes, including the transcriptional downregulation of Tyms and profound inhibition of ATIC and XOR. Inhibiting purine salvage using Hgprt knockout or inhibition alters the expression of many stimulation-induced genes, suppresses macrophage migration and phagocytosis, and increases the proliferation of the intracellular parasite Toxoplasma gondii. Together, these results thoroughly uncover the dynamic reprogramming of macrophage nucleotide metabolism upon classical activation and elucidate the regulatory mechanisms and functional significance of such reprogramming.

Toxoplasma gondii is an intracellular protozoan found worldwide that is capable of infecting nearly all warm-blooded animals, including humans. Its parasitic success lies in its capacity to create chronic infections while avoiding immune detection, altering host immune responses, and disrupting programmed cell death pathways. This review examines the complex relationship between T. gondii and host immunity, focusing on how the parasite influences innate and adaptive immune responses to survive in immune-privileged tissues. We present recent findings on the immune modulation specific to various parasite strains, the immunopathology caused by imbalanced inflammation, and how the parasite undermines host cell death mechanisms such as apoptosis, necroptosis, and pyroptosis. These immune evasion tactics enable prolonged intracellular survival and pose significant challenges for treatment and vaccine development. We also review advancements in therapeutic strategies, including host-directed approaches, nanoparticle drug delivery, and CRISPR-based technologies, along with progress in vaccine development from subunit and DNA vaccines to live-attenuated candidates. This review emphasizes the importance of T. gondii as a model for chronic infections and points out potential avenues for developing innovative therapies and vaccines aimed at toxoplasmosis and similar intracellular pathogens.