Biology subjects

Hehl, A. B.

Publications and source records attributed to Hehl, A. B..

6 recordsLinked to original sources

bioRxiv2025

Genome-wide Transcriptomic Analysis of Toxoplasma gondii Reveals Stage-specific Regulatory Programs and Metabolic Adaptations Driving Oocyst Sporulation

The sporulation of Toxoplasma gondii oocysts is a critical developmental transition transforming non-infective environmental stages into highly resilient and infective forms capable of zoonotic transmission. Despite significant advances, the molecular regulation underlying this complex process remains poorly understood. We generated comprehensive RNA-Seq datasets capturing gene expression dynamics at three timepoints during oocyst sporulation, to illuminate essential regulatory mechanisms and metabolic adaptations. Employing stringent variance filtering, unsupervised clustering, and differential expression analyses, we identified transcriptional patterns associated with key developmental transitions, notably the involvement of T. gondii Apetala2 (TgAP2) transcription factors and other DNA-binding proteins. We highlight significant shifts in metabolic pathways essential for oocyst environmental resilience and infectivity, including lipid metabolism, amino acid biosynthesis, and specialized secondary metabolism. Furthermore, we investigated expression profiles of genes associated with structural development of oocyst and sporocyst walls, environmental persistence, and host transmission. Importantly, combining these datasets with prior life cycle datasets covering intermediate and final host stages, we close an important knowledge gap by providing a genome-wide mRNA expression profile spanning the entire T. gondii life cycle. Integration with previous foundational transcriptomic and proteomic work provides refined resolution and identifies novel candidate genes and pathways. Collectively, these findings substantially advance our understanding of T. gondii sporulation in the wider context of the development in the cat intestine, highlighting critical molecular events underpinning parasite transmission and environmental adaptation.

cell biology↗

bioRxiv2023

In vitro production of cat-restricted Toxoplasma pre-sexual stages by epigenetic reprogramming

Summary paragraphSexual reproduction of Toxoplasma gondii, which is restricted to the small intestine of felids, is sparsely documented, due to ethical concerns surrounding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1. In this study, we found that transcription factors AP2XII-1 and AP2XI-2, expressed in tachyzoite stage that causes acute toxoplasmosis, can silence genes necessary for merozoites, a developmental stage critical for sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a drastic change in the transcriptional program, promoting a complete transition from tachyzoites to merozoites. Pre-gametes produced in vitro under these conditions are characterized by specific protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit the epigenitors MORC and HDAC31, which in turn restrict the accessibility of chromatin to the transcriptional machinery. Thus, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. This effective in vitro culture of merozoites paves the way to explore Toxoplasma sexual reproduction without the need to infect kittens and has potential for the development of therapeutics to block parasite transmission.

microbiology↗

bioRxiv2022

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Fornicata

Giardia lamblia, a parasitic protist of the metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically-related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC) across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Taken together, this provides the first comprehensive nanometric view of Giardias endocytic system architecture and sheds light on the evolution of GLACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

cell biology↗

bioRxiv2021

Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata

Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how the evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardias pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the Endoplasmic Reticulum, and for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia, and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, and associating to multiple cellular locations and presenting changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

evolutionary biology↗

bioRxiv2019

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

Phosphorylated derivatives of phosphatidylinositol (PIPs), are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown.\n\nHere we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico annotation techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

microbiology↗

bioRxiv2018

Mitochondrial targeting of glycolysis in a major lineage of eukaryotes

Glycolysis is a major cytosolic catabolic pathway that provides ATP for many organisms1. Mitochondria play an even more important role in the provision of additional cellular ATP for eukaryotes2. Here, we show that in many stramenopiles, the C3 part of glycolysis is localised in mitochondria. We discovered genuine mitochondrial targeting signals on the six last enzymes of glycolysis. These targeting signals are recognised and sufficient to import GFP into mitochondria of a heterologous host. Analysis of eukaryotic genomes identified these targeting signals on many glycolytic C3 enzymes in a large group of eukaryotes found in the SAR supergroup3, in particular the stramenopiles. Stramenopiles, or heterokonts, are a large group of ecologically important eukaryotes that includes multi- and unicellular algae such as kelp and diatoms, but also economically important oomycete pathogens such as Phytophthora infestans. Confocal immunomicroscopy confirmed the mitochondrial location of glycolytic enzymes for the human parasite Blastocystis. Enzyme assays on cellular fractions confirmed the presence of the C3 part of glycolysis in Blastocystis mitochondria. These activities are sensitive to treatment with proteases and Triton X-100 but not proteases alone. Our work clearly shows that core cellular metabolism is more plastic than previously imagined and suggests new strategies to combat stramenopile pathogens such as the causative agent of late potato blight, P. infestans.

evolutionary biology↗