Biology subjects

Klotz, C.

Publications and source records attributed to Klotz, C..

9 recordsLinked to original sources

bioRxiv2026

Cryptosporidium sex employs a complex male fertilization apparatus that is required for continued infection

Most apicomplexan parasites replicate asexually to colonize their mammalian hosts, and sexually in the context of transmission. In contrast, for Cryptosporidium sex is essential to sustain intestinal infection in vivo and therefore is a promising opportunity to prevent and treat the disease. However, the mechanism of sex, which occurs within host enterocytes, is poorly understood, limiting the discovery of targets for such intervention. Here we identify an elaborate fertilization apparatus and numerous of its molecular components in male gametes. We discern two elements, one displayed on the apex, that makes intimate contact with host cells containing female parasites, and a second internal structure, that harbors the membrane fusogen HAP2. Ablation of HAP2 by conditional mutagenesis blocked fertilization and oocyst production, rapidly cured infected mice and left them with enhanced resistance to subsequent challenge. Our studies provide insights into a remarkable fertilization mechanism and highlight specific molecules as targets for translation.

microbiology↗

bioRxiv2026

Cryptosporidium sequentially remodels its single rhoptry into the host interface

The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease in young children. Within the intestinal epithelium, Cryptosporidium establishes a unique intracellular niche in the apical brush border of enterocytes. A structurally complex interface between host and parasite acts as a holdfast and enables protein and metabolite transport. How the parasite invades its host cell and builds the interface is poorly understood. Here, we reveal parasite invasion with high temporal and spatial resolution using rigorous molecular markers. We find that sequential discharge of specialized secretory organelles anchors the parasite within the host prior to internalization. The single Cryptosporidium rhoptry persists beyond its initial pre-invasion discharge, acting as a conduit for the secretion of multiple waves of effector proteins. Ultimately, the rhoptry membrane gives rise to the feeder organelle that separates host from parasite cytoplasm. These findings lead us to propose a mechanistic model of Cryptosporidium invasion and intracellular parasitism.

microbiology↗

bioRxiv2025

Human airway organoids as a versatile model to study BSL-4 virus replication and pathogenesis

Research with BSL-4 viruses such as Ebola, Marburg, and Nipah presents significant challenges due to their high virulence and the stringent containment measures required. A major limitation in studying viral pathogenesis and developing therapeutic strategies is the absence of suitable animal models that accurately replicate human disease. In this context, 3D cell culture systems offer significant advantages over traditional 2D monolayer cultures, mimicking native physiological conditions including cell polarization and composition. Human airway organoids, derived from pluripotent or adult stem cells, closely replicate the structure and function of the human respiratory system, providing a relevant and accessible environment for studying viral replication and pathogenesis. In contrast to conventional cell lines, airway organoids enable investigation of virus-host interactions within a human tissue context, providing insights that are more directly translatable to human disease. In our study, we generated airway organoids from both clinical donor tissues and commercially available nasal epithelial cells and showed in comparative analyses with whole lung tissue that these organoids are comparable in terms of cell composition. Despite donor-specific variations due to genetic factors, airway organoids derived from different sources and donors exhibit a remarkably similar cellular make-up. We further demonstrated that organoids derived from nasal swabs can effectively replicate BSL-4 viruses, establishing them as a standardized 3D model for broader research applications and advancing our understanding of these pathogens, especially in the absence of reliable animal models. Author SummaryThis study establishes human airway organoids as a robust model for investigating BSL-4 pathogens, such as Ebola, Marburg, and Nipah virus. Airway organoids represent reliable systems due to their ability to replicate the complexity of human respiratory epithelia and support viral infection. These organoids exhibit high susceptibility to these viruses, allowing for subsequent analysis of infection kinetics, immune evasion, and tissue-specific tropism within a controlled environment. This platform provides a powerful tool for antiviral testing and studying virus-host interactions, thus helping bridge critical gaps in high-containment virus research.

cell biology↗

bioRxiv2025

Arginine metabolism has a pivotal function for the encystation of Giardia duodenalis

Arginine metabolism plays a key role in the energy metabolism of the intestinal parasite Giardia duodenalis, an amitochondrial protozoan that infects humans and animals and causes significant morbidity. An arginine deiminase (ADI) has been implicated in virulence, but it is currently unknown if ADI allele variants from the different genetic G. duodenalis subgroups (assemblages) differ in function. Here, the hypothesis was tested that sequence variation detected between G. duodenalis ADI alleles from the two G. duodenalis assemblage types found in humans affects functional parameters of the enzyme with potential consequences in life cycle progression. The ADI enzyme affinity for arginine was drastically reduced in sub-assemblage AII isolates, a human specific assemblage, in comparison to zoonotic sub-assemblage AI and B isolates. We identified the two amino acid residues responsible for the lower substrate affinity of ADIAII variant. Using genetic approaches to generate ADI knockout mutants, biochemical approaches to unravel substrate affinity as well as cellular approaches to determine efficiency of life cycle progression, we show that ADI is essential for efficient encystation of the parasite and that the lower substrate affinity in ADIAII correlates with lower encystation efficiency. We further demonstrate that arginine is essential for efficient encystation, and by generating ADI knock-out parasites we present evidence that ADI is the functional correlate for this arginine dependence. Thus, our data describe ADI as a quantitative trait that affects life cycle progression of G. duodenalis with putative clinical and epidemiological relevance. Author summaryIn the human pathogenic parasite Giardia duodenalis, arginine deiminase (ADI) mediates the first step in the arginine dehydrolase pathway (ADH), metabolizing arginine to provide chemical energy in form of ATP. The bacterial-derived ADH pathway had been inherited by horizontal gene transfer, and ADI has been proposed as a virulence factor. We show here by biochemical and genetic approaches with ADI knock-out mutants that arginine and its metabolizing enzyme ADI are essential for efficient life cycle progression (encystation) to form infectious cysts. Furthermore, we show a drastically impaired arginine substrate affinity for the human-specific G. duodenalis genotype AII in comparison to the zoonotic genotypes AI and B and identified the molecular entities responsible for this altered substrate affinity. This lower substrate affinity also correlated with lower cyst formation in the AII genotype.

microbiology↗

bioRxiv2023

Comparative genomics reveals the emergence of an outbreak-associated Cryptosporidium parvum population in Europe and its spread to the USA

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g. IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water-and food-borne outbreaks. In this study, we generated new whole genome sequence (WGS) data from 123 human-and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China and the USA (n=72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analysed a dataset from 141 isolates from the zoonotic groups IIa (n=119) and IId (n=22). Based on 28,047 high quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: isolates from China (IId) and Egypt (IIa and IId) formed population 1, a minority of European isolates (IIa and IId) formed population 2, while the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the USA (IIa) clustered in population 3. Based on analyses of the population structure, population genetics and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the UK, reach the USA where it also expanded. In addition, genetic exchanges between different populations led to the formation of mosaic genomes. The reason(s) for the successful spread of population 3 remained elusive, although genes under selective pressure uniquely in this population were identified.

genomics↗

bioRxiv2023

Dynamic ventral disc contraction is necessary for Giardia attachment and host pathology

Giardia lamblia is a common parasitic protist that infects the small intestine and causes giardiasis, resulting in diarrhea, vomiting, weight loss, and malabsorption. Giardiasis leads to cellular damage, including loss of microvilli, disruption of tight junctions, impaired barrier function, enzyme inhibition, malabsorption, and apoptosis. In the host, motile Giardia trophozoites attach to the duodenal microvilli using a unique microtubule organelle called the ventral disc. Despite early observations of disc-shaped depressions in microvilli after parasite detachment, little is known about disc-mediated attachment mechanisms and there little direct evidence showing that parasite attachment causes cellular damage. However, advancements in in vitro organoid models of infection and genetic tools have opened new possibilities for studying molecular mechanisms of attachment and the impact of attachment on the host. Through high-resolution live imaging and a novel disc mutant, we provide direct evidence for disc contraction during attachment, resolving the long-standing controversy of its existence. Specifically, we identify three types of disc movements that characterize contraction, which in combination result in a decrease in disc diameter and volume. Additionally, we investigate the consequences of attachment and disc contractility using an attachment mutant that has abnormal disc architecture. In a human organoid model, we demonstrate that this mutant has a limited ability to break down the epithelial barrier as compared to wild type. Based on this direct evidence, we propose a model of attachment that incorporates disc contraction to generates the forces required for the observed "grasping" of trophozoites on the host epithelium. Overall, this work highlights the importance of disc contractility in establishing and maintaining parasite attachment, leading to intestinal barrier breakdown.

microbiology↗

bioRxiv2023

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 {micro}m in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened "crescent" or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

microbiology↗

bioRxiv2022

Oncogenic signalling is coupled to colorectal cancer cell differentiation state

Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC/Wnt, KRAS, TP53 and SMAD4 stepwisely enable an oncogenic signalling network. Here, we developed a novel mass cytometry antibody panel to analyse colorectal cancer cell differentiation and signalling in human isogenic colorectal cancer organoids and in patient-derived cultures. We define a differentiation axis following EphrinB2 abundance in all tumour progression states from normal to cancer. We show that during colorectal cancer progression, oncogenes decrease dependence on external factors and shape distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell-promoting or restricting effects. Individual nodes of the signalling network remain coupled to the differentiation state, regardless of the presence of oncogenic signals. Our work underscores the key role of cell plasticity as a hallmark of cancer that is gradually unlocked during colorectal cancer progression.

cancer biology↗

bioRxiv2020

Dissection of barrier dysfunction in organoid-derived human intestinal epithelia induced by Giardia duodenalis

Background and aimsThe protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed to overcome these limitations and to recapitulate the authentic series of events in the primary human duodenal tissue by using the human organoid system. MethodsWe established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological and tight junction components. ResultsOrganoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of epithelial barrier upon G. duodenalis infection. Transcriptomic analysis suggested major expression changes in genes contributing to ion transport and tight junction structure. SLC12A2/NKCC1- and CFTR-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. ConclusionData suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection ignited by alterations of cellular ion transport followed by breakdown of the tight junctional complex and loss of epithelial integrity. The newly established organoid-derived model to study G. duodenalis infection will help enable further molecular dissection of the disease mechanism and, thus, can help to find new options treating disease and infection, in particular relevant for chronic cases of giardiasis.

microbiology↗