Biology subjects

Striepen, B.

Publications and source records attributed to Striepen, B..

At least 19 recordsLinked to original sources

bioRxiv2026

A defined multi-kingdom microbial consortium naturalizes the immune system of laboratory mice

Dirty mice experiments suggest that laboratory mice fail to model certain human immune responses due to their limited microbial exposure. The identity, nature, and number of microbes required to normalize the immune system are unknown. Through systematic screening, we uncovered unexpected cross-kingdom microbial interactions and identified four enteric microbes spanning viruses, fungi, and protists sufficient to reproduce key immunological features of pet store mice and adult humans. This consortium also altered cancer outcomes, shifting tumorigenesis toward the anatomical pattern of human familial adenomatous polyposis in ApcMin/+ mice and recapitulating the imperfect response of many patients to chimeric antigen receptor (CAR) T cell therapy. Thus, surprisingly few microbes can profoundly reshape immune states and disease responses, providing a tractable approach to naturalize laboratory mouse immunity.

immunology↗

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↗

bioRxiv2026

The role of integrins in T cell-mediated resistance to Cryptosporidium parvum

Cryptosporidium is a protozoan that infects epithelial cells of the small intestine and is a cause of diarrhea and death in immunocompromised individuals and malnourished children. Immunity to this parasite is mediated by an intestinal T cell response, which is generated in gut-associated lymphoid tissues and dependent on type 1 conventional dendritic cells (cDC1s). The initial priming of T cells is accompanied by changes in integrin expression and subsequent trafficking to the site of infection. The role of specific integrins in trafficking to the ileum during cryptosporidiosis is largely unknown. The development of a transgenic Cryptosporidium strain that expresses MHCI and MHCII-restricted model antigens provides the ability to track T cell responses to this parasite. Our studies in this system revealed marked changes in the integrin profile of parasite-specific T cells as they are activated and traffic to the gut, and demonstrate that cDC1s contribute to the expression of the integrins 4, {beta}7, {beta}1, and L. Surprisingly, blockade of the canonical gut-homing integrin 4{beta}7 does not impact the ability of parasite-specific T cells to access the gut. However, blockade of integrin L decreases the parasite-specific T cell frequency at the site of infection and delays control of parasite burden. These datasets highlight an 4{beta}7-independent mechanism of T cell trafficking to the small intestine and indicate that L is an integrin required for T cell-mediated resistance to Cryptosporidium.

immunology↗

bioRxiv2025

Diverse infections transcriptionally reprogram the intestinal epithelium and epithelial-immune cell interactions

The distal small intestine plays vital roles in host physiology by regulating nutrient and fluid homeostasis. Despite being impacted in Crohns disease and a major target for a range of infections, we know relatively little about the complexity of cellular responses and cell-cell communication in the ileum during infection. Single cell and spatial transcriptomics have emerged as powerful technologies to study tissue heterogeneity in the gut, but these tools have focused on the large intestine, in part due to the accessibility of this tissue for biopsies and its importance in cancer. Here we present GutPath, an atlas of over 500,000 single cells with RNA and protein expression profiles for 91 cell states in the ileum across diverse infectious archetypes. We show that GutPath accurately captures established immune responses to infection while revealing pathogen-specific responses in enterocytes. To highlight the discovery potential of this atlas, we identify a novel enterocyte cell state present during Yersinia pseudotuberculosis infection that is spatially linked to bacterial load and tissue pathology. GutPath establishes a much-needed resource for the immunology community that will accelerate the study of the transcriptional diversity of cellular landscapes in the small intestine.

immunology↗

bioRxiv2025

Widespread genomic heterogeneity at the type II NAD(P)H dehydrogenase locus predisposes Cryptosporidium to clofazimine resistance

The parasite Cryptosporidium is a leading cause of life-threatening diarrheal disease, and effective treatment is not available. The discovery of potent anti-Cryptosporidium activity of clofazimine, offered the opportunity to repurpose a drug already used to treat leprosy and tuberculosis. However, clofazimine failed in a human trial, which was attributed to poor bioavailability. Here, we observed differential susceptibility among parasite isolates which we exploit to map the mode of action to type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of NDH2 resulted in profound clofazimine resistance, and biochemical studies demonstrated NDH2 mediated electron transfer to clofazimine. Through genomic analyses we uncovered heterogeneity at the NDH2 locus for C. parvum and C. hominis and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.

microbiology↗

bioRxiv2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual lifecycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

genetics↗

bioRxiv2023

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-gamma on parasite restriction

The production of IFN-{gamma} is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. The use of single cell RNA sequencing to profile IEC during infection revealed induction of IFN-{gamma}-dependent gene signatures that was comparable between uninfected and infected cells, and IEC expression of the IFN-{gamma} receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-{gamma} demonstrated the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-{gamma} on IEC and suggest a model in which IFN-{gamma}-mediated bystander activation of uninfected enterocytes is important for control of Cryptosporidium. AUTHOR SUMMARYThe cytokine interferon-gamma (IFN-{gamma}) plays an important role in the control of intracellular infections by a wide variety of bacteria, viruses and parasites. While the impact of IFN-{gamma} on immune cells has been a major research focus, how it impacts intestinal epithelial cells remains poorly understood. Cryptosporidium parasites are an important cause of morbidity in a variety of epidemiological settings and exclusively infect intestinal epithelial cells (IEC). Recent advances in the ability to genetically modify and study Cryptosporidium in wild-type hosts provides a useful model to investigate IEC-intrinsic mechanisms of pathogen control. In this study, single cell RNA-sequencing was used to analyze the IEC response to infection and IFN-{gamma} signalling. We demonstrate broad changes in the epithelial compartment during infection that include the induction of an IEC population with robust induction of IFN-{gamma}-stimulated genes. In addition, we show that infected IEC remain responsive to IFN-{gamma} signalling, and that this cytokine causes a delayed reduction in parasite burden that correlates with the kinetics of IEC responsiveness to IFN-{gamma} stimulation. Together, our work uncovers how Cryptosporidium infection impacts the IEC compartment and helps define the relationship between the kinetics of IFN-{gamma} responsiveness and pathogen control in IEC.

immunology↗

bioRxiv2023

Intestinal cDC1s provide IL-12 dependent and independent functions required for CD4+ T cell mediated resistance to Cryptosporidium

Cryptosporidium is an enteric pathogen that is a prominent cause of diarrheal disease. Control of this infection requires CD4+ T cells, though the processes that lead to T cell-mediated resistance have been difficult to assess. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to dissect the early events that influence CD4+ T cell priming and effector function. These studies highlight that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node (mesLN) and differentiate into Th1 cells in the gut, where they mediate IFN-{gamma}-dependent control of the infection. Although type 1 conventional dendritic cells (cDC1s) were not required for initial priming of CD4+ T cells, cDC1s were required for CD4+ T cell expansion and gut homing. cDC1s were also a major source of IL-12 that was not required for priming but promoted full differentiation of CD4+ T cells and local production of IFN-{gamma}. Together, these studies reveal distinct roles for cDC1s in shaping CD4+ T cell responses to enteric infection: first to drive early expansion in the mesLN and second to drive effector responses in the gut. SummaryCryptosporidium parasites that express model antigens were generated to dissect how parasite-specific CD4+ T cells are primed and mediate effector functions required to control this enteric pathogen. cDC1s produced IL-12p40 and were required for early expansion and gut homing of CD4+ T cells. However, IL-12p40 was only required for the development of Th1 CD4+ T cell effector function in the gut.

immunology↗

bioRxiv2023

Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8+ T cell responses

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of IFN-{gamma} that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8+ T cell responses to Cryptosporidium.

immunology↗

bioRxiv2023

Genetic crosses within and between species of Cryptosporidium

Parasites and their hosts are engaged in rapid coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing C. parvum, a parasite of cattle and humans, and C. tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity. Significance statementThe parasite Cryptosporidium is a leading cause of diarrheal disease. While infection is common all around the world, young children experiencing malnutrition are impacted most profoundly, and the disease is an important contributor to early childhood mortality. This study experimentally demonstrates that different strains and even species of Cryptosporidium can recombine their genomes through sex. The progeny of such genetic crosses shows combined features of both parents, with resistance to multiple drugs being one example. Sex thus provides a critical mechanism for the parasite to rapidly adapt to changing environments and hosts. Genetic crosses as an experimental tool may also be harnessed in the future to discover the genes underlying differences in virulence, drug sensitivity, and immunogenicity between parasite isolates.

microbiology↗

bioRxiv2022

Genetic ablation of a female specific Apetala 2 transcription factor blocks oocyst shedding in Cryptosporidium parvum

The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire lifecycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in C. parvum we develop and validate a small molecule inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knock out. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro, but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission.

microbiology↗

bioRxiv2022

An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. In contrast to previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associated with the exocytic site - prior to invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarity to GPCR proteins. We propose that the CRMP complex acts as host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

cell biology↗

bioRxiv2021

Live imaging of the Cryptosporidium parvum lifecycle reveals direct development of male and female gametes from type one meronts

Cryptosporidium is a leading infectious cause of diarrhea around the world associated with waterborne outbreaks, community spread, or zoonotic transmission. The parasite has significant impact on early childhood mortality, and infection is both consequence and cause of malnutrition and stunting. There is currently no vaccine, and treatment options are very limited. Cryptosporidium is a member of the Apicomplexa, and as typical for this protist phylum relies on asexual and sexual reproduction. In contrast to other Apicomplexa, like malaria parasite Plasmodium, Cryptosporidiums entire lifecycle unfolds in a single host in less than three days. Here we establish a model to image lifecycle progression in living cells, and observe, track, and compare nuclear division of asexual and sexual stage parasites. We establish the length and sequence of the cell cycles of all stages and map the developmental fate of parasites across multiple rounds of invasion and egress. We determine that the parasite executes an intrinsic program of three generations of asexual replication, followed by a single generation of sexual stages that is independent of environmental stimuli. We find no evidence for a morphologically distinct intermediate stage (the tetraploid type II meront) but demonstrate direct development of gametes from 8N type I meronts. The progeny of each meront is collectively committed to either asexual or sexual fate, but importantly, meronts committed to sexual fate give rise to both males and females. We define a Cryptosporidium lifecycle matching Tyzzers original description and inconsistent with the coccidian lifecycle now shown in many textbooks.

microbiology↗

bioRxiv2021

A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes required for Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFN{lambda} reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFN{lambda} production critical to early protection against this infection. Author SummaryCryptosporidium infection is an important contributor to global childhood mortality. There are currently no vaccines available, and the only drug has limited efficacy in immunocompromised individuals and malnourished children who need it most. To discover which host proteins are essential for Cryptosporidium infection, we conducted a genome wide knockout screen in human host cells. Our results confirm the importance of glycosaminoglycans on the surface of epithelial cells for attachment and invasion of the parasite. We also found that host GPI anchor biosynthesis and interferon signaling pathways were enriched by our screen. Examining the role of interferon signaling further we found a type III interferon response, IFN{lambda}, was generated in response to infection and shown to be initiated in the infected cell. Utilizing mouse models of infection, we found that the type III interferon response was important early during infection with its induction likely preceding IFN{gamma}, a key cytokine for the control of this infection. We also determined that TLR3 was the pattern recognition receptor responsible for IFN{lambda} production during Cryptosporidium infection. Our work shows that IFN{lambda} acts directly on the enterocyte and its use in treating immunocompromised mice produced striking reductions in infection.

microbiology↗

bioRxiv2021

The enteric pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected cell

The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the C. parvum MEDLE protein family to be translocated into the cytoplasm of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response that was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering pathogenesis factors into the infected enterocyte.

microbiology↗

bioRxiv2021

Crosstalk between enterocytes and innate lymphoid cells drives early IFN-g-mediated control of Cryptosporidium

The intestinal parasite, Cryptosporidium, is a major contributor to global child mortality and causes opportunistic infection in immune deficient individuals. Innate resistance to Cryptosporidium, which specifically invades enterocytes, is dependent on the production of IFN-{gamma}, yet whether enterocytes contribute to parasite control is poorly understood. In this study, utilizing the natural mouse pathogen, Cryptosporidium tyzzeri, we show that epithelial-derived IL-18 synergized with IL-12 to stimulate innate lymphoid cell (ILC) production of IFN-{gamma}. This innate IFN-{gamma} was required for early parasite control. Loss of STAT1 in enterocytes, but not dendritic cells or macrophages, antagonized early parasite control. Transcriptional profiling of enterocytes from infected mice identified an IFN-{gamma} signature and enrichment of anti-microbial effectors like IDO, GBP and IRG. Deletion experiments identified a role for Irgm1/m3 in parasite control. Thus, enterocytes promote ILC production of IFN-{gamma} that acts on enterocytes to restrict the growth of C. tyzzeri.

immunology↗

bioRxiv2021

Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal new biological insights

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community still relies on a fragmented reference genome sequence from 2004. Incomplete reference sequences hamper experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, C. hominis and C. tyzzeri. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers 13 of 16 possible telomeres and raises a new hypothesis for the remaining telomeres and associated subtelomeric regions. Comparative annotation revealed that most "missing" orthologs are found suggesting that species differences result primarily from structural rearrangements, gene copy number variation and SNVs in C. parvum, C. hominis and C. tyzzeri. We made >1,500 C. parvum annotation updates based on experimental evidence. They included new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete DNA methylase Dnmt2 ortholog. 190 genes under positive selection including many new candidates were identified using the new assembly and annotation as reference. Finally, possible subtelomeric amplification and variation events in C. parvum are detected that reveal a new level of genome plasticity that will both inform and impact future research.

genomics↗