Biology subjects

Sateriale, A.

Publications and source records attributed to Sateriale, A..

10 recordsLinked to original sources

bioRxiv2025

Cryptosporidium modifies intestinal microvilli through an exported virulence factor

Cryptosporidium is a common intestinal infection of vertebrates and a significant threat to public health. Within the epithelial layer of the intestine, the parasite invades and replicates. Infected cells are readily detected under the microscope by the presence of elongated microvilli, particularly around the vacuole where the parasite resides. Here, we identify a family of Cryptosporidium virulence factors that are exported into the host cell during infection and localise to the microvilli. We examine the trafficking and function of the most highly expressed family member, MVP1, which appears to control the elongation of microvilli through engagement of host EBP50 and CDC42. Remarkably, this mechanism closely mirrors that of an enteropathogenic Escherichia coli virulence factor, MAP, which is also known to drive host microvilli elongation during infection. This highlights a unique instance where eukaryotic and prokaryotic virulence factors have convergently evolved to modulate host actin structures through a similar mechanism.

microbiology↗

bioRxiv2024

The essential host genome for Cryptosporidium intracellular survival exposes metabolic dependencies that can be leveraged for treatment

Mapping how pathogens interact with their host cells can reveal unexpected pathogen and host cell biology, paving the way for new treatments. Cryptosporidium is an intracellular parasite of intestinal epithelial cells, and a leading cause of diarrheal death and disease in infants worldwide. Despite this, very little is known about the cell biology of infection of this eukaryotic pathogen. Here, we designed and implemented a unique microscopy-based arrayed CRISPR-Cas9 screen to interrogate the effects of the loss of every protein-coding human gene on a Cryptosporidium infection. As the experimental readout is image-based, we extracted multiple phenotypic features of infection, including parasite growth, progression of the parasite to its sexual life stage, and recruitment of host actin to pedestals beneath the parasite vacuole. Using this dataset, we discovered a tipping point in the host cholesterol biosynthesis pathway that controls Cryptosporidium infection. Parasite growth can either be inhibited or promoted by the intermediary metabolite squalene. A build-up of squalene in epithelial cells creates a reducing environment, with more reduced host glutathione available for uptake by the parasite. Because Cryptosporidium has lost the ability to synthesise glutathione, this uptake from the host cell is required for growth and progression through its life cycle. We demonstrate that this dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that has efficacy against Cryptosporidium in vitro and in vivo.

cell biology↗

bioRxiv2024

Targeted CRISPR Screens Reveal Genes Essential for Cryptosporidium Survival in the Host Intestine

The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited eKicacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method we examine the parasites pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group we determined the parasite gene known as Cp23 to be essential for survival, which was confirmed through inducible knockout in vitro and in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is essential for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.

microbiology↗

bioRxiv2023

Dietary environmental factors shape the defence against infection with Cryptosporidium

Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. While it is known from human cases and animal models that CD4+ T-cells play a role in curbing Cryptosporidium, the role of CD8+ cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express, and are dependent on, the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduced CD8+ IELs, decreased their cytotoxicity, and worsened infection. Transfer of CD8+ IELs rescued severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol to new-born mice promoted resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

immunology↗

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↗

bioRxiv2021

Spontaneous selection of Cryptosporidium drug resistance in a calf model of infection

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last [~]10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50S of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence. ImportanceCryptosporidium is a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance. Cryptosporidium parvum infected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emerging Cryptosporidium drug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential. One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calf C. parvum infection model.

microbiology↗

bioRxiv2020

Stage-Specific Long Non-coding RNAs in Cryptosporidium parvum as Revealed by Stranded RNA-Seq

Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs 86% of which are differentially expressed. Nearly 10% of annotated mRNAs have an antisense lncRNA. lncRNAs also appear to occur most often at the 3 end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation trend between lncRNA and the upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvum, C. hominis and C. baileyi. Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. We provide an initial characterization of the C. parvum non-coding transcriptome to facilitate further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

genomics↗