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

  1. Jennifer E Dumaine
  2. Adam Sateriale
  3. Alexis R Gibson
  4. Amita G Reddy
  5. Jodi A Gullicksrud
  6. Emma N Hunter
  7. Joseph T Clark
  8. Boris Striepen  Is a corresponding author
  1. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, United States
  2. Franklin College of Arts and Science, University of Georgia, United States

Abstract

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 Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol 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, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.

Editor's evaluation

Apicomplexa parasites export proteins into their infected cells to modulate/co-opt signaling pathways for their intracellular development. This study demonstrates for the first time the export of a protein, MEDLE2, from Cryptosporidium parvum, and characterizes its targeting signal as well as its effector function in the infected host cell.

https://doi.org/10.7554/eLife.70451.sa0

Introduction

The Apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease worldwide. Young children are highly susceptible to infection, and cryptosporidiosis is an important contributor to child mortality (Khalil et al., 2018; Kotloff et al., 2013). Children in resource-poor settings carry a disproportionate burden of severe disease (Choy and Huston, 2020). Malnutrition enhances the risk of severe cryptosporidiosis, and at the same time, the disease impacts the nutritional state of children, which can lead to impaired growth (Costa et al., 2011; Mondal et al., 2009). Infection with the parasite results in protective immunity, but this immunity is not sterile and may require multiple exposures to develop (Chappell et al., 1999; Okhuysen et al., 1998). Most human diseases are due to infection with Cryptosporidium hominis, which only infects humans, and Cryptosporidium parvum, which can be zoonotically transmitted (Feng et al., 2018; Nader et al., 2019). The emergence of Cryptosporidium species is driven by host adaptation resulting in specialization and narrowing host specificity; however, the sexual life cycle of the parasite allows for recombination and can lead to rapid convergent evolution of host specificity (Guo et al., 2015; Nader et al., 2019).

Cryptosporidium infects the epithelium of the small intestine, where it lives in a unique intracellular, but extracytoplasmic niche (Elliott and Clark, 2000). The mechanism by which this niche is established during invasion is still debated but involves the rearrangement of the host actin cytoskeleton, the formation of tight junction-like structures between host and parasite membranes, and a dense band of unknown composition at the host-parasite interphase (Bonnin et al., 1999; Elliott and Clark, 2000). Cryptosporidium has severely reduced metabolic capabilities and relies heavily upon the host cell for nutrients and metabolites (Abrahamsen et al., 2004; Xu et al., 2004). A number of specialized uptake mechanisms have been proposed to fill this need, many of which are believed to be localized to the so-called feeder organelle at the host-parasite interface (Guérin and Striepen, 2020). In summary, Cryptosporidium remodels the host cell in significant ways that include its cytoskeleton (Bonnin et al., 1999; Elliott and Clark, 2000), cellular physiology and metabolism (Argenzio et al., 1990; Kumar et al., 2018), as well as aspects of immune restriction and regulation (Laurent and Lacroix-Lamandé, 2017).

Many bacterial, protozoan, and fungal pathogens use translocated effectors to manipulate their hosts to secure nutrients and to block host immunity. In Plasmodium falciparum, exported effectors form adhesive structures on the surface of red blood cells to alter tissue distribution and mechanical properties to prevent clearance (Crabb et al., 1997; Leech et al., 1984) and install new nutrient and ion uptake mechanisms (Baumeister et al., 2006). In Toxoplasma gondii, translocated effectors disarm critical elements of interferon-induced cellular restriction (Gay et al., 2016; Olias et al., 2016), establish access to cellular nutrients, and rewire signaling and transcriptional networks to antagonize immune responses to promote a favorable environment for parasite growth (Bougdour et al., 2013; Braun et al., 2013; Braun et al., 2019; Gold et al., 2015). Cryptosporidium has been hypothesized to use exported effector proteins to ensure its survival (Pellé et al., 2015); however, to date, no such factors have been identified. Here, we report that the polymorphic protein MEDLE2 is exported to the host cell cytoplasm by all stages of the C. parvum life cycle in vitro and in vivo. This protein is not injected during invasion, but rather is transported into the host cell following the initial establishment of infection. We carefully mapped the requirements for export and found a signal that includes a proteolytic cleavage site, and we demonstrate that exported proteins undergo processing. Overall, we demonstrate the presence of a robust translocation mechanism established by intracellular parasites that delivers parasite proteins to the host cell.

Results

The C. parvum protein MEDLE2 is exported into the host cell

The genome of C. parvum encodes multiple families of paralogous proteins that carry N-terminal signal peptides and share conserved amino acid repeat motifs among family members (Abrahamsen et al., 2004). These genes are often found within clusters (homologous and heterologous), many of which are located proximal to the telomeres of multiple chromosomes (Figure 1A). Comparing strains and species, these genes are highly polymorphic and vary in copy number, which has been interpreted as a sign of rapid evolution driven by their roles in invasion, pathogenesis, and host cell specificity (Guo et al., 2015; Nader et al., 2019; Xu et al., 2019) We hypothesized that such roles might be reflected in the targeting of presumptive effectors to the host cell and selected representatives from each polymorphic gene family for initial localization studies (Table 1). Selected loci were modified in the C. parvum IOWAII isolate using CRISPR/Cas9-driven homologous recombination (Vinayak et al., 2015) to append three hemagglutinin epitopes (3XHA) in translational fusion to the C-terminus (Figure 1A). Drug-resistant parasites were recovered for four of six initial candidates, and successful genomic insertion was mapped by PCR (Figure 1B and C, Figure 1—figure supplement 1). We next infected human ileocecal colorectal adenocarcinoma cell cultures (HCT-8) with transgenic parasites and assessed the localization of the tagged proteins by immunofluorescence assay (IFA, Figure 1D and E). For most candidates, the tagged protein (red) appeared to coincide with the parasite and/or the parasitophorous vacuole (cgd8_3560, Figure 1D, Figure 1—figure supplement 1). In contrast, upon infection with parasites tagged in the MEDLE2 locus (cgd5_4590), HCT-8 cells showed HA staining in the cytosol (Figure 1E). We note that the cells that stain for HA (red) were those that were infected with parasites, labeled with Vicia villosa lectin (VVL, green), and conclude that MEDLE2-HA is exported by the parasite into the host cell during or following invasion.

Figure 1 with 3 supplements see all
MEDLE2 is exported to the host cell cytoplasm.

(A) Schematic overview of the chromosomal location for polymorphic gene families in the C. parvum genome. (B) Map of the MEDLE2 locus targeted in C. parvum for insertion of a 3× hemagglutinin (HA) epitope tag, a nanoluciferase reporter gene (Nluc), and neomycin phosphotransferase selection marker (Neo). (C) PCR mapping of the MEDLE2 locus using genomic DNA from wild type (WT) and transgenic (MEDLE2-HA) sporozoites, corresponding primer pairs are shown in (B), and thymidine kinase (TK) gene used as a control. Note the presence of two bands in the 5′–3′ amplification, indicating the presence of a transgene (3081 bp) and persistence of an unmodified copy (1174 bp), suggesting multiple copies of MEDLE2 in the C. parvum genome; also see Figure 1—figure supplement 2. (D, E) HCT-8 cultures were infected with WYLE4-HA (D) or MEDLE2-HA (E) transgenic parasites and fixed after 24 hr for immunofluorescence assay (IFA). Red, antibody to HA; green, Vicia villosa lectin stain, VVL (Gut and Nelson, 1999); blue, Hoechst DNA dye. Additional genes targeted and the localizations of their products are summarized in Table 1 and Figure 1—figure supplements 1 and 3.

Table 1
Members of multigene families for which localization of protein product was initially attempted in this study.
Gene familyGene IDResult
MEDLEcgd5_4590Exported to host cell
FLGNcgd4_4470Transgenic unsuccessful
GGCcgd5_3570Transgenic unsuccessful
SKSRcgd8_30Not in host cell
SKSRcgd8_40Not in host cell
WYLEcgd8_3560Not in host cell

We investigated whether other members of the MEDLE gene family are similarly exported and selected MEDLE1 (cgd5_4580) and MEDLE6 (cgd6_5490) for epitope tagging. In IFAs, both tagged MEDLE1 and MEDLE6 localized around the parasite, as well as to the host cell cytoplasm, but expression and export was less than what was observed for MEDLE2 (Figure 1—figure supplement 3). To explore this difference, we engineered a chimeric mutant carrying an extra copy of MEDLE1-HA driven by the MEDLE2 promoter. Upon infection with these parasites, we observed increased MEDLE1 expression making export into the host cell easier to appreciate (Figure 1—figure supplement 3). We conclude that multiple members of the MEDLE family are host-targeted proteins. Of these, MEDLE2 was expressed and exported most robustly and was selected for further study.

MEDLE2 is exported across the C. parvum life cycle in culture and infected animals

To better understand export of MEDLE2 to the host cell, we next engineered a reporter parasite in which the endogenous locus of MEDLE2 was 3XHA epitope tagged, and these parasites expressed a tandem mNeon green fluorescent protein in their cytoplasm to allow for identification and quantification of parasites (MEDLE2-HA-tdNeon, Figure 2—figure supplement 1). These parasites were then used in time-course experiments across the 72 hr of infection afforded by the HCT-8 culture model. Following inoculation with sporozoites, cultures were fixed in 12 hr increments and processed for IFA. MEDLE2 was observed at all time points (Figure 2A) and quantification showed that the number of HA positive host cells (Figure 2B, red) increased over time, closely matching the increase in the number of parasites (blue). 94 ± 1.83% (mean ± SD, n = 3695) of the cells showing HA staining also showed parasite infection. Importantly, this high level of correlation between host cell HA expression and infection (r2 = 0.9) remained constant over 72 hr. Previous work has shown synchronous life cycle progression over this time span. Initially, all parasites represent asexual stages replicating by merogony followed by a dramatic development switch at 40 hr, and later cultures are dominated by male and female gametes (Tandel et al., 2019). We staged parasites at 48 hr and identified female gametes and male gamonts using antibodies for the markers COWP1 and tubulin, respectively. We found the host cells infected with all these stages to be positive for MEDLE2-HA (Figure 2D).

Figure 2 with 1 supplement see all
Infected cells express MEDLE2-HA across the parasite life cycle.

(A) 200,000 MEDLE2-HA-tdNeon transgenic parasites were used to infect HCT-8 cells and fixed at intervals across a 72 hr time period. Data shown are representative images from triplicate coverslips processed for immunofluorescence assay (IFA). Red, hemagglutinin (HA)-tagged protein; green, parasites (mNeon); blue, Hoechst. (B, C) Quantification of MEDLE2-expressing cells (red) versus intracellular parasites (blue) for 3695 host cells evaluated across a 72 hr time course. 20 fields of view quantified using ImageJ to identify host cells and parasites (B). The percentage of cell exhibiting MEDLE2-HA and mNeon staining is constant across the time course with a cumulative 94 ± 1.83% (mean ± SD) (C). (D) HCT-8 cultures infected with MEDLE2-HA parasites were fixed for IFA at 48 hr when sexual life stages were present. Cells were stained with stage-specific antibodies for female (COWP1) and male (α- tubulin), demonstrating MEDLE2 is exported across the parasite life cycle. Red, HA-tagged protein; green, parasites (stage-specific antibody); blue, Hoechst. (E) IFA of cryosections from the small intestine of Ifng-/- mice infected with MEDLE2-HA-tdNeon C. parvum (images representative of samples from three mice). Red, HA-tagged protein; green, parasites (tdNeon); blue, Hoechst; gray, Phalloidin (actin).

Figure 2—source data 1

Numerical data used for the quantification of HA positive host cells and the intracellular parasites.

https://cdn.elifesciences.org/articles/70451/elife-70451-fig2-data1-v2.xlsx

We also tested whether MEDLE2 export occurs in vivo. Susceptible Ifng-/- mice were infected with 10,000 oocysts of the reporter strain, and after 12 days, mice were euthanized, the ileum was resected, fixed, and processed for histology. As shown by immunohistochemistry of sections of infected intestines in Figure 2E, MEDLE2 was exported to infected cells in vivo and exhibited cytoplasmic localization.

MEDLE2 is expressed and exported by trophozoites once infection has been established

Two broadly conserved temporal patterns have been described for host-targeted proteins of Apicomplexa (Figure 3A). Those involved in early aspects of the infection are packaged into the rhoptry organelle and injected during invasion (Rastogi et al., 2019). A second wave of proteins is translated and exported after the parasite has established its intracellular niche, and they are delivered to the host cell by a translocon-based mechanism (de Koning-Ward et al., 2009; Franco et al., 2016). For this reason, we next determined the timing of MEDLE2 expression using IFA of wild type (WT) and transgenic sporozoites mounted to cover glass with poly-lysine. We readily observed labeling for the sporozoite antigen Cp23 but did not detect any HA staining in transgenic sporozoites, indicating that MEDLE2 is not prepackaged into secretory organelles (Figure 3B). We then stained intracellular stages at different time points following invasion to determine the kinetics of MEDLE2 expression and export. At 4 hr, HA is first detectable, with labeling being associated with the parasite (parasite nuclei stained with Hoechst highlighted by white arrowheads). Beginning at 5.5 hr, MEDLE2-HA staining was observed throughout the host cell cytoplasm and continued to accumulate over time (Figure 3C).

MEDLE2 is expressed by trophozoites and passes through the secretory pathway.

(A) Schematic representation of hypothetical patterns of MEDLE2 export in C. parvum. (B) Immunofluorescence assay (IFA) of wild type (WT) and MEDLE2-HA sporozoites fixed on poly-L-lysine-treated coverslips. We note that MEDLE2-HA is not observed in sporozoites. Red, hemagglutinin (HA)-tagged protein; green, sporozoite antigen Cp23; blue, Hoechst. (C) HCT-8 cells infected with MEDLE2-HA parasites were fixed in 30 min increments and processed for IFA. Data shown are representative images from a time-course bridging 3 hr (no observed MEDLE2-HA) and 6 hr (MEDLE2-HA abundant in host cell). White arrowheads denote parasite nuclei. Red, HA-tagged protein; blue, Hoechst. (D) MEDLE2-HA parasites were excysted and used to infect HCT-8 and after 3 hr media were supplemented with brefeldin A (BFA) (10 µg/mL). 10 hr post infection, cells were fixed and processed for IFA. Red, HA-tagged protein; green, parasites (VVL); blue, Hoechst (D). (E) The impact of BFA treatment on MEDLE2-HA export was quantified showing a significant reduction in MEDLE2 export when comparing BFA-treated (red) and untreated cells (blue) (n = 191 untreated, n = 98 treated; mean ± SEM; p<0.0001; unpaired t test with Welch’s correction).

Figure 3—source data 1

Numerical data used for the quantification of HA positive host cells in the absence and presence of Brefeldin A (BFA).

https://cdn.elifesciences.org/articles/70451/elife-70451-fig3-data1-v2.xlsx

MEDLE2 is predicted to be a 209 amino acid protein with a putative N-terminal signal peptide suggesting trafficking through the parasite’s secretory pathway. To test this, we used brefeldin A (BFA), which blocks passage through the secretory pathway between the ER and Golgi. Cells were infected with MEDLE2-HA parasites, treated with BFA beginning 3 hr post infection, and fixed for IFA at 10 hr to assess MEDLE2 localization. BFA treatment initiated after invasion ablated export and resulted in the accumulation of MEDLE2-HA within the parasite (Figure 3D). Image analysis and quantification showed this reduction in export to be significant when comparing treated (red) to untreated cells (blue) (n = 98 treated; n = 198 untreated; p<0.0001; unpaired t test with Welch’s correction; Figure 3E). We conclude that MEDLE2 is not injected by the sporozoite during invasion; rather, it is expressed and exported by the trophozoite and arrives in the host cell about 5 hr post infection (note that the length of the intracellular lytic cycle of asexual stages is 11.5 hr; Guérin et al., 2021). BFA does block parasite growth, and that treatment was therefore restricted to a single merogony cycle.

MEDLE2 is an intrinsically disordered protein, and its export is blocked by ordered reporters

To further investigate the export of MEDLE2, we sought to develop a reporter assay to follow trafficking using three previously established systems. We fused the fluorescent reporter mScarlet to the C-terminus of MEDLE2 in its endogenous locus (Figure 4A, Figure 4—figure supplement 1). Transgenic parasites showed robust expression of the reporter when used to infect HCT-8 cultures. However, this fluorescence (red) was associated with parasites, highlighted by staining with VVL (Figure 4B, green), suggesting that MEDLE2-mScarlet remained trapped within or close to the parasite. We considered that export may occur, but that we lack the sensitivity to detect it. Thus, we engineered two highly sensitive assays that employ enzymes to amplify the signal.

Figure 4 with 3 supplements see all
Ordered reporters disrupt MEDLE2 export.

(A) Schematic map of the MEDLE2 locus targeted for insertion of three different reporter genes (mScarlet, beta-lactamase, or Cre recombinase), nanoluciferase (Nluc), and the selection marker (Neo). The guide RNA and flanking sequences used here were the same as those employed to generate MEDLE2-HA transgenic parasites (see Figure 2—figure supplement 1, Figure 4—figure supplement 1 for more detail). (B) MEDLE2-mScarlet parasites were used to infect HCT-8 cells and fixed for immunofluorescence assay (IFA) across a time course. Data shown are from 10 hr post infection, which is representative of the MEDLE2 localization observed at all time points. Red, Medle2-mScarlet; green, parasites (VVL); blue, Hoechst. (C) HCT-8 cells were infected with MEDLE2-BLA C. parvum for 24 hr before incubation with the CCF4-AM beta-lactamase substrate and visualization by live microscopy. This experiment was repeated three times. Red, parasites (tdTomato); green, uncleaved CC4F-AM; blue, cleaved CCF4-AM; gray, DIC. We attribute lack of CCF4-AM cleavage to failure of MEDLE2-BLA to export (Figure 4—figure supplement 1). (D) MEDLE2-Cre parasites were used to infect loxGFP/RFP color switch HCT-8 cells ((Figure 4—figure supplement 2) for schematic representation). After 48 hr, cells were subjected to flow cytometry. Live, single cells were gated based upon forward and side scatter, and green fluorescence (GFP) and red fluorescence (RFP) were measured to detect Cre recombinase activity. Despite robust infection, MEDLE2-Cre-infected cultures did not express RFP (Figure 4—figure supplement 2) compared to the positive control that was transiently transfected to express Cre recombinase.

First, we tagged MEDLE2 with beta-lactamase, which has been used to reveal effector export in bacteria and protozoa (Charpentier and Oswald, 2004; Lodoen et al., 2010). These parasites also expressed a cytoplasmic red fluorescent protein (Figure 4—figure supplement 1B). Cells infected with MEDLE2-BLA sporozoites were incubated with CCF4-AM, a cell-permeable substrate of beta-lactamase and imaged by live microscopy. Infected and uninfected cells accumulated CCF4-AM (Figure 4C, green); however, we did not detect substrate cleavage, resulting in blue fluorescence (Figure 4C). This could be due to the lack of MEDLE-BLA expression or export. To visualize localization of the MEDLE2-BLA fusion protein during infection, we performed IFA on MEDLE2-BLA-infected cells using a BLA antibody and observed that MEDLE2-BLA (green) was expressed but remained with the parasite (Figure 4—figure supplement 1C, red). Next, we generated a MEDLE2 fusion with Cre recombinase and used these parasites to infect an HCT-8 host cell line we engineered to carry a floxed GFP/RFP color switch reporter (see Figure 4—figure supplement 2). After 48 hr, cells were subjected to flow cytometry (Figure 4D). Using a gate for live, single cells, uninfected cells showed green (95.1%) but not red fluorescence (2.51%). Transfection of host cells with a Cre recombinase expression plasmid resulted in a pronounced shift to red fluorescence (+ control, 32%, Figure 4D). Cells infected with either WT parasites or MEDLE2-Cre parasites remained green, despite robust infection (Figure 4—figure supplement 2). Therefore, we tested three reporters, none of which resulted in detectable export to the host cell. We note that multiple algorithms predict MEDLE2 to be a highly disordered protein (Figure 4—figure supplement 3; low-complexity regions are indicated in light blue in Figure 5B) and conclude that translocation is blocked when folded reporters are fused to the protein.

Figure 5 with 3 supplements see all
MEDLE2 contains a host-targeting motif that is processed during export.

(A) Map showing the strategy used to engineer an ectopic copy of MEDLE2-HA in the thymidine kinase (TK) locus. Expression of an ectopic copy of MEDLE2-HA was driven by the MEDLE2 promoter. All point mutations were confirmed by Sanger sequencing (Figure 5—figure supplement 1). (B) Schematic representation of the MEDLE2 mutants generated using the strategy outlined in (A). The signal peptide (SP) is represented by dark blue, and low-complexity regions are shown in light blue. Candidate motifs targeted for mutagenesis are indicated with black triangles, and mutagenized amino acids are shown in red for two representative mutants. (C, D) Mutant parasites were used to infect HCT-8 cells and fixed for immunofluorescence assay (IFA) after 24 hr. For mutants shown in (C), the entire candidate motif was replaced with a matching number of alanine residues (e.g., KDVSLI/6A → AAAAAA). For mutants shown in (D), each individual amino acid in the KDVSLI sequence was changed to alanine. Red, hemagglutinin (HA)-tagged protein; green, parasites (VVL); blue, Hoechst. We note that SP and leucine 35 within the KDVSLI sequence are required for MEDLE2 export. (E, F) 5 × 106 transgenic oocysts were used to infect HCT-8 cells for 48 hr before preparation of whole-cell lysates. Proteins were separated by for SDS-PAGE and analyzed by western blot. The resulting blots for infections with whole motif mutants (E) and individual amino acid point mutants (F) are shown. Red, neomycin; green, HA. Note that when mutants are expressed in mammalian cells and not C. parvum the resulting proteins do not show any size differences (Figure 5—figure supplement 2).

MEDLE2 export depends upon N-terminal sequence features

We then sought to determine whether MEDLE2 contains sequence-specific information for host targeting. Using previously established host-targeting motifs from P. falciparum and T. gondii as models (Coffey et al., 2015; Marti et al., 2004), we searched the MEDLE2 amino acid sequence to identify candidate export motifs. Preference was given to regions with a basic amino acid, followed by one or two random amino acids, and a leucine residue (Pellé et al., 2015). While Plasmodium host-targeting motifs are typically found in close proximity to the signal peptide, T. gondii exhibits less rigid distance requirements (Coffey et al., 2015). We identified four motifs, three sites in proximity to the N-terminus and one C-terminal candidate for mutational analysis (Figure 5A). As folded reporters are not tolerated, we engineered parasite lines to express an ectopic copy of MEDLE2 marked by an HA tag. A cassette driven by the MEDLE2 promoter was inserted into the locus of the dispensable thymidine kinase gene (TK, Figure 5B), and expression level and export of ectopic WT protein was indistinguishable from protein tagged within the native locus.

Removal of the sequence encoding the N-terminal signal peptide (ΔSP) prevented MEDLE2-HA export, and the resulting protein accumulated within the parasite (Figure 5C). Next, we constructed a series of parasite strains in which each of the candidate motifs was replaced by a matching number of alanine residues (all mutants were confirmed by PCR mapping and Sanger sequencing; Figure 5—figure supplement 1). Mutagenesis of three of these candidate motifs had no impact on MEDLE2 translocation to the host cell (Figure 5C, Figure 5—figure supplement 1). In contrast, when the most N-terminal sequence KDVSLI was changed to six alanines, HA staining accumulated in the parasite and host cell staining was lost (Figure 5C). We conclude that in this mutant MEDLE2-HA is made but export is ablated. Next, we constructed six additional strains using the same strategy to change each amino acid position of the KDVSLI motif to alanine 1 residue at a time (Figure 5—figure supplement 1). Mutation of residue leucine 35 to alanine (L35A) ablated export and instead MEDLE2-HA remained with the parasite (Figure 5D). Changing the remaining five amino acids individually did not alter MEDLE2 localization in the host cell (Figure 5D). We conclude leucine 35 to be critical for export.

MEDLE2 export is linked to proteolytic processing

For both P. falciparum and T. gondii, leucine residues serve as crucial sites for proteolytic processing events that license proteins to leave the parasite and enter the host cell (Coffey et al., 2015; Hiller et al., 2004; Marti et al., 2004). To test whether such processing occurs during export of MEDLE2 in C. parvum, we performed western blot analysis on whole-cell lysates of infected cell cultures using antibodies to HA (MEDLE2-HA, green) and the drug resistance marker Neo (loading control, red). For WT MEDLE2-HA, we observed a single band with an apparent molecular weight of 31 kDa (Figure 5E, the predicted molecular weight for full-length MEDLE2-HA is 26.9 kDa but the abundance of positive charges is likely to result in reduced electrophoretic mobility). Protein KPVLKN/6A, carrying a mutation that did not affect trafficking to the host cell cytoplasm, was of identical size to WT MEDLE2-HA. In contrast, the KDVSLI/6A mutant, which is no longer exported, appeared to be of a larger molecular weight (Figure 5E). The mutant lacking the 22 amino acid signal peptide (ΔSP) produced an intermediate size band larger than the exported WT but smaller than the retained ΔKDVSLI mutant. We found a very similar pattern when analyzing the single amino acid mutants, where the L35A change caused the mutant protein to migrate more slowly when compared to WT or the ΔSP mutant (Figure 5F). To ensure the observed differences in apparent molecular weight were due to processing by the parasite and not the consequence of folding or subsequent host processing, we also expressed WT and mutants in mammalian cells (see Materials and methods for detail). In this context, the proteins are the same size (Figure 5—figure supplement 2). Overall, we interpret the relative sizes of the mutated proteins to indicate processing of MEDLE2 at a point beyond the signal peptide, a position that would be consistent with leucine 35. We note that this processing appears to require translocation into the ER as it does not occur in mutants lacking a signal peptide. Furthermore, the L35A mutation apparently prevented removal of the signal peptide, suggesting that processing at L35 could replace the canonical signal peptidase activity.

MEDLE2 induces an ER stress response in the host cell

To begin to understand the consequence of MEDLE2 export on the host cell, we expressed the protein in human cells. MEDLE2 omitting the N-terminal signal peptide (aa 2–20) was codon optimized for human cell expression, appended to the N-terminus of GFP, and the resulting plasmid introduced into HEK293T cells by Lipofectamine transfection (we note that this protein contains 15 amino acids [aa 21–35] that are likely missing in the parasite exported protein due to N-terminal processing). A GFP-only parent plasmid served as control for the impact of transfection on cellular responses. Transfection with both constructs resulted in cytoplasmatic green fluorescence in roughly 40% of cells 24 hr post transfection (Figure 6A). GFP-positive cells were enriched by flow cytometry and the resulting populations were subjected to mRNA sequencing (three biological repeats for each sample, Figure 6B). Differential gene expression analysis revealed 413 upregulated genes and 487 genes with lower transcript abundance in MEDLE2-GFP-expressing cells compared to cells expressing GFP alone, with an adjusted p-value less than 0.05 (Figure 6C). Gene set enrichment analysis (GSEA) detected changes in multiple pathways following introduction of MEDLE2 to host cells (Figure 6—source data 1). Notably, many of these pathways converged upon translation, ribosomes, amino acid metabolism, and associated signaling (Figure 6—source data 1). Functional annotation clustering using the Database for Annotation, Visualization and Integrated Discovery (DAVID) of the GSEA results revealed multiple responses contributing to ER stress, including changes in genes linked to the unfolded protein response (UPR). Genes that are part of the core enrichment of the ER stress response are highlighted (red) in the volcano plot, and the most upregulated genes in the ER stress signaling pathway identified by GSEA are identified by name (Figure 6D). Genes that were differentially expressed at an adjusted p-value less than 0.01 were used to derive a MEDLE2 response signature from the transfected cell dataset (234 genes). We next sought to explore whether the MEDLE2 gene signature observed following cell transfection was also present during Cryptosporidium infection using a published mRNA sequencing dataset generated from C. parvum infection of a homeostatic mini-intestine model (Nikolaev et al., 2020). We found enrichment for this MEDLE2 response signature with 51 genes of the MEDLE2 response present, and 22 of them contributing to the core enrichment of this response (Figure 6E, Figure 6—source data 2). As further validation that ER stress responses also occur during in vivo infection, we performed qPCR on ileal segments resected from mice infected with C. parvum or those that were uninfected. We measured the RNA abundance for the four genes highlighted in the volcano plot and found three to be upregulated in infected mice compared to uninfected controls (NUPR1, CHAC1, DDIT3, Figure 6F).

Figure 6 with 2 supplements see all
MEDLE2-expressing cells exhibit upregulation of genes involved in the unfolded protein response.

(A) HEK293T cells were transfected with plasmids encoding MEDLE2-GFP or GFP alone. After 24 hr, cells were fixed and processed for immunofluorescence assay (IFA). GFP is shown in green, Hoechst in blue. (B) 24 hr post transection, HEK293T cells were trypsinized and double sorted for live, GFP+ singlets directly into RNA lysis buffer and subjected to RNA sequencing. (C) Heat map depicting the differential gene expression between MEDLE2-GFP (top panel) and GFP control expressing cells (bottom panel). Upregulated gene expression is shown in red (row Z score > 0), while blue shows genes that are downregulated in expression (row Z score < 0). Expressing cells compared to GFP control cells. 413 transcripts showed upregulation in MEDLE2-GFP-expressing cells (right) and 487 genes had lower transcript abundance (left). The horizontal dashed line indicates p-value = 0.05. Gene set enrichment analysis (GSEA) performed on the 900 differentially expressed genes from the MEDLE2 transfection dataset identifies core enrichment of 20 genes that belong to ER stress response signaling pathways, which are indicated on the volcano plot in red. The most upregulated genes are identified by their gene ID. (E) The 234 genes with the greatest differential expression (p<0.01, log fold change absolute value > 1.5) were used to define a MEDLE2 gene set from the MEDLE2-GFP transfection dataset. This signature was used to perform GSEA using data from single-cell RNA sequencing on C. parvum-infected organoid-derived cultures, which showed enrichment of 51 genes with 22 genes in the core enrichment for the MEDLE2 response set highlighted in solid red. We note that we did not detect the MEDLE2 response signature in datasets from other enteric infections including rotavirus (Figure 6—figure supplement 1). (F) Ileal sections were removed from C. parvum-infected Ifng-/- mice and uninfected controls (each n = 3), and expression levels for the four differentially upregulated genes in the MEDLE2 response set (NUPR1, CHAC1, DDIT3, and TRIB3) were measured by qPCR. (G) HCT-8 cultures were pretreated for 2 hr with inhibitors (GSK2606414 and KIRA6) of ER stress signaling pathways prior to infection with 10,000 MEDLE2-HA parasites. After 24 hr, cells were lysed and nanoluciferase assay was performed as a measure of parasite growth. Inhibition of the IRE1 signaling pathway with KIRA6 significantly reduced parasite growth (one-way ANOVA, Dunnett’s multiple comparisons test p=0.0303; a representative experiment is shown [n = 6]). This experiment was repeated three times. (H) Ddit3-/- and C57BL/6J mice were treated with anti-mouse-IFN gamma antibody 1 day prior to infection with 10,000 MEDLE2-HA-tdNeon oocysts, and again at day 2 of infection. Fecal luminescence was determined by nanoluciferase activity to calculate the area under the curve for the duration of the infection. Ddit3-/- mice exhibited a 56% reduction in infection (1,416,227 ± 44,850; total peak area ± standard error) compared to control mice (3,189,123 ± 69,887; unpaired t test, p<0.001; n = 4 mice per group). One representative experiment is shown, which was repeated two more times, with a 54% reduction and no change in infection being observed.

We next wanted to explore whether the observed ER stress response impacts on parasite survival. HCT-8 cell cultures were treated with chemical inhibitors of the PERK (GSK2606414) and IRE1 (KIRA6) UPR signaling pathways and infected with nanoluciferase expressing C. parvum parasites. After 24 hr, cultures treated with KIRA6 showed reduced parasite growth when compared to the DMSO vehicle control (one-way ANOVA, Dunnett’s multiple comparisons test p=0.0303). Thapsigargin and GSK2606414 treatment did not affect parasite growth (Figure 6G). To test whether manipulation of the ER stress signaling pathways impacts parasite growth in vivo, we infected Ddit3-/- mice with MEDLE2-HA C. parvum parasites and monitored fecal relative luminescence as a measure of parasite shedding. C. parvum infection of Ddit3-/- mice resulted in a 56% reduction in the area under the curve compared to infection in C57BL/6J control mice (3,189,123 ± 69,887, 1,416,227 ± 44,850; total peak area ± standard error; unpaired t test, p<0.001; Figure 6H). Taken together, the ER stress response triggered by ectopic expression of MEDLE2 in mammalian cells is also observed during parasite infection in culture and mice and may contribute to parasite survival.

Discussion

Intestinal cryptosporidiosis in animals and humans is caused by parasites that are morphologically indistinguishable, and therefore were initially described as a single taxon, C. parvum. Extensive population genetic studies have since replaced C. parvum with a multitude of species, subspecies, and strains (Checkley et al., 2015; Xiao et al., 1999). The genomes of these parasites reflect the overall high degree of similarity in their conservation of gene content and synteny; nonetheless, these parasites show pronounced differences in their host specificity (Feng et al., 2017; Feng et al., 2018; Nader et al., 2019). The genomic differences observed are focused on families of predicted secretory proteins that have been proposed to contribute to host specificity, prominently among them the MEDLE family (Fei et al., 2018; Li et al., 2017; Su et al., 2019). Previous studies found that treatment of sporozoites with antisera raised against recombinant MEDLE1 or MEDLE2 diminished the efficiency of infection by 40%. This led the authors to consider that MEDLE proteins may play a role in invasion and that host specificity is rooted in receptor specificity (Fei et al., 2018; Li et al., 2017). In this study, we screened polymorphic genes for the localization of the proteins they encode by epitope tagging the endogenous loci. We found that C. parvum exports multiple members of the MEDLE protein family into the cytosol of the host cell. This is particularly robust for the highly expressed MEDLE2 protein, where the observation is based on more than 15 independent transgenic strains using different epitope tags and antibodies, in locus and ectopic tagging, and held true in cultured cells and infected animals. Importantly, mutation of the tagged gene changed the localization and molecular weight of the protein, highlighting the specificity of the reagents used.

Apicomplexa evolved multiple mechanisms to deliver proteins to their host cells during and following invasion. The timing of MEDLE2 expression and export and its sensitivity to BFA treatment argues for a mechanism that becomes active after the parasite has established its intracellular niche, in contrast to injection during invasion from organelles already poised to secrete. In P. falciparum, such export relies on the translocon complex PTEX (de Koning-Ward et al., 2009), which uses the ATPase activity of a heat shock protein to unfold and then extrude cargo through a transmembrane channel into the red blood cell (Ho et al., 2018; Matthews et al., 2019). Genetic ablation of this complex results in loss of export and loss of parasite viability (Beck et al., 2014; Elsworth et al., 2014). In T. gondii, proteins are exported during intracellular growth via the MYR complex, which is independent of ATP hydrolysis and requires intrinsically disordered cargo (Hakimi et al., 2017). Translocon and export are dispensable for growth of this parasite in culture, but loss profoundly impacts virulence in vivo (Hakimi et al., 2017; Marino et al., 2018). With the exception of a putative HSP101 chaperone, genome searches do not readily identify C. parvum homologs of the components that make up these two previously characterized translocons (Pellé et al., 2015), but nonetheless, there are important parallels that suggest conserved features of Apicomplexan export.

We demonstrate that MEDLE2 export depends on an N-terminal signal peptide and a leucine residue at position 35. Point mutation of this residue ablates export and results in a higher apparent molecular weight of the protein, which we interpret as a lack of processing at the mutated site. Disorder may be critical for MEDLE2 export as fusion of well-folded domains potently blocked its export into the host cell. This is consistent with an export mechanism unable to unfold proteins as proposed for T. gondii and Plasmodium liver stages (Beck and Ho, 2021; Gehde et al., 2009; Marino et al., 2018).

Export of proteins in P. falciparum and T. gondii similarly requires a host-targeting motif (Coffey et al., 2015; Hiller et al., 2004; Marti et al., 2004). While the sequence of the motifs varies between species, all share a required leucine residue that has been linked to processing by an aspartyl protease to license export (Boddey et al., 2010; Coffey et al., 2015; Hammoudi et al., 2015; Russo et al., 2010). This protease localizes to the ER in P. falciparum where it appears to replace the activity of signal peptidase for exported proteins, while the T. gondii homolog acts in the Golgi (Coffey et al., 2015; Marapana et al., 2018). Our mutational analysis in C. parvum suggests that cleavage of L35 could replace the activity of signal peptidase similar to P. falciparum. The C. parvum genome encodes six putative aspartyl proteases, three of which share significant similarity with the Plasmepsin V/ASP5 enzymes responsible for processing exported proteins in P. falciparum and T. gondii. Ablating these genes may provide an opportunity to block export to the host cell.

Pathogens inject a wide array of factors into the cells of their hosts to remodel cellular architecture, hijack death and survival pathways, change cell physiology and metabolism, and rewire transcriptional networks. Many of them aid immune evasion and the evolutionary arms race between host and pathogen and are potent modulators of host specificity (Long et al., 2019; van der Does and Rep, 2007). Here, we have identified the first examples of host-targeted effector proteins for intracellular stages of C. parvum. We introduced the protein into its target compartment, the mammalian cytosol, by transient transfection, and mRNA sequencing revealed a transcriptional signature of ER stress and UPR upon expression of MEDLE2, but not in matched control transfection with a GFP-only-expressing plasmid. We found that an ER stress response signature was also detectable when cells or mice were infected with C. parvum, which supports previous work identifying a UPR response during C. parvum infection in an in vitro model of infection (Morada et al., 2013).

ER stress is a common feature of intracellular infection that greatly modulates host cell survival and inflammatory response. Bacteria, viruses, and protozoa alike have evolved strategies to trigger or block the UPR during infection, suggesting the response not to be a by-product of infection, but rather an active participant (Abhishek et al., 2018; Alshareef et al., 2021; Perera et al., 2017). This relationship is complex, and depending on cellular context, the UPR can aid pathogens or restrict them. Modulating the UPR is pivotal for intracellular bacteria that reside in vacuoles, including Chlamydia, Brucella, Listeria, and Mycobacterium, to build their intracellular niche and to acquire nutrients (Celli and Tsolis, 2015). Cryptosporidium lost the ability to synthesize most required metabolites and depends on an elaborate membranous feeder organelle at the host-parasite interface. This reduction includes biosynthesis of many lipids (Coppens, 2013), and we note modulation of lipid synthesis and mobilization as one important consequence of the UPR (Moncan et al., 2021). The UPR is also important in the context of recognition of intracellular pathogens and the subsequent cytokine-mediated immune responses. Cryptosporidium triggers an enterocyte intrinsic inflammasome leading to the release of IL-18 from the infected cell (McNair et al., 2018), and infection also results in pronounced production of interferon λ (Ferguson et al., 2019). We also tested GRA16, a disordered effector protein of T. gondii in similar experiments and found it to similarly upregulate the ER stress response. This may indicate that such upregulation is inherent to the structure of these proteins (Figure 6—figure supplement 2). However, we note that manipulation of ER stress signaling using pharmacological inhibition of IRE1 in culture or genetic ablation of DDIT3 in mice resulted in reduced growth of C. parvum, suggesting that this response may contribute to parasite survival. We deem it likely that MEDLE2 has additional roles yet to be determined.

The MEDLE genes are highly polymorphic and encoded by multiple loci, but our current view of the family likely underestimates its true plasticity. The recent reannotation of the C. parvum genome using long read sequencing found evidence for multiple ‘alternative’ telomeres carrying different MEDLE gene clusters (Baptista et al., 2021). Our experimental observations support this. When we ablated the MEDLE2 gene by homologous recombination, additional MEDLE2 copies remained in the genome (Figure 1—figure supplement 2). Similar observations were made when tagging the gene (Figure 1B, Figure 2—figure supplement 1). It is important to note that Cryptosporidium has a single host life cycle and undergoes sexual exchange throughout the infection, providing near constant opportunity for recombination (Tandel et al., 2019). This is reminiscent of the importance of chromosomal position and recombination for the antigenically varied VSG and var genes in Trypanosoma brucei and P. falciparum, respectively (Dreesen et al., 2007; Zhang et al., 2019). The fact that MEDLE proteins are delivered into the host cell makes them prime targets for immunity, and immune evasion may drive chromosomal organization, expression, and evolution of these genes.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Cryptosporidium parvum)MEDLE2Li et al., 2017cgd5_4590
Gene (C. parvum)MEDLE1Fei et al., 2018cgd5_4580
Gene (C. parvum)MEDLE6This papercgd6_5490Named according to the spatial localization in the genome similarly to MEDLE2 and MEDLE1
Gene (C. parvum)WYLE4This papercgd8_3560Named for the gene family and according to the spatial localization in the genome
Gene (C. parvum)SKSR7This paperCgd8_30Named for the gene family and according to the spatial localization in the genome
Strain, strain background (C. parvum)C. parvum oocysts, IOWAII strain (WT)Bunchgrass
Strain, strain background (Mus musculus)Ifng-/-, C57BL/6JThe Jackson LaboratoryJax 002287;
RRID:IMSR_JAX:002287
Strain, strain background (M. musculus)Ddit3tm2.1DronThe Jackson LaboratoryJax 005530;
RRID:IMSR_JAX:005530
Strain, strain background (M. musculus)C57BL/6JThe Jackson LaboratoryJax 000664;
RRID:IMSR_JAX:000664
Genetic reagent (C. parvum)MEDLE2-HAThis papercgd5_4590 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)WYLE4-HAThis papercgd8_3570 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)SKSR7-HAThis papercgd8_30 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)MEDLE2 KOThis paperCgd5_4590Stable transgenic parasite line with one copy of MEDLE2 knocked out
Genetic reagent (C. parvum)MEDLE1-HAThis papercgd5_4580 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)MEDLE6-HAThis papercgd6_5490 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)Medle2 MEDLE1-HAThis papercgd5_4440 modifiedStable transgenic parasite line expressing HA
Genetic reagent (C. parvum)MEDLE2-HA-tdNeonThis papercgd5_4590 modifiedStable transgenic parasite line expressing HA and tdNeon
Genetic reagent (C. parvum)MEDLE2-mScarletThis papercgd5_4590 modifiedStable transgenic parasite line expressing mScarlet
Genetic reagent (C. parvum)MEDLE2-Bla-2A-tdTomatoThis papercgd5_4590 modifiedStable transgenic parasite line expressing BLA and tdTomato
Genetic reagent (C. parvum)MEDLE2-CreThis papercgd5_4590 modifiedStable transgenic parasite line expressing Cre recombinase
Genetic reagent (C. parvum)Ectopic MEDLE2-HAThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2
Genetic reagent (C. parvum)ΔSPThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 (aa 21–209)
Genetic reagent (C. parvum)KDVSLI/6AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 31–36) mutated to six alanines
Genetic reagent (C. parvum)KPVLKN/6AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with KPVLKN (aa 73–78) mutated to six alanines
Genetic reagent (C. parvum)KNVNLS/6AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 77–82) mutated to six alanines
Genetic reagent (C. parvum)RGLLRGLSG/9AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 191–199) mutated to six alanines
Genetic reagent (C. parvum)K31AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with K31 mutated to alanine
Genetic reagent (C. parvum)D32AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with D32 mutated to alanine
Genetic reagent (C. parvum)V33AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with V33 mutated to alanine
Genetic reagent (C. parvum)S34AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with S34 mutated to alanine
Genetic reagent (C. parvum)L35AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with L35 mutated to alanine
Genetic reagent (C. parvum)I36AThis papercgd5_4440 modifiedStable transgenic parasite line expressing extra copy of MEDLE2 with I36 mutated to alanine
Cell line (human)HCT-8ATCCCCL-224;
RRID:CVCL_2478
Cell line (human)HEK293TATCCCRL-3216;
RRID:CVCL_0063
Cell line (Escherichia coli)GC5Genesee
Scientific
42-653Electrocompetent cells
Cell line (E. coli)One Shot
Topo10,
InvitrogenC404003Electrocompetent cells
Transfected construct (human)loxP GFP/RFP color switch lentivirusGenTarget IncCat#: LVP460-NeoTransfected construct (human)
Biological sample (M. musculus)Isolated sections of ileumIfng-/- miceJax 002287;
RRID:IMSR_ JAX:002287
6-week-old male mice
AntibodyAnti-HA (rat monoclonal)MilliporeSigmaCat# 11867431001; RRID:AB_390919IF (1:500), WB (1:500), IHC (1:500)
AntibodyAnti-Cp23 (mouse monoclonal)LS BioCat# LS-C137378; RRID:AB_10947007IF (1:100)
AntibodyAnti-alpha tubulin (mouse monoclonal)Developmental Studies Hybridoma BankCat#12G10; RRID:AB_1157911IF (1:1000)
AntibodyAnti-COWP1 (rat monoclonal)This paper, produced by GenScriptThis paperIF (1:100)
AntibodyAnti-neomycin phosphotransferase II (rabbit polyclonal)MilliporeSigmaMillipore Cat# 06-747; RRID:AB_310234WB (1:1000)
AntibodyAnti-mouse IFN gammaBio X CellClone: XMG1.2;
Cat# BE0055; RRID:AB_1107694
In vivo 100 µg
AntibodyGoat anti-rat polyclonal Alexa Fluor 594Thermo Fisher ScientificCat# A-21213; RRID:AB_2535799IFA (1:500)
AntibodyGoat anti-mouse polyclonal Alexa Fluor 488Thermo Fisher ScientificCat# A-11001; RRID:AB_2534069IFA (1:500)
Strain, strain backgroundAlexa Fluor 647 PhalloidinThermo Fisher ScientificA22287;
RRID:AB_2620155
IFA (1:1000)
AntibodyIRDye 800CW goat anti-rat IgGLI-COR926-32219;
RRID:AB_1850025
WB (1:10,000)
AntibodyIRDye 680RD goat anti-rabbit IgGLI-COR926-68071;
RRID:AB_2721181
WB (1:10,000)
Recombinant DNA reagentCas9 cgd5_4590 (plasmid)This paperGuide targeting C terminus of MEDLE2
Recombinant DNA reagentCas9 cgd8_3560 (plasmid)This paperGuide targeting C terminus of WYLE4
Recombinant DNA reagentCas9 cgd8_30 (plasmid)This paperGuide targeting C terminus of SKSR7
Recombinant DNA reagentCas9 cgd5_4580 (plasmid)This paperGuide targeting C terminus of MEDLE1
Recombinant DNA reagentCas9 Cgd6_5490 (plasmid)This paperGuide targeting C terminus of MEDLE6
Recombinant DNA reagentCas9 Tk guide int (plasmid)Tandel et al., 2019Guide targeting internal cgd5_4440
Recombinant DNA reagentCas9 MEDLE2
KO (plasmid)
This paperGuide targeting internal MEDLE2
Recombinant DNA reagentLic HA
(plasmid)
This paperCrypto expression vector for HA tagging
Recombinant DNA reagentLic tdTomato
KO (plasmid)
This paperCrypto expression vector for replacing gene KO with tdTomato
Recombinant DNA reagentLic medle2-MEDLE1 HA (plasmid)This paperCrypto expression vector with medle2 promoter driving MEDLE1-HA expression
Recombinant DNA reagentLic HA-2A-
TdNeon
(plasmid)
This paperCrypto expression vector for HA tagging and cytoplasmic tdNeon
Recombinant DNA reagentLic mScarlet (plasmid)This paperCrypto expression vector for mScarlet tagging
Recombinant DNA reagentLic Bla-2A-TdTomato
(plasmid)
This paperCrypto expression vector for BLA tagging and cytoplasmic tdTomato
Recombinant DNA reagentLic Cre
(plasmid)
This paperCrypto expression vector for Cre tagging
Recombinant DNA reagentLic Extra
MEDLE2-HA (plasmid)
This paperCrypto expression vector for extra copy of MEDLE2-HA (MEDLE2 promoter)
Recombinant DNA reagentLic ΔSP MEDLE2-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA (aa 21–209)
Recombinant DNA reagentLic KDVSLI/6A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with KDVSLI (aa 31–36) mutated to six alanines
Recombinant DNA reagentLic KPVLKN/6A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with KPVLKN (aa 73–78) mutated to six alanines
Recombinant DNA reagentLic KNVNLS/6A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with KNVNLS (aa 77–82) mutated to six alanines
Recombinant DNA reagentLic RGLLRGLSG/9A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with RGLLRGLS (aa 191–199) mutated to six alanines
Recombinant DNA reagentLic K31A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with K31 mutated to alanine
Recombinant DNA reagentLic D32A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with D32 mutated to alanine
Recombinant DNA reagentLic V33A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with V33 mutated to alanine
Recombinant DNA reagentLic S34A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with S34 mutated to alanine
Recombinant DNA reagentLic L35A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with L35 mutated to alanine
Recombinant DNA reagentLic I36A-HA (plasmid)This paperCrypto expression vector for extra copy of MEDLE2-HA with I36 mutated to alanine
Recombinant DNA reagentmEGFP-Lifeact-7 (plasmid)Addgene# 54610Used as a mammalian expression vector to clone codon optimized MEDLE2 into
Recombinant DNA reagentGFP-onlyThis paperRemoved Lifeact domain from Addgene plasmid #54610 for a GFP-only control plasmid
Recombinant DNA reagentRecod MEDLE2-GFP (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a GFP tag
Recombinant DNA reagentGRA16-GFPThis paperT. gondii GRA16 (aa 24–505) with GFP tag.
Recombinant DNA reagentRecod MEDLE2-HA (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a HA tag.
Recombinant DNA reagentRecod KDVSLI/6A-HA (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a HA tag and KDVSLI (aa 31–36) mutated to six alanines
Recombinant DNA reagentRecod KPVLKN/6A-HA (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a HA tag and KPVLKN (aa 73–78) mutated to six alanines
Recombinant DNA reagentRecod KNVNLS/6A-HA (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a HA tag and KNVNLS (aa 77–82) mutated to six alanines
Recombinant DNA reagentRecod RGLLRGLSG/9A-HA (plasmid)This paperHuman codon optimized MEDLE2 (aa 21–209) with a HA tag and RGLLRGLS (aa 191–199) mutated to six alanines
Sequence-based
reagent
Recodonized MEDLE2Integrated DNA TechnologiesMEDLE2 (aa 21–209) codon optimized for human expressionSee Supplementary file 1 for sequence
Sequence-based
reagent
PCR primersThis paperPlease see Supplementary file 1
Commercial assay or kitDNeasy Blood & Tissue KitQIAGENCat# 69504
Commercial assay or kitZymoPureII Plasmid Maxiprep KitZymo ResearchCat# 11-555B
Commercial assay or kitNano-Glo Luciferase Assay SystemPromegaCat# N1130
Commercial assay or kitSF Cell Line 4D X Kit LLonzaCat# V4XC-2024
Commercial assay or kitLiveBLAzer FRET-B/G Loading KitThermo FisherCat# K1095
Commercial assay or kitZeroBlunt TopoTA KitInvitrogenCat# 450245
Commercial assay or kitRNeasy MicrokitQIAGENCat# 74004
Commercial assay or kitSMART cDNA synthesis kitTakaraCat# 635040
Commercial assay or kitNextera XT DNA Library Prep KitIlluminaCat# FC-131-1096
Commercial assay or kitRNeasy MiniKitQIAGENCat# 74104
Commercial assay or kitQIAshredderQIAGENCat# 79656
Commercial assay or kitSuperScript First Strand Synthesis kitThermo FisherCat#18091050
Commercial assay or kitLipofectamine 3000Thermo FisherCat# L3000015
Chemical compound, drugParomomycinGeminiCat# 400-155PUsed 16 g/L water
Chemical compound, drugBrefeldin A (BFA)BioLegendCat# 420601Used 10 µg/mL
Chemical compound, drugThapsigarginMedChemExpressHY-13433Used 1 µM
Chemical compound, drugGSK2606414MedChemExpressHY-18072Used 30 nm
Chemical compound, drugKIRA6MedChemExpressHY-19708Used 500 nm
Software, algorithmPrism 8GraphPadRRID:SCR_002798
Software, algorithmImageJFijiRRID:SCR_003070
Software, algorithmFlowJo v10, LLCTreeStarRRID:SCR_008520
Software, algorithmKallisto v0.44.0BioConductor (Bray et al., 2016)Pachter Lab
Software, algorithmLimma-VoomBioConductor (Law et al., 2014; Ritchie et al., 2015)
Software, algorithmBioconductor tximportBioConductor (Robinson et al., 2010)DOI: 10.18129/B9.bioc.tximport
Software, algorithmMolecular Signatures Database (MSigDB)UC San Diego and Broad Institute (Mootha et al., 2003; Subramanian et al., 2005)https://www.gsea-msigdb.org/gsea/msigdb
Software, algorithmCryptoDBVEuPathDBcryptodb.org
OtherFluorescin Vicia villosa lectin stainVector LabsCat# FL-1231-2IF (1:1000)
OtherDAPI stainInvitrogenCat# D1306Flow cytometry (1 µg/mL)
OtherHoechst 33342Thermo FisherCat# H3570IF (1:10,000)
OtherAlexa Fluor 647 PhalloidinThermo FisherCat# A22287IF (1:1000)

Contact for reagent and resource sharing

Request a detailed protocol

For access to reagents or parasite strains used in this study, please contact Dr. Boris Striepen: Tel.: 1-215-573-9167; fax: 1-215-746-2295; e-mail: striepen@upenn.edu.

Mouse models of infection

Request a detailed protocol

Ifng-/- (stock no: 002287; RRID:IMSR_JAX:002287) were purchased from Jackson Laboratory and maintained as a breeding colony at the University of Pennsylvania. All mice used in this study ranged from 4 to 12 weeks of age. We note that both male and female Ifng-/- mice were used to generate and propagate C. parvum transgenic parasite lines, without a difference being noted in parasite shedding. Ddit3-/- (stock no: 005530) and C57BL/6J (stock no: 000664) mice were purchased from Jackson Laboratory. All protocols for animal care were approved by the Institutional Animal Care and Use Committee of the University of Georgia (protocol A2016 01-028-Y1-A4) and the Institutional Animal Care and Use Committee of the University of Pennsylvania (protocol #806292).

Cell lines

Request a detailed protocol

HCT-8 (ATCC: CCL-224TM; RRID:CVCL_2478) and HEK293T (ATCC: CRL-3216TM; RRID:CVCL_0063) cell lines were purchased from ATCC, mycoplasma tested, and used for all experiments once confirmed to be mycoplasma negative.

Parasite strains

Request a detailed protocol

C. parvum transgenic strains were made and propagated in Ifng-/- mice (stock no: 002287; RRID:IMSR_JAX:002287). Oocysts were then purified from fecal collections using sucrose flotation followed by a cesium chloride gradient (see Materials and methods). All C. parvum oocysts used in this study as WT controls, as well as to generate transgenic strains, are on the IOWAII strain background, purchased from Bunchgrass Farms (Dreary, ID).

Plasmid construction

Request a detailed protocol

Guide oligonucleotides (Sigma-Aldrich, St. Louis, MO) were introduced into the C. parvum Cas9/U6 plasmid by restriction cloning, as detailed in Pawlowic et al., 2017. All plasmids encoding epitope tags, as well as for ectopic MEDLE2 expression, were constructed by Gibson assembly using NEB Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA). A linear repair template was generated by PCR. See Supplementary file 1 for a complete list of primers used for this study.

Generation of transgenic parasites

Request a detailed protocol

Transgenic parasites were derived as previously described (Sateriale et al., 2020). Briefly, 5 × 107 C. parvum oocysts were bleached on ice, washed in 1× PBS, and incubated in sodium taurodeoxycholate. Excysted sporozoites were resuspended in transfection buffer supplemented with a total of 100 µg DNA (comprising 50 µg of Cas9/gRNA plasmid and 50 µg of repair template generated by PCR) and nucleofected using an Amaxa 4D nucleofector (Lonza, Basel, Switzerland). Transfected parasites were resuspended in PBS and administered to Ifng-/- mice. Mice were pretreated with antibiotics for 1 week preceding infection and with sodium bicarbonate immediately before parasite administration (Sateriale et al., 2020). Mice received 16 mg/mL paromomycin in drinking water for selection. Transgenic parasites were detected by measuring fecal nanoluciferase activity and purified from feces using sucrose flotation followed by a cesium chloride gradient and stored in PBS at 4°C (Sateriale et al., 2020).

Nanoluciferase assay to monitor parasite shedding

Request a detailed protocol

20 mg of fecal material was dissolved in nanoluciferase lysis buffer and mixed 1:1 with nanoluciferase substrate/nanoluciferase Assay Buffer (1:50) in a white-bottom plate. Relative luminescence was read using a Promega GloMax Plate Reader.

Integration PCR to confirm generation of transgenic parasites

Request a detailed protocol

DNA was purified from excysted sporozoites using the QIAGEN DNeasy Blood and Tissue kit (QIAGEN 69504). PCR primers were designed to anneal outside of the 5′ and 3′ homology arms used to direct homologous recombination and matched with primers annealing to the nanoluciferase reporter gene or the neomycin selection marker, respectively. Primers for the thymidine kinase gene served as control, unless otherwise noted. Where indicated, amplicons were cloned using the ZeroBlunt TopoTA kit (Invitrogen 450245) and transformed into One Shot Topo10 Chemically Competent Escherichia coli (Invitrogen C404003). Individual colonies were miniprepped and sequenced.

In vitro infection and immunofluorescence assay

Request a detailed protocol

Coverslips seeded with human ileocecal adenocarcinoma cells (HCT-8) (ATCC CCL-244; RRID:CVCL_2478) were infected when 80% confluent with 200,000 purified oocysts (bleached, washed, and resuspended in RPMI medium containing 1% serum). For time-course infections, parasites were allowed to invade for 3 hr, then medium was removed, and the cells were washed with PBS to remove unexcysted oocysts and replaced with fresh RPMI medium with 1% serum. At indicated time points, cells were washed with PBS, and successively fixed and permeabilized with PBS supplemented with 4% paraformaldehyde or 0.1% Triton X-100 for 10 min each (Sigma). Coverslips were blocked with 1% bovine serum albumin (BSA) (Sigma). Antibodies were diluted in blocking solution. The was rat monoclonal anti-HA (MilliporeSigma, Burlington, MA; RRID:AB_390919) and was used as primary antibody (1:500) and goat anti-rat polyclonal Alexa Fluor 594 (Thermo Fisher, Waltham, MA; RRID:AB_2535799) as secondary along with V. villosa lectin (Vector Labs Burlingame, CA). Host and parasite nuclei were stained with Hoechst 33342 (Thermo Fisher). Slides were imaged using a Zeiss LSM710 Confocal microscope or a Leica Widefield microscope. Experimental slides were prepared and imaged in duplicate for a minimum of two biological replicates.

Immunohistochemistry on infected intestine

Request a detailed protocol

Infected Ifng-/- mice (RRID:IMSR_JAX:002287) were euthanized at day 12 during peak infection, and the distal 1/3 of the small intestine was dissected. The tissue was washed with PBS and ‘swiss-rolled’ and fixed overnight in 4% paraformaldehyde at 4°C, placed in 30% sucrose in PBS for cryoprotection, and mounted with OCT compound (Tissue-Tek, Sakura Finetek, Japan) and frozen. Cryomicrotome sections were permeabilized and blocked and labeled as described above (Alexa Fluor 647 Phalloidin [RRID:AB_2620155; Thermo Fisher] was used in addition). Sections were prepared in duplicate and imaged using a Zeiss LSM710 Confocal Microscope.

Poly-L-lysine treatment of coverslips and sporozoite IFA

Request a detailed protocol

Sterile coverslips were treated with poly-L-lysine (Sigma), washed with water for 5 min, and airdried. Sporozoites suspended in PBS were allowed to settle on treated coverslip for 1 hr prior to fixing and IFA. Primary antibodies used were mouse anti-Cp23 (1:100) (LS Bio Seattle, WA; RRID:AB_10947007) and rat monoclonal anti-HA (RRID:AB_390919) (1:500). Secondary antibodies include goat anti-rat polyclonal Alexa Fluor 594 (RRID:AB_2535799)and goat anti-mouse polyclonal Alexa Fluor 488 (RRID:AB_2534069) (both 1:1000, Thermo Fisher).

BFA treatment during C. parvum infection

Request a detailed protocol

Excysted oocysts were allowed to invade HCT-8 coverslip cultures (RRID:CVCL_2478) for 3 hr, unexcysted oocysts were removed by PBS wash, and cultures were replaced with medium supplemented with 1% serum and 10 µg/mL BFA from a 1000× stock in DMSO. Medium supplemented with carrier alone served as control. Cultures were fixed and processed 10 hr post infection.

Live imaging of beta-lactamase reporter assay

Request a detailed protocol

1 × 106 WT and MEDLE2-BLA oocysts were used to infect HCT-8 cells (RRID:CVCL_2478) in a 35 mm glass-bottom dish (MatTek Life Sciences, Ashland, MA). After 24 hr, the medium was replaced with RPMI medium containing CCF4-AM substrate from the LiveBLAzer FRET-B/G Loading Kit (Thermo Fisher Scientific). Cells were incubated in the dark at 37°C for 1 hr, washed with PBS three times, and live imaged using a Leica SP5 Confocal Microscope using a water immersion lens.

Cre recombinase reporter assay by flow cytometry

Request a detailed protocol

Pre-made lentivirus was used to transform HCT-8 cells (RRID:CVCL_2478) with a loxP GFP/RGP color switch cassette (GenTarget Inc, San Diego, CA). Cells were selected with 400 mg/mL neomycin (MilliporeSigma) for 14 days and validated by transfection with 5 µg Cre recombinase plasmid using Lipofectamine P3000 (Thermo Fisher Scientific). After 24 hr and 48 hr, cells were trypsinized and flow sorted using a LSRFortessa (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo v10 software (TreeStar) (RRID: SCR_008520).

1 × 106 WT and MEDLE2-Cre oocysts were used to infect six-well cultures of Lox GFP/RFP color switch cells. After 48 hr, cells were trypsinized and resuspended in 1 mL PBS. 300 µL were used for nanoluciferase assay and 700 µL cells for flow cytometry. Forward and side scatter was used to gate viability, untransfected uninfected cells to establish the green gate, and Cre recombinase transfected cells for the red gate (three biological replicates for each condition).

Western blot on C. parvum infected cells

Request a detailed protocol

HCT-8 cultures (RRID:CVCL_2478) infected with 5 × 106 oocysts for 48 hr were treated with Trypsin 0.25% EDTA (Thermo Fisher Scientific), pelleted, and flash frozen in liquid nitrogen. Cell pellets were lysed in Pierce IP Lysis Buffer (Thermo Fisher Scientific), supplemented 1:100 with both protease inhibitor cocktail (Sigma) and benzonase nuclease (MilliporeSigma). Lysates were incubated on ice for 15 min, sonicated (80% amplitude, 10 s pulses, rest on ice for 1 min between three times), cleared by centrifugation (20,000 × g, 10 min, 4°C), mixed with freshly prepared Laemmli Sample buffer (MilliporeSigma) + β-mercaptoethanaol (1:20) (Sigma), boiled and loaded on a 12% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad, Hercules, CA) run at 70 V for 2.5 hr. Gels were transferred to 0.45 µm pore size nitrocellulose membrane (Thermo Fisher Scientific) overnight at 0.02 A at 4°C. The membrane was blocked for 1 hr with Intercept (TBS) Protein-Free Blocking Buffer (LI-COR, Lincoln, NE), antibodies we diluted in blocking solution with 0.01% Tween20 (Sigma) using rat monoclonal anti-HA 1:500 (MilliporeSigma; RRID:AB_390919), and rabbit anti-neomycin phosphotransferase II 1:1000 (MilliporeSigma; RRID:AB_310234) as primary and IRDye 800CW goat anti-rat IgG (RRID:AB_1850025) and IRDye 680RD goat anti-rabbit IgG (RRID:AB_2721181) (both 1:10,000, LI-COR) as secondary antibody. Washed membranes were imaged using an Odyssey Infrared Imaging System v3.0 (LI-COR).

Generation of MEDLE2 mutant plasmids for host cell transfection

Request a detailed protocol

Human codon optimized MEDLE2 lacking the N-terminal signal peptide (aa 21–209) was synthesized by Integrated DNA Technologies (IDT, Coralville, IA) and cloned into the mEGFP-Lifeact-7 mammalian expression plasmid (Addgene #54610), replacing Lifeact-GFP and appending a 3× HA tag. Point mutations were engineered by Gibson cloning. HEK293T cells (ATCCCRL-3216; RRID:CVCL_0063) were transfected with 5 µg of each plasmid using Lipofectamine P3000 (Thermo Fisher Scientific). 24 hr post transfection, cells were harvested and processed for western blot analysis. Additionally, a MEDLE2-EGFP plasmid was cloned by Gibson cloning to introduce human codon optimized MEDLE2 lacking the N-terminal signal peptide (aa 21–209) into the same mEGFP-Lifeact-7 mammalian expression plasmid, replacing Lifeact. A GFP-only-expressing plasmid was engineered removing the Lifeact from Addgene plasmid #54610. Similarly, T. gondii GRA16 omitting the sequence encoding the N-terminal signal peptide (aa 24–505) was amplified from gDNA of T. gondii strain ME49 parasites and cloned into the mEGFP-Lifeact-7 mammalian expression plasmid in place of Lifeact.

Flow cytometry analysis of transfected cells

Request a detailed protocol

HEK293T cells (RRID:CVCL_0063) were subjected to lipofection with 25 µg GFP-only plasmid or MEDLE2-GFP plasmid, grown for 24 hr, trypsinized, washed, and resuspended in PBS with DAPI and passed through a 40 µM filter (BD Biosciences). Cell viability was gated based upon DAPI staining. Untransfected HEK293T served as negative control and GFP-expressing HEK293T cells as positive control to establish gates. 10,000 green, single cells were double sorted using an Aria C flow cytometer first into PBS then into lysis buffer (three biological replicates for each condition).

RNA extraction sequencing and data analysis

Request a detailed protocol

Total RNA was extracted using the QIAGEN RNeasy Microkit (QIAGEN, Germantown, MD) and input RNA was quality controlled and quantified using a Tape Station 4200 (Agilent Technologies, Santa Clara, CA). cDNA synthesis was performed following the clonTechSMART-seq cDNA synthesis protocol (15 cycles). Following DNA cleanup, a Nextera library was prepared and nucleic acid was quantified using the Qubit 3 Fluorometer (Thermo Fisher Scientific). Samples were pooled for RNA-sequencing of 4 nM of total cDNA, and sequencing was performed using a NextSeq 500 Instrument (Illumina Inc, San Diego, CA).

RNAseq reads were pseudo-aligned to the Ensembl Homo sapiens reference transcriptome v86 using kallisto v0.44.0 (Bray et al., 2016). In R, transcripts were collapsed to genes using Bioconductor tximport (Robinson et al., 2010) and differentially expressed genes were identified using Limma-Voom (Law et al., 2014; Ritchie et al., 2015). The MEDLE2 transcription response dataset can be found under GEO accession number GSE174117. GSEA was performed using the GSEA software and the annotated gene sets of the Molecular Signatures Database (MSigDB) (Mootha et al., 2003; Subramanian et al., 2005). The MEDLE2 signature was generated from the differentially expressed genes and read into GSEA to evaluate its presence in published datasets of C. parvum infection (Nikolaev et al., 2020; Saxena et al., 2017).

qPCR for MEDLE2 response genes from infected mice

Request a detailed protocol

8-week-old Ifng-/- mice (RRID:IMSR_JAX:002287) were infected with 10,000 C. parvum oocysts, and the infection was tracked by fecal nanoluciferase activity. Infected mice (n = 3) and uninfected controls (n = 3) were euthanized after 10 days, and the distal 1/3 of the small intestine was removed. The tissue was washed with 1× PBS until clear of fecal material and then cut longitudinally. 5 mm diameter gut punches were made and preserved in RNAlater solution (Thermo Fisher Scientific). RNA was extracted from tissue samples using the QIAGEN RNeasy MiniKit (QIAGEN) following homogenization with a bead beater and passage through a QIAshredder (QIAGEN). 5 µg of cDNA was reverse transcribed using the SuperScript First Strand Synthesis kit following the manufacturer’s instructions for use with OligoDT (Thermo Fisher Scientific). qPCR was performed using a Viia7 Real-time PCR System (Thermo Fisher Scientific), and relative gene expression was determined using the ΔΔCT method.

qPCR for MEDLE2 response genes in transfected cells

Request a detailed protocol

5 µg plasmid (GFP-only, MEDLE2-GFP, GRA16-GFP) was introduced to HEK293T cells (RRID:CVCL_0063) by Lipofectamine transfection. Cells were grown for 24 hr and then the medium was removed and replaced with RLT lysis buffer from the QIAGEN RNeasy MiniKit (QIAGEN) and passaged through a QIAshredder (QIAGEN) to begin the RNA extraction protocol. Untransfected HEK293T served as negative control. 5 µg of cDNA was reverse transcribed using the SuperScript First Strand Synthesis kit, and qPCR was performed using the methods described above with primers specific for GAPDH (control), GFP, CHAC1, DDIT3, NUPR1, and TRIB3.

In vitro growth assay in the presence of UPR inhibitors

Request a detailed protocol

HCT-8 cells (RRID:CVCL_2478) were grown to 80% confluency in 96-well plates. 2 hr prior to infection, the medium was removed from the plate and replaced with RPMI medium containing DMSO (vehicle), 1 µM thapsigargin, 30 nM GSK2606414, or 500 nM KIRA6, all from MedChemExpress (Monmouth Junction, NJ). Nanoluciferase expressing C. parvum parasites were excysted, and 10,000 parasites were used to infect each well. After 24 hr, medium was removed from the wells and replaced with nanoluciferase lysis buffer and incubated for 5 min. The lysate was then mixed 1:1 with nanoluciferase substrate/nanoluciferase Assay Buffer (1:50) in a white-bottom plate and relative luminescence read using a Promega GloMax Plate Reader.

Infection of DDIT3 KO mice

Request a detailed protocol

6–9-week-old Ddit3-/- (stock no: 005530; RRID:IMSR_JAX:005530) and C57BL/6J (stock no: 000664; RRID:IMSR_JAX:000664) mice were purchased from Jackson Laboratory. Mice were treated with 100 mg of InVivo Mab anti mouse-IFN gamma antibody Bio X Cell (Lebanon, NH; RRID:AB_1107694) 1 day prior to infection and again at day 2 of infection. Mice were infected with 10,000 MEDLE2-HA-tdNeon oocysts, and feces were collected every 2 days to measure fecal luminescence by nanoluciferase activity as described above.

Quantification and statistical methods

Request a detailed protocol

GraphPad Prism (RRID:SCR_002798) was used for all statistical analyses. When measuring the difference between two populations, a standard t-test was used. For datasets with three or more experimental groups, a one-way ANOVA with Dunnett’s multiple comparison test was used. Simple linear regression was used to determine the goodness-of-fit curve for the number of MEDLE2-expressing cells and intracellular parasites. Quantification of imaging experiments was performed using ImageJ (RRID:SCR_003070) macros programmed to count both parasites and host cell nuclei in blinded images that were captured using a scanning function to avoid bias during acquisition.

Data availability

The RNA sequencing dataset generated from the MEDLE2 transfection experiment has been deposited in GEO under accession number GSE174117. Source code and data files for this dataset were provided. Furthermore, numerical source data used for imaging quantification experiments in Figures 2 and 3 were provided.

The following data sets were generated
    1. Dumaine JE
    2. Sateriale A
    3. Gibson AR
    4. Reddy AG
    5. Gullicksrud JA
    6. Hunter EN
    7. Clark JT
    8. Striepen B
    (2021) NCBI Gene Expression Omnibus
    ID GSE174117. The enteric pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected host cell.
The following previously published data sets were used

References

    1. Gut J
    2. Nelson RG
    (1999)
    Cryptosporidium parvum: synchronized excystation in vitro and evaluation of sporozoite infectivity with a new lectin-based assay
    The Journal of Eukaryotic Microbiology 46:56S–57S.

Decision letter

  1. Olivier Silvie
    Reviewing Editor; Sorbonne Université, UPMC Univ Paris 06, INSERM, CNRS, France
  2. Dominique Soldati-Favre
    Senior Editor; University of Geneva, Switzerland
  3. Daniel E Goldberg
    Reviewer; Washington University School of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "The enteric pathogen Cryptosporidium parvum exports proteins into the cytoplasm of the infected cell" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Dominique Soldati-Favre as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Daniel E Goldberg (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

All three reviewers agree that this is an interesting and important study, and that the work is elegant and clearly presented. However they also raised a number of points, as detailed below in the recommendations to the authors, which have to be addressed to strengthen the manuscript. Specifically, the following key points need your full attention:

1) A key question is whether the observed ER stress response is a byproduct of infection or is triggered by the parasite to promote its growth and/or survival. Some key additional experiments are required to address this issue (for example transfection of GRA protein, testing the effects of UPR inhibitors on infection).

2) The host cell transcriptome data should be presented in more details, including a list of differentially expressed genes, and a more thorough discussion of which UPR pathways are affected.

Importantly beside the Essential Revisions, please consider the recommendations made by the individual reviewers and address all the specific points as far as possible in the revision.

Reviewer #1:

This is an interesting study reporting that the intracellular parasite Cryptosporidium, an understudied yet medically relevant apicomplexan pathogen, exports proteins into the cytoplasm of infected cells. The manuscript is very well written and describes a large amount of work based on state of the art genetic approaches.

The authors used genetic tagging to investigate the cellular localization of members of multigene families, and identified MEDLE2 as a protein that is efficiently exported into the cytoplasm of infected cells, both in cultured cells and infected mice. The authors found that MEDLE2 export occurs after invasion, indicative of a translocon-mediated process, and is blocked when the protein is fused to ordered reporters. Using a series of mutants, the authors identify a N-terminal host targeting motif and a critical Leucine residue required for export of MEDLE2. They also document MEDLE2 protein cleavage during export. Finally, RNAseq analysis of cells transfected with MEDLE2 revealed a subset of differentially expressed host genes, pointing at a possible ER stress response of the host cell induced by expression of MEDLE2. These data were confirmed on a small subset of genes using qPCR in infected mice.

While the concept of protein export has been extensively studied in other apicomplexan parasites, this study offers additional insights into host-parasite interactions and a counterpoint to protein translocation mechanisms in Plasmodium and Toxoplasma. MEDLE2 export shares some aspects with protein export in these parasites, but also points at possible divergence, as the translocon elements present in Plasmodium spp or T. gondii are not found in Cryptosporidium. Although the export machinery remains to be identified, this study opens perspectives to study protein export as a target for therapeutic strategies against cryptosporidiosis. Initial characterization of the host cell response revealed a transcriptional signature of ER stress and unfolded protein response, but this aspect of the manuscript is rather superficial.

In summary, this well conducted study provides a comprehensive characterization of protein export in Cryptosporidium-infected cells. This work will stimulate future studies to identify the export machinery. A key question at this stage is the function of MEDLE2 and other exported proteins, and whether the host ER stress response is a bystander effect or a specific response induced by the parasite to promote its growth or survival.

The host cell response could be explored in more details. There are several UPR pathways and it is not clear whether canonical UPR proteins are affected by MEDLE2. A previous study showed that C. parvum induces an ER response in HCT-8 cells (PMID 23986438), and this paper should be discussed here.

The RNAseq data (Figure 6D) shows that only a small fraction of ER stress response genes are significantly upregulated, and there are other genes that are even more upregulated. What are these genes? A full list of DGE should be provided as a supplemental table and discussed in the text.

A key question is whether the observed ER stress response is a byproduct of infection or is triggered by the parasite to promote its growth and/or survival. There are a number of simple experiments, based on knockout or knockdown of key UPR components, or using agonists or chemical inhibitors, which could provide elements of response.

Another open question is the function of MEDLE2. Ectopic expression via transfection of MEDLE2 in host cells may not reflect the natural responses during infection. While the presence of multiple copies of MEDLE genes is probably a major obstacle for genetic studies, the authors could test whether the host cell response differs when MEDLE2 is exported (wildtype protein) or not (mutant versions). This would provide stronger evidence that in the context of infection MEDLE2 is directly responsible (at least in part) for the observed host cell response. Related to this point, line 28 and 75: there is no evidence that MEDLE2 is a pathogenesis factor

MEDLE2 processing shares common features with processing in Plasmodium/Toxoplasma, where aspartyl proteases are involved. Did the authors try to block MEDLE2 export with aspartyl protease inhibitors?

The image quality of Fig5D is not optimal, the MEDLE2-HA signal is very weak.

Reviewer #2:

This comprehensive study elegantly demonstrates that like other parasites of the phylum Cryptosporidium parvum exports proteins into the cytosol of infected cells by a sophisticated mechanism. The authors investigate in great detail the maturation, transit and export of the MEDLE2 protein across the C. parvum lifecycle in culture and infected animals. Briefly, they showed that MEDLE2 passes through the secretory pathway and holds a host targeting motif that is processed during export. MEDLE2 is a bona fide intrinsically disordered protein (IDP), i.e., the protein has no single well-defined tertiary structure under native conditions. This feature is also shared by the growing family of exported GRA proteins in Toxoplasma gondii and like them, MEDLE2 exports is blocked by ordered reporters.

While there is substantial evidence presented that supports the delivery of MEDLE2 in the cytosol of the infected enterocytes, the transit and secretion of other MEDLE family members is less clear. This does not detract from the discovery of a highly regulated export system of parasite-derived effectors in C. parvum, of which MEDLE2 is the flagship.

Based on a wide variety of approaches, the experiments were well designed with appropriate controls and biological replicates, but further clarification is needed, especially with respect to transcriptional analyses based on expression in a heterologous system of the parasite effector. MEDLE2 does not accumulate in the nucleus of infected cells, yet its ectopic expression in mammalian cells is altering the expression of genes involved in ER stress response, including changes in genes linked to the unfolded protein response. One would have to rule out the possibility that the induction of this pathway is not simply a byproduct of the transient overexpression of a disordered protein. This argument is to some extent mitigated by the demonstration that alteration of genes in this pathway has also been observed during infection with C. parvum, yet definitive evidence with a medle2 KO is still missing.

Overall, all the molecular and cell biological analyses and data presented are technically strong and quite convincing and support the notion of an important and novel example of a pathogen-derived modulator of the host response in C. parvum.

– Lines 368-371: the authors state that "It remains to be determined whether the UPR is a byproduct of host detection of the disordered protein MEDLE2 following its translocation during C. parvum infection, or if it is an active process deliberately triggered to promote parasite survival." A key experiment to address this dilemma rightly formulated by the authors would be to express in 293 cells in parallel to MEDLE2, instead of GFP, a mock protein with an intrinsically disordered region (e.g. a Tg exported GRA protein), and monitor by qRT-PCR the expression levels of the genes mentioned in Figure 6F.

– Ectopic expression of MEDLE1 at the TK locus (Figure 1—figure supplement 3E-F) under the control of a fragment of the MEDLE2 promoter only greatly enhances extra-vacuolar MEDLE1 secretion. How were the functional boundaries of MEDLE2 promoter/enhancer (if any) determined? It would have been wise to keep the genomic context of MEDLE2 by making an in-locus promoter fusion (i.e. replacing MEDLE2 ORF by a tagged version of MEDLE1). This would provide insight into whether MEDLE2 protein is better optimized for transport or its expression is strong enough to see accumulation in the cell.

– In Figure 6F, we would expect to see quantification of target gene expression under both uninfected and infected conditions.

Reviewer #3:

The manuscript demonstrates that MEDLE2 is exported by diverse parasite stages and fails to export when tethered to folded reporters. Targeted mutagenesis shows that Leucine 35 is required for the correct processing of MEDLE2, and is also a prerequisite for successful export. The authors go on to show that mammalian protein expression of MEDLE2 leads to an unfolded protein response and that this correlates fairly well with the response seen in vivo. The work is well done and the manuscript is clearly written. This paper opens up the field for exploration of effector proteins and their export mechanism.

– Brefeldin A experiments suggest that transit through the secretory pathway is required, though viability of the treated parasites needs to be demonstrated. A poison like azide would also block transit without demonstrating that vesicular transport is involved.

– Endogenous tagging of MEDLE2, as well as trials of knocking out the gene showed that the wild type locus is present in the transfected population even after the integration of the transgene cassette. Based on that, the author concluded that there are multiple copies of the MEDLE2 gene in C. parvum genome. However, might an alternative explanation be that the transfected population is a mixture of wild type and genetically modified parasites?

– The MEDLE2-GFP fusion that was used to determine the effect of the protein on host cells has 15 residues (aa 21 to 35) more than what would be expected to remain on the protein exported from the parasite. This is very likely not an issue, but the caveat should be noted.

– In figure 5D, it would be good to have both infected and uninfected cells to show the background fluorescence.

– Lines 93-94 refer to Figure 1B and C.

– Is there some parasite retention of the I36 mutant? Looks like there might be from the one example shown.

– The most highly upregulated gene is not identified by name, contrary to what is said in the text.

https://doi.org/10.7554/eLife.70451.sa1

Author response

Reviewer #1:

The host cell response could be explored in more details. There are several UPR pathways and it is not clear whether canonical UPR proteins are affected by MEDLE2. A previous study showed that C. parvum induces an ER response in HCT-8 cells (PMID 23986438), and this paper should be discussed here.

Thank you for bringing this to our attention. It was an oversight not to cite this paper. Morada et al., 2013 showed C. parvum induces an ER stress response in HCT-8 cells, which was characterized by increases in stress response proteins (Calreticulin, GRP78, Nrf2, phosphoEIF2, and CHOP) by Western Blot. These findings align with our experimental observations of an ER stress response present both in the MEDLE2 transfection and C. parvum infection experiments (and we now test a CHOP KO mouse). We now specifically mention the findings of Morada et al., in the discussion (lines 373 -375).

The RNAseq data (Figure 6D) shows that only a small fraction of ER stress response genes are significantly upregulated, and there are other genes that are even more upregulated. What are these genes? A full list of DGE should be provided as a supplemental table and discussed in the text.

The full list of DGE were provided as in the Supplementary files submitted for both the MEDLE2 cell transfection dataset and the C. parvum infection dataset. We agree this data would be clearer if mentioned directly in the text instead of just providing the data in the supporting information. We have made this change as suggested and referenced a new table, Figure 6- source data 1. This table contains the full list of GSEA results with the NES, p value and gene names in the pathways. We also explained in more detail in the text how we narrowed in to study the ER stress response from the GSEA results through functional annotation clustering performed in DAVID on the full list of DEGs (lines 271-277).

A key question is whether the observed ER stress response is a byproduct of infection or is triggered by the parasite to promote its growth and/or survival. There are a number of simple experiments, based on knockout or knockdown of key UPR components, or using agonists or chemical inhibitors, which could provide elements of response.

Thank you for this suggestion. In response we have tested the impact of chemical inhibition of the UPR in HCT8 cells during Cryptosporidium infection using inhibitors. We chose inhibitors specific for 2 UPR signaling pathways for which we observed changes in our RNA sequencing datasets from MEDLE2 transfection and infection. We found that KIRA6, which targets the IRE1 branch of the UPR, reduces infection in vitro. Interestingly, GSK2606414 which inhibits the PERK signaling pathway had little impact on parasite growth. In support of these observations, infection of Ddit3 -/-mice (also n=known as CHOP, n = 12) resulted in reduced parasite burden when compared to infection of BL6 control mice (n = 12), which we interpret to suggest that the ER stress response does contribute to parasite survival. The results from both the in vitro inhibitor infections and Ddit3-/- mouse experiments have been added to Figure 6, as panels G and H, with corresponding figure legends added (Lines 1052-1065).

Another open question is the function of MEDLE2. Ectopic expression via transfection of MEDLE2 in host cells may not reflect the natural responses during infection. While the presence of multiple copies of MEDLE genes is probably a major obstacle for genetic studies, the authors could test whether the host cell response differs when MEDLE2 is exported (wildtype protein) or not (mutant versions). This would provide stronger evidence that in the context of infection MEDLE2 is directly responsible (at least in part) for the observed host cell response. Related to this point, line 28 and 75: there is no evidence that MEDLE2 is a pathogenesis factor

We agree that ablating the gene(s) would be the most direct avenue to understand their function. However, to date, this has not been possible due to multiple technical limitations. Genetic modifications that ablate export (DSP and L35A) were conducted using ectopic copies of MEDLE2, while the remaining endogenous cop(ies) remain intact and translocated to the host cell. We successfully engineered a parasite strain in which MEDLE2 was replaced with a marker (Figure 1—figure supplement 2); however, additional wild type copies remained. The recent reannotation of the C. parvum genome revealed MEDLE2 genes at multiple telomeric sites (Baptista et al., 2021) and we discuss this further in our response to reviewer 3. We did not observe an overt loss of fitness in ectopic mislocalization mutants that we engineered. They produce infections of similar parasite burden in culture and in animals.

We believe it likely that MEDLE proteins are pathogenesis factors, they are exported into the host cell, and they are under significant selection and encoded in complex highly malleable loci (see below), however, the reviewer is absolutely right that this has not been formally demonstrated. We have thus removed the phrase “pathogenesis factor” from lines 28 and 75 and replaced it with the neutral phrase “parasite proteins”.

MEDLE2 processing shares common features with processing in Plasmodium/Toxoplasma, where aspartyl proteases are involved. Did the authors try to block MEDLE2 export with aspartyl protease inhibitors?

We had attempted to block MEDLE2 export with aspartyl protease inhibitors synthesized for use in Plasmodium falciparum (WEHI916, WEHI842) and Toxoplasma gondii (WEHI586). None of the compounds tested fully ablated export (it is our understanding that this is true for Plasmodium as well), but treatment with WEHI842 resulted in accumulation of MEDLE2 and did impact parasite growth. We recognize several potential limitations to these experiments. The specificity and drug sensitivity of the Cryptosporidium enzyme may be different for that of these other parasites, as may be compound access due to difference in intracellular localization. For these reasons, we decided not to include this data in the current manuscript. We are conducting experiments that seek to explore genetically which C. parvum aspartyl protease is responsible for processing exported proteins; however, there are multiple candidates, and these are complex time-consuming experiments that we have not yet concluded, as some of the genes appear to be essential.

The image quality of Fig5D is not optimal, the MEDLE2-HA signal is very weak.

We apologize for the poor quality. We have improved the contrast and very much hope the reviewer finds it easier now to appreciate the staining. We cannot redo the full microscopy off all mutants and controls show here. Unfortunately, cryopreservation for Cryptosporidium oocysts is not fully robust yet. As a result, the parasites need to be passaged through mice to maintain viability every 6 months. Unfortunately, we had to triage what we could keep alive during the pandemic due to our limited access the lab and our mouse facilities.

Reviewer #2:

– Lines 368-371: the authors state that "It remains to be determined whether the UPR is a byproduct of host detection of the disordered protein MEDLE2 following its translocation during C. parvum infection, or if it is an active process deliberately triggered to promote parasite survival." A key experiment to address this dilemma rightly formulated by the authors would be to express in 293 cells in parallel to MEDLE2, instead of GFP, a mock protein with an intrinsically disordered region (e.g. a Tg exported GRA protein), and monitor by qRT-PCR the expression levels of the genes mentioned in Figure 6F.

As suggested, we cloned TgGRA16 (aa 24-505) into the GFP expression plasmid and performed transfection of HEK293T cells in parallel with MEDLE2-GFP and the GFP only control. qPCR revealed changes in the ER stress response genes CHAC1, DDIT3, NUPR1, and TRIB3. We include these new experiments as Figure 6—figure supplement 2. The experiment shows that multiple unfolded parasite proteins can trigger the response upon transfection. It does not address whether Cryptosporidium, or Toxoplasma for that matter, benefits from that response to their exported unfolded proteins. We find reduced Cryptosporidium growth with pharmacological inhibition and genetic ablation experiments – but it’s not a complete block (see response to reviewer one). We agree with the reviewer, that divining pathogen intent is difficult, and thus have taken care in the discussion not to overextend our claim, and to focus on our findings and measurements.

– Ectopic expression of MEDLE1 at the TK locus (Figure 1—figure supplement 3E-F) under the control of a fragment of the MEDLE2 promoter only greatly enhances extra-vacuolar MEDLE1 secretion. How were the functional boundaries of MEDLE2 promoter/enhancer (if any) determined? It would have been wise to keep the genomic context of MEDLE2 by making an in-locus promoter fusion (i.e. replacing MEDLE2 ORF by a tagged version of MEDLE1). This would provide insight into whether MEDLE2 protein is better optimized for transport or its expression is strong enough to see accumulation in the cell.

Little is known about Cryptosporidium promoters, nothing about whether there are enhancers. Intergenic regions are usually small in this very compact genome, and in contrast to e.g. T. gondii, 5’UTRs are extremely short. For most genes the transcription initiation site appears to be in close proximity of the initiation codon used for translation. By now we have tested upstream regions of about ten different genes, and empirically found these small regions to be sufficient to drive expression of a reporter gene, and to reproduce the stage specificity and strength of the native locus (see. e.g. Tandel et al., 2019). We did not conduct mechanistic studies into what exactly defines promoters (Cryptosporidium is a rather poor model for such studies for numerous technical reasons). There are several published studies exploring this question using Toxoplasma, which found epigenetic marks and transcription factor binding sites in proximity of the transcription initiation site. We do have extensive transcriptional data and using multiple transcriptomic datasets we found the transcription initiation site of MEDLE2 to fall within a few bp of the start of the coding region.

Maybe more importantly, we have experimentally tested the presumptive promoter by using it to drive ectopic (medle2)MEDLE2-HA expression in the TK locus. We found expression timing and targeting to be indistinguishable from MEDLE2 tagged in the native locus. We used the same sequence and strategy to construct all of our point mutations with highly consistent results. And thus again used this strategy to engineer the chimeric (medle2)MEDLE1-HA (as so often, experiment shown early in the article, in real-life were done towards the end). In planning the point mutation studies, we actually considered performing “in locus” experiments similar to the one the reviewer discussed here. In the end we decided against this, however. The main reason was that Cryptosporidium genetic engineering relies exclusively on double stranded break repair by homologous recombination. Painful past experience taught us that even short stretches of sequence identity of 10-20 bp can suffice to enable unwanted cross overs (see e.g. Vinayak et al., 2020). As a result, when targeting a locus with identical or highly similar sequences, the repair DNA construct has to be extensively recodonized. Changing the primary sequence and codon usage comes with its own costs and caveats and may impact transcription and translation efficiency. We thus opted to use ectopic expression here. We have made this point more explicitly now.

– In Figure 6F, we would expect to see quantification of target gene expression under both uninfected and infected conditions.

We apologize for the confusion in how the data is being displayed. The graph in 6F is displayed as the fold change for each the genes of interest compared to the uninfected control. The left most panel represents the fold change of the uninfected, which is 1 for each of the genes being tested. Showing the fold change of the uninfected for each gene may make this clearer and we have updated the graph to reflect this now.

Reviewer #3:

The manuscript demonstrates that MEDLE2 is exported by diverse parasite stages and fails to export when tethered to folded reporters. Targeted mutagenesis shows that Leucine 35 is required for the correct processing of MEDLE2, and is also a prerequisite for successful export. The authors go on to show that mammalian protein expression of MEDLE2 leads to an unfolded protein response and that this correlates fairly well with the response seen in vivo. The work is well done and the manuscript is clearly written. This paper opens up the field for exploration of effector proteins and their export mechanism.

– Brefeldin A experiments suggest that transit through the secretory pathway is required, though viability of the treated parasites needs to be demonstrated. A poison like azide would also block transit without demonstrating that vesicular transport is involved.

We fully agree with the reviewer that pharmacological experiments have their caveats. BFA has more limited effects than azide, as it targets ER to Golgi trafficking and does not cause generalized cellular asphyxiation. However, it is likely to block parasite growth as they depend on BFA sensitive pathways to build secretory organelles required for invasion. For this reason, we also conducted genetic experiments that ablated the signal peptide, and these produced very similar results. We note the extensive short-term use of BFA to study protein trafficking in apicomplexan parasites over the last 20 years (e.g. Soldati et al., 1998 studying rhoptry protein maturation, or Wickham et al., 2001 studying the export of PFEMP1, Tonkin et al., 2006 studying apicoplast targeting). Not every pathway is immediately ablated by BFA toxicity. While targeting to e.g. the rhoptry, the food vacuole, or the host cell is susceptible to BFA, targeting of the apicoplast or the mitochondrion is not (a small number of specific apicoplast proteins does traffic via Golgi, and those are susceptible). We limited our experiments to a single merogony cycle and added BFA 3h following invasion with 7h incubation prior to fixation. Nonetheless, we now formally acknowledge that BFA is likely to inhibit long-term parasite growth in the segment that describes this experiment.

– Endogenous tagging of MEDLE2, as well as trials of knocking out the gene showed that the wild type locus is present in the transfected population even after the integration of the transgene cassette. Based on that, the author concluded that there are multiple copies of the MEDLE2 gene in C. parvum genome. However, might an alternative explanation be that the transfected population is a mixture of wild type and genetically modified parasites?

We considered this alternative, however, our mapping studies indicated that the locus was properly modified, but an additional copy (copies) of WT MEDLE2 persisted independently (Figure 1- Supplemental Figure2). We note that this was the same for all our experiments targeting this locus (even when we did not disrupt the gene). In experiments where we introduced an epitope tag or a fluorescent reporter, we could observe the presence of the cassette by following the protein(s) it encoded. All parasites uniformly expressed the endogenously tagged MEDLE2-HA arguing against heterogeneity due to transgene loss. Furthermore, while passaging transgenic oocysts through mice, we maintain selection with paromomycin. The MEDLE2-HA-tdNeon strain has been passaged through mice 8 times, and the presence of the transgene and WT bands remained unchanged. The subtelomeric locus that is home to MEDLE2 has long puzzled us, however, a recent genome reannotation effort by the Kissinger lab (Baptista et al., 2021) found numerous copies of the MEDLE gene family in multigene cassettes at different telomeres. This is not due to assembly artefacts as it is based on nanopore long read, single molecule sequencing. Please refer to Figure 6 in https://doi.org/10.1101/2021.01.29.428682 which shows complex expression sites with numerous copies of MEDLE genes, and the detailed discussion provided in that article. The overall conclusion was that the population of C. parvum parasites infecting an animal maintains multiple alternative telomeres among its individuals which contains members of multigene families with MEDLE being most prominent. These parasites engage in constant sexual recombination, and it appears that different members of the population may carry different cassettes. Note that the rest of the genome is very stable. Similar cassettes are known from antigenically varied pathogenesis factors in other organisms. We believe it likely that this organization reflects the overall importance of this gene family in host-parasite interaction, but more work is needed to fully understand this. The Baptista article and the complex telomere architecture is mentioned directly in the discussion now.

– The MEDLE2-GFP fusion that was used to determine the effect of the protein on host cells has 15 residues (aa 21 to 35) more than what would be expected to remain on the protein exported from the parasite. This is very likely not an issue, but the caveat should be noted.

Thank you for pointing this out. We now have made note of this in the text to ensure that the reader is aware of what exactly this construct includes: “we note that this protein contains 15 amino acids (aa 21 to 35) that are likely missing in parasite exported MEDLE2 due to N-terminal processing” (lines 262-264).

– In figure 5D, it would be good to have both infected and uninfected cells to show the background fluorescence.

We apologize that we did not include images of these controls. The images provided in Figure 5D are cropped to show only a single parasite containing infected cell. We now provided uncropped images for each of the representative cells shown in panel 5D to include uninfected cells to demonstrate the background fluorescence as a supplemental figure (Figure 5—figure supplement 3).

– Lines 93-94 refer to Figure 1B and C.

Thank you for this suggestion. We have incorporated this citation for figure 1B and C in the text (lines 93-94).

– Is there some parasite retention of the I36 mutant? Looks like there might be from the one example shown.

This is an astute observation by the reviewer, and we did not initially notice this ourselves. We do agree that the image provided appears to have accumulated MEDLE2 near the site of infection. We looked at additional images and believe the image here may represent MEDLE2 being exported within a normal context, as some images show MEDLE2 accumulation, while others do not. We cannot exclude the possibility that mutation of I36A may lead to accumulated MEDLE2 in the parasite; however, it does not ablate MEDLE2 export as is the observation for L35A.

– The most highly upregulated gene is not identified by name, contrary to what is said in the text.

We apologize for the confusion. To address this concern, we have added an additional source data file which includes a table (Figure 6-source data 1) that contains a full list of the GSEA results for the MEDLE2 transfection experiment. We also have provided a source data file which contains the core enrichment genes for both the transfection and infection datasets (Figure 6- source data 2). We also have added a statement about what most of the GSEA pathways involve and address how we narrowed in upon studying the ER stress response (Lines 273 -277).

References

Baptista, R.P., Li, Y., Sateriale, A., Sanders, M.J., Brooks, K.L., Tracey, A., Ansell, B.R.E., Jex, A.R., Cooper, G.W., Smith, E.D., et al. (2021). Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal new biological insights. bioRxiv, 2021.2001.2029.428682.

Tandel, J., English, E.D., Sateriale, A., Gullicksrud, J.A., Beiting, D.P., Sullivan, M.C., Pinkston, B., and Striepen, B. (2019). Life cycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum. Nat Microbiol 4, 2226-2236.

Vinayak, S., Jumani, R.S., Miller, P., Hasan, M.M., McLeod, B.I., Tandel, J., Stebbins, E.E., Teixeira, J.E., Borrel, J., Gonse, A., et al. (2020). Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase. Science Translational Medicine 12, eaba8412.a

https://doi.org/10.7554/eLife.70451.sa2

Article and author information

Author details

  1. Jennifer E Dumaine

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5975-4523
  2. Adam Sateriale

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Present address
    The Francis Crick Institute, London, United Kingdom
    Contribution
    Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Alexis R Gibson

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1078-4841
  4. Amita G Reddy

    Franklin College of Arts and Science, University of Georgia, Athens, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  5. Jodi A Gullicksrud

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  6. Emma N Hunter

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  7. Joseph T Clark

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  8. Boris Striepen

    Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Conceptualization, Funding acquisition, Project administration, Supervision, Writing - original draft, Writing – review and editing
    For correspondence
    striepen@upenn.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7426-432X

Funding

National Institute of Allergy and Infectious Diseases (R01AI127798)

  • Boris Striepen

National Institute of Allergy and Infectious Diseases (R01AI112427)

  • Boris Striepen

National Institute of Allergy and Infectious Diseases (T32AI007532)

  • Jennifer E Dumaine

National Institute of Allergy and Infectious Diseases (K99AI137442)

  • Adam Sateriale

National Institute of Allergy and Infectious Diseases (T32A1055400)

  • Jodi A Gullicksrud

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported in part by funding from the National Institutes of Health through grants to BS (R01AI127798 and R01AI112427), and fellowships and career awards to JED (T32AI007532), AS (K99AI137442), and JAG (T32A1055400). We thank Andrea Stout, Jasmine Zhao (Cell and Developmental Biology Microscopy Core), Gordon Ruthel (Penn Vet Imaging Core), Dan Beiting (Center for Host Microbial Interactions), and Patty Costello (Comparative Pathology Core) for help with microscopy, RNA sequencing, and histology, respectively, and Jacek Gaertig and Phillip Scott for sharing reagents.

Ethics

All animals used in this study were handled and cared for in accordance with approved Institutional Animal Care and Use Committee protocols at the University of Georgia (protocol A2016 01-028-Y1-A4) and the University of Pennsylvania (protocol #806292).

Senior Editor

  1. Dominique Soldati-Favre, University of Geneva, Switzerland

Reviewing Editor

  1. Olivier Silvie, Sorbonne Université, UPMC Univ Paris 06, INSERM, CNRS, France

Reviewer

  1. Daniel E Goldberg, Washington University School of Medicine, United States

Publication history

  1. Received: May 17, 2021
  2. Preprint posted: June 4, 2021 (view preprint)
  3. Accepted: November 19, 2021
  4. Accepted Manuscript published: December 6, 2021 (version 1)
  5. Version of Record published: December 20, 2021 (version 2)

Copyright

© 2021, Dumaine et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,206
    Page views
  • 203
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Jennifer E Dumaine
  2. Adam Sateriale
  3. Alexis R Gibson
  4. Amita G Reddy
  5. Jodi A Gullicksrud
  6. Emma N Hunter
  7. Joseph T Clark
  8. Boris Striepen
(2021)
The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell
eLife 10:e70451.
https://doi.org/10.7554/eLife.70451

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Atanas Radkov et al.
    Research Article

    Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

    1. Immunology and Inflammation
    2. Microbiology and Infectious Disease
    Paola Kučan Brlić, Ilija Brizić
    Insight

    A new study sheds light on how SARS-CoV-2 influences the way natural killer cells can recognize and kill infected cells.