PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of mbioJournal InfoAuthorsReviewersBoard of EditorsJournals ASM.orgmBiomBio Article
 
mBio. 2013 Sep-Oct; 4(5): e00387-13.
Published online 2013 September 3. doi:  10.1128/mBio.00387-13
PMCID: PMC3760245

Evidence for a Structural Role for Acid-Fast Lipids in Oocyst Walls of Cryptosporidium, Toxoplasma, and Eimeria

ABSTRACT

Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin).

IMPORTANCE

Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.

Introduction

Coccidian parasites make infectious walled oocysts that are spread by the fecal-oral route (1). Toxoplasma gondii, a zoonotic coccidian of worldwide distribution, makes oocysts with a double-layered wall that are shed by cats. Once shed in the environment, Toxoplasma makes a sporulated oocyst that contains two-walled sporocysts, each of which contains four sporozoites that infect humans and other warm-blooded animals (2). In immunocompetent persons, acute Toxoplasma infections are controlled, but the parasite remains within cysts in brain and muscle, which are not symptomatic. In contrast, Toxoplasma causes disseminated infections in fetuses and in AIDS patients who lack cellular immunity (3). Eimeria spp. are a large group of parasites infecting the gut that make oocysts and sporocysts similar to those of Toxoplasma (4). However, Eimeria is limited to a specific animal and specific region of the gut. For example, Eimeria tenella is confined to ceca of chickens, where it causes dysentery and costs billions of dollars worldwide (5).

Cryptosporidium parvum causes diarrhea in people and in livestock. Recently Cryptosporidium has been found to be among the four most important causes of moderate to severe diarrhea in children in the developing world (6). Cryptosporidium makes a different oocyst than those of Toxoplasma and Eimeria, which does not contain sporocysts and has a simpler wall (7).

We recently showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria contains fibrils of β-1,3-glucan that form a porous scaffold (8). A parasite glucan hydrolase has a unique glucan-binding domain and is present in the inner layer of the oocyst wall. Echinocandins, which are inhibitors of fungal glucan synthases, arrest development of the Eimeria oocyst wall and inhibit release of oocysts into the intestinal lumen of chickens. The presence of the β-1,3-glucan fibrils can explain the strength but not the impermeability of oocyst walls. Dityrosines, which are present in tyrosine-rich oocyst wall proteins, may contribute to the impermeability of oocyst walls of Toxoplasma and Eimeria, but the oocyst wall of Cryptosporidium lacks dityrosines and is missing the scaffold of β-1,3-glucan (9, 10).

Prior to the identification of the human immunodeficiency virus (HIV), AIDS was diagnosed by the presence of opportunistic infections, such as Cryptosporidium, which was detected in stools by an acid-fast stains (Fig. 1A) (11). The goal here was to determine the structural role, if any, of acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. As background, the cell walls of mycobacteria are acid-fast (i.e., retain lipophilic dyes following washing with hydrochloric acid in ethanol) due to the presence of high-molecular-weight lipids that form a waxy coat (see Fig. S1 in the supplemental material) (12, 13). Among the best-characterized mycobacterial wall lipids are mycolic acids, which are synthesized in part by polyketide synthases (14). The plant cuticle on the surface of leaves and stems, which also labels with lipophilic dyes, is composed of wax esters and cutin (a polymer of glycerol and ω–hydroxy and mid-chain hydroxy fatty acids) (15).

FIG 1
Oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria label with acid-fast stains. (A) Bright-field (Kinyoun in Cryptosporidium and Toxoplasma, Ziehl-Neelsen in Eimeria) acid-fast stains bind to oocysts (11). Filled arrowheads mark mature Eimeria oocysts ...

We became interested in the lipid content of oocyst walls when we identified by mass spectrometry an extraordinarily abundant polyketide synthase (PKS1, also known as type 1 fatty acid synthase) in Toxoplasma and Eimeria oocysts, which resembles mycobacterial polyketide synthases. To explore the potential importance of acid-fast lipids in oocyst walls, we treated isolated walls with organic solvents, which made the walls fall apart. We analyzed released lipids with high-resolution and high-accuracy mass spectrometry. The most abundant oocyst wall lipids were triglycerides that have polyhydroxy fatty acyl chains like those of plant cutin but different than mycolic acids.

RESULTS

Oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria all label with acid-fast stains.

The oocyst walls of each parasite label with carbol-fuchsin, a lipophilic dye used for bright-field acid-fast stains (Kinyoun or Ziehl-Neelsen), and with auramine-O, a fluorescent acid-fast stain (Fig. 1A) (16). Developing Eimeria oocysts have acid-fast vesicles in their periphery (Fig. 1B). Sporocyst walls of Toxoplasma are acid-fast, while those of Eimeria are not. Instead acid-fast stains localize to “refractile bodies” of Eimeria sporozoites, an organelle of unknown function. The latter result suggests that acid-fast lipids are not an important component of sporocyst walls of Eimeria, which distinguishes this parasite from Toxoplasma. Plant cuticles also stain with auramine-O (17). Additional acid-fast stains are shown in Fig. S1 in the supplemental material.

Polyketide synthases are among the most abundant proteins in oocysts of Toxoplasma and Eimeria.

Coccidian parasites each have two predicted polyketide synthases that resemble those of mycobacteria (Fig. 2A) (10, 18, 19). In contrast, Plasmodium, which is related to coccidian parasites but is not spread by the fecal-oral route, has no polyketide synthases (10). The coccidian polyketide synthases are very large, since each enzyme contains four modules (Toxoplasma and Eimeria) or three modules (Cryptosporidium) of catalytic domains. Each module contains six catalytic domains that add two carbons to the growing chain by a series of reactions that includes oxygenated intermediates (20). The PKS1 of Toxoplasma (encoded by the TGVEG_013030 gene) was very abundant in tryptic digests of oocyst proteins, as shown by 263 unique peptides and 36% sequence coverage (Fig. 2B). For comparison, the number of unique peptides and sequence coverage for the 10 most abundant cytosolic proteins of Toxoplasma are shown in Table S1 in the supplemental material. The PKS1 of Eimeria (encoded by the ETH_00015480 gene) showed 9% sequence coverage and 69 unique peptides. Mass spectrometry of Cryptosporidium proteins was not performed here. However, messenger RNAs of a Cryptosporidium polyketide synthases (type 1 fatty acid synthase encoded by cgd3_2180) peak at 48 h of culture when oocyst walls are being made (21). Reverse transcription-PCR (RT-PCR) showed that oocysts of Toxoplasma and Eimeria express PKS1 and PKS2, as well as a 4′-phosphopantetheine transferase (PPTase), which is essential for PKS activity (see Fig. S1 in the supplemental material) (22).

FIG 2
Polyketide synthases are extraordinarily abundant in oocysts of Toxoplasma and Eimeria. (A) Parasite polyketide synthases have domain structures like those of mycobacterial PKS12, except that the parasite enzymes may have three modules (Cryptosporidium ...

Lipids appear to be an important component of the rigid bilayer present in the oocyst wall of Cryptosporidium.

To explore further the possible role of acid-fast lipids in the structure of oocyst walls, we treated isolated walls with reagents that remove proteins or lipids. The oocyst wall of Cryptosporidium, which does not contain β-glucan, is simpler than the oocyst wall of Eimeria and so will be described first. Sonicated and washed walls of Cryptosporidium form scrolls that have a moderately electron-dense inner layer that is rich in glycoproteins (Fig. 3A) (7). There is also a rigid bilayer (as shown by scrolling) that is thicker than a cell membrane. Pronase, which digests proteins, removes the inner layer of the oocyst wall but leaves the rigid bilayer intact (Fig. 3B). Pronase-treated oocyst walls of Cryptosporidium remain acid-fast in a quantitative assay (Fig. 3D). In contrast, chloroform-methanol (2:1), which extracts lipids, completely disrupts the oocyst walls of Cryptosporidium and prevents acid-fast staining (Fig. 3C and D), while chymotrypsin, which degrades proteins, reduces acid-fast staining. Treatment with 1 N NaOH, which deproteinates yeast walls and Eimeria oocyst walls (see next section), dissolved Cryptosporidium oocyst walls (data not shown). These results suggest a simple, if incomplete, model of the Cryptosporidium oocyst wall, in which acid-fast lipids are likely an important component of the rigid bilayer, while glycoproteins are present in the inner layer (see Fig. 6) (7).

FIG 3
Lipids appear to be an important component of the rigid bilayer in the oocyst wall of Cryptosporidium. (A) Sonicated oocyst walls of Cryptosporidium, which curl into scrolls, have an outer bilayer and an inner glycoprotein layer (7). (B) The rigid bilayer, ...
FIG 6
Coccidia, mycobacteria, and plants, which are deeply divergent organisms, each have a lipid-rich coat that makes them resistant to environmental stress. The rigid bilayer of the Cryptosporidium oocyst wall is composed of acid-fast lipids, while glycoproteins, ...

Organic solvents remove the outer layer of the oocyst wall of Eimeria.

Because of issues of availability, these studies were performed with unsporulated oocysts of Eimeria from euthanized chickens rather than Toxoplasma from euthanized cats. Previously we have used Eimeria oocyst walls for transmission electron microscopic (TEM) studies of fibrils of β-1,3-glucan in the inner layer of the oocyst wall (8). The control for these studies was the wall of Saccharomyces, which is composed of a single layer that contains fibrils of β-1,3-glucan and chitin (Fig. 4A) (23). The wall of Saccharomyces, which does not contain lipids, is resistant to chloroform-methanol. In contrast, sodium hydroxide removes proteins from the Saccharomyces walls, so that only fibrils remain.

FIG 4
In contrast to fungal walls, the oocyst wall of Eimeria is very sensitive to organic solvents. (A) Transmission electron microscopy (TEM) shows that walls of Saccharomyces cerevisiae, which have a single layer (between the hollow arrowheads), remain relatively ...

The outer layer of the Eimeria oocyst wall, which is relatively electron dense, has linear structures that extend from the bilayer to the external surface of the wall (Fig. 4B). The outer layer is uninterrupted, as shown by en face negative staining of intact oocysts, and so forms the permeability barrier in the oocyst wall. In contrast, the inner layer of the Eimeria oocyst wall, which is less electron dense, is composed of a porous scaffold of fibrils of β-1,3-glucan. The macrophage lectin dectin-1 binds to fibrils of β-1,3-glucan in the inner layer of the oocyst wall (Fig. 4C) (8, 24). Organic solvents disrupt the outer layer of the Eimeria oocyst wall and markedly reduce the acid-fast staining and UV fluorescence of dityrosines (Fig. 4B to D).

Treatment with sodium hydroxide, which extracts proteins and breaks ester bonds within triglycerides (see next section), disrupts the outer layer of the Eimeria oocyst wall that develops a “soap bubble” appearance by negative staining (Fig. 4C). Sodium hydroxide does not reduce dectin-1 binding or acid-fast staining, but it decreases dityrosine fluorescence (consistent with removal of proteins) (Fig. 4D). Together, these data suggest a model for the Eimeria oocyst wall in which the inner layer contains β-1,3-glucan like fungal walls, while the outer layer and the rigid bilayer contain acid-fast lipids like those of mycobacterial walls (Fig. 6). Because oocyst walls of Toxoplasma and Eimeria share common components, including proteins cross-linked with dityrosines, homologs of Cryptosporidium oocyst wall proteins, glucan hydrolases, β-1,3-glucan, and acid-fast lipids (see next section), it is likely that this model also applies to oocyst walls of Toxoplasma (1, 2, 4, 811, 25). We do not presently have a model for sporocyst or tissue cyst walls of Toxoplasma.

Triglycerides, many with polyhydroxy fatty acyl chains, are the most abundant lipids in oocyst walls.

High-resolution Fourier transform ion cyclotron resonance mass spectrometry, which has an accuracy of better than 1 part per million, allowed us to determine the elemental composition of lipids extracted with chloroform-methanol from oocyst walls (Fig. 5A; see Table S2 in the supplemental material) (26). For example, the chemical formula for the lipid with [M + Na]+ 953.7419 m/z is C57H102O9. Cryptosporidium oocyst wall lipids also include phosphatidylcholines, which may represent membrane contamination. Because the triglycerides vary in the lengths of the fatty acyl chains and their degrees of unsaturation and/or oxidation, oocyst wall lipids are a complex mix for each organism (Fig. 5B). The hydroxyl groups but not the double bonds can be localized by low-energy collision-induced dissociation (CID) of some of the triglycerides (26).

FIG 5
Triglycerides are the most abundant lipids in chloroform-methanol extracts of oocyst walls. (A) High-accuracy and high-resolution mass spectrometry makes it possible to determine the m/z and assign the chemical composition to the complex set of lipids ...

Eimeria triglycerides included numerous species with polyhydroxy acyl chains, while Toxoplasma and Cryptosporidium triglycerides included numerous species with longer fatty acyl chains (Fig. 5C and D; see Table S2 in the supplemental material). While it is not possible to estimate the relative abundance of each triglyceride in a complex mixture, multiple biological repeats of Eimeria lipids showed that triglycerides with polyhydroxy acyl chains, which contain 7 to 12 oxygens per triglyceride where glycerol contains six oxygens, are predominant in the higher-molecular-weight range. In the same way, Cryptosporidium triglycerides with elongated fatty acyl chains containing as many as 24 carbons are predominant in the higher-molecular-weight range. Triglycerides with polyhydroxy acyl chains and elongated fatty acyl chains are relatively less abundant in Toxoplasma.

Consistent with the presence of triglycerides in oocyst walls, mRNAs for diacylglycerol acyltransferases (DGAT1 and DGAT2) (27), as well as a putative acyl coenzyme A (acyl-CoA):cholesterol acyltransferase (ACAT), are expressed in Eimeria oocysts (see Fig. S1 in the supplemental material). While Toxoplasma tachyzoites (an asexual wall-less stage that can be propagated in vitro) make fatty acids with 14 to 26 carbons and zero to one carbon double bonds (28), they are missing the hydroxyl groups present in oocyst wall triglycerides. In contrast, fatty acyl chains containing multiple hydroxyl groups are present in cutin polymers in the plant cuticle (15). Finally, although oocyst walls are acid-fast and oocysts strongly express a polyketide synthase, we did not identify lipids that resemble mycolic acids.

DISCUSSION

These observations suggest structural roles for lipids in parasite walls and appear to broaden our understanding of what lipids make walls acid-fast (Fig. 6). The evidence for the importance of triglycerides in the oocyst walls of coccidian parasites includes the following. The oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria are each acid-fast. The oocyst walls of Cryptosporidium and Eimeria fall apart when treated with organic solvents. Each oocyst wall contains a rigid bilayer that is reminiscent of the outer membrane of mycobacteria (13). By far the most abundant lipids in extracts of the oocyst walls of all three parasites are triglycerides, which contain fatty acyl chains that vary in length and in the degree of unsaturation and/or oxidation. At least 250 species of triglycerides are made by mycobacteria and may contribute to the acid-fast walls (12). Previously triglycerides have been considered only as storage lipids in Toxoplasma tachyzoites, parallel to their role in host cells (27).

Because the gene knockout methodology is not available (29), we are unable to prove the link between the abundant polyketide synthase identified by mass spectrometry in Toxoplasma and Eimeria and the triglycerides extracted from the oocyst walls. Because we were unable to extract lipids from oocyst walls without killing the parasites inside, we were unable to prove that lipids are essential for the impermeability of the oocyst wall and for pathogenicity. The two-layered oocyst walls of Cryptosporidium (glycoproteins and acid-fast lipids) and Toxoplasma and Eimeria (glucan and acid-fast lipids), if this is the case, resemble two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plant cuticles (cellulose and waxes/cutin) (Fig. 6). In addition to chitin and proteins, nematode eggs contain an inner layer rich in lipids (30). Because coccidia, mycobacteria, and plants are deeply divergent, the use of lipid coats to protect these organisms from environmental challenges appears to be the result of convergent evolution. In contrast, walls of fungi and of other parasites transmitted by the fecal-oral route (e.g., Entamoeba and Giardia) are missing the lipid layer (31). Finally, these results may help explain why Eimeria oocysts are destroyed in vitro by essential oils (32).

MATERIALS AND METHODS

Parasites and animals.

All animal work was approved by Institutional Animal Care and Use Committees at Boston University and at the USDA. Unsporulated oocysts of Eimeria tenella and Toxoplasma gondii (VEG and ME49 strains) were prepared from infected chickens and cats, respectively, using previously described methods (8). Eimeria oocysts at various stages of development were prepared from homogenized ceca by centrifugation in the absence of high salt. Oocysts of Eimeria and Toxoplasma were sporulated by incubation for 48 to 72 h at 30°C. Oocysts of Cryptosporidium parvum (Iowa strain), which had been passaged through newborn calves, were purchased from Bunch Grass Farm, Dury, ID.

Acid-fast staining and fluorescence microscopy.

Oocysts were washed extensively in phosphate-buffered saline (PBS) and applied to glass slides, which were then heat fixed. Alternatively, cryosections of ceca of chickens infected with Eimeria were applied to glass slides. For bright-field acid-fast stains, slides were incubated in carbol-fuchsin for 45 min at room temperature (Kinyoun method), washed, and destained with 3% HCl in ethanol for 5 s (11). Mycobacterium smegmatis, a gift of Eric Rubin of the Harvard School of Public Health, was a positive control, while Saccharomyces cerevisiae was a negative control. Histology slides were acid-fast stained by the Ziehl-Neelsen method, using methylene blue as a counterstain. For fluorescent acid-fast stains, heat-fixed slides were stained with auramine-O (Polysciences kit 24665) for 30 min at room temperature and destained in ethanol-HCl solution for 30 s at room temperature (16). Slides were examined with a DeltaVision deconvolving microscope (Applied Precision, Issaquah, WA), using the filters for fluorescein. Images were taken at 100× primary magnification and deconvolved using Applied Precision’s softWoRx software. Broken oocysts of Toxoplasma and Eimeria were incubated with Alexa Fluor-labeled dectin-1, as previously described (8). Dityrosine autofluorescence of oocysts of Toxoplasma and Eimeria was observed in the UV channel and photographed.

Electron microscopy of oocysts treated with proteases and organic solvents.

Oocysts of Cryptosporidium were washed and broken with glass beads, and walls were isolated as previously described (8). Walls were left untreated, extracted in chloroform-methanol (2:1) for 3 h, or treated with 10 µg/ml pronase or 1 mg/ml chymotrypsin, for 3 h at 37°C. Sonicated treated or untreated Cryptosporidium oocyst walls were washed in PBS, fixed in aldehydes containing ruthenium red, and prepared for transmission electron microscopy (TEM), as previously described (8). Unsporulated oocyst walls of Toxoplasma and Eimeria were broken with glass beads, isolated by centrifugation, and deproteinated with 1 N sodium hydroxide for 60 min at 80°C. Alternatively, pelleted broken walls of Toxoplasma and Eimeria were extracted with 50 volumes of chloroform-methanol (2:1) or hexane isomers overnight at room temperature. As a control, intact Saccharomyces cells were treated with chloroform-methanol or sodium hydroxide. Treated and untreated walls of the parasites and fungi were prepared for TEM and negative staining, as previously described (8).

Quantitative fluorescence assays.

Treated and untreated broken oocyst walls of Cryptosporidium in PBS were pipetted into wells of black 96-well plates (Greiner Bio-One), left to dry overnight at 37°C, heat fixed, and acid-fast stained with auramine-O. Auramine-O acid-fastness of triplicate samples of oocyst walls was measured with a fluorimeter using 410-nm excitation and 500-nm emission wavelengths, and the experiment was repeated 3 times. For quantitation of binding of auramine-O, dectin-1, and UV autofluorescence, treated and untreated Eimeria walls were fixed to 96-well plates and stained or labeled, and fluorescence was measured for auramine-O using methods described above. The excitation/emission wavelengths were 495/519 nm for Alexa Fluor 488-labeled dectin-1 and 360/457 nm for autofluorescence.

Mass spectrometry of oocyst proteins.

Sporulated and unsporulated oocysts of Toxoplasma (VEG strain) and Eimeria (1 to 2 million oocysts each) were extensively washed and broken with glass beads. Oocyst proteins were extracted by breaking unsporulated oocysts in 2% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} with complete protease inhibitor cocktail lacking EDTA (Roche). Tryptic peptides were prepared and analyzed with the LTQ-Orbitrap Discovery ETD hybrid tandem mass spectrometer (Thermo-Fisher Scientific, Inc., Waltham, MA), as previously described (21). The predicted proteins of Toxoplasma and Eimeria at EupathDB and Mascot were used to identify tryptic peptides, the bulk of which will be reported elsewhere. Data acquisition and analysis were performed with XCalibur software (Thermo, Fisher Scientific). In Table S1 in the supplemental material, the number of unique peptides and percentage of coverage are shown for the 10 most abundant cytosolic proteins of Toxoplasma oocysts.

RT-PCR of oocyst mRNAs.

RNA was extracted from Toxoplasma (ME49 strain) and Eimeria unsporulated and sporulated oocysts using PureLink RNA minikit (Life Technologies) by breaking the oocysts with glass beads in the extraction buffer. Reverse transcription-polymerase chain reactions (RT-PCR) were performed using SuperScript III kit (Life Technologies) with 30 ng of total RNA per sample, according to the manufacturer’s instructions. Primers were designed to produce products that span several exons to distinguish RNA from potential DNA products. Toxoplasma primers were to the PKS1 (TGME49_294820), PKS2 (TGME49_204560), PPTase (TGME49_214440), and actin (TGME49_209030) genes (shown in Table S3 in the supplemental material) (10). Eimeria primers were to the PKS1 (ETH_00015480), PKS2 (ETH_00005790), PPTase (ETH_00040195), DGAT1 (ETH_00032635), DGAT2 (ETH_00034355), ACAT (ETH_00032235), and actin (ETH_00009555) genes (see Table S3). Products were analyzed on agarose gels with ethidium staining. There was no attempt at quantitation.

Extraction of lipids from oocyst walls and analysis with high-resolution and high-accuracy mass spectrometry.

Oocysts were broken using glass beads in a bead beater and washed extensively in PBS and high-performance liquid chromatography (HPLC)-grade water. Oocyst walls were dried, extracted in 2:1 chloroform-methanol overnight, and centrifuged to remove insoluble material. Extracted wall lipids were analyzed with a 12-T solariX hybrid Qq-FTICR mass spectrometer (Bruker Daltonics, Billerica, MA) (26). The collision voltage was varied between 18 V and 30 V for fragmentation of the selected triglycerides, and argon was used as the collision gas. DataAnalysis 4.0 (Bruker Daltonics) was used for data analysis. The lipids were manually identified by use of elemental composition and CID fragmentation patterns. Six biological replicates of Eimeria lipids, four of Toxoplasma, and three of Cryptosporidium were examined by mass spectrometry.

SUPPLEMENTAL MATERIAL

Figure S1

Acid-fast stains of sporulated Toxoplasma and Eimeria and RT-PCRs for selected enzymes. This figure adds to data shown in Fig. 1 and 2 of the main text. (A) Bright-field acid-fast stains (Kinyoun method) of Mycobacterium smegmatis and sporulated Toxoplasma. Bacteria and sporocyst walls stain red and so contain acid-fast lipids. Black size bars represent 10 µm. (B) Permeabilized sporocyst of Toxoplasma shows auramine-O staining of sporocyst wall (S), as well as refractile bodies (RB). In contrast, a sporulated oocyst of Eimeria shows staining only in oocyst wall (O) and in refractile bodies (RB). Size bars represent 5 µm. (C) RT-PCR was used to detect mRNAs for polyketide synthases and PPTase in unsporulated (U) and sporulated (S) oocysts of Toxoplasma and Eimeria. (D) RT-PCR was used to detect mRNAs for diacylglycerol acyltransferase (DGAT1 and DGAT2) and acetyl-CoA:cholesterol acyltransferase (ACAT) in unsporulated (U) and sporulated (S) oocysts of Eimeria. Primers for RT-PCR are shown in Table S3 in the supplemental material. Download

Figure S2

Hexane treatment of oocyst walls of Eimeria. This figure adds to data shown in Fig. 4 of the main text. (A) Untreated, broken, and washed Eimeria oocyst walls are acid-fast with auramine-O. Hexane-treated, broken, and washed Eimeria oocyst walls stain with dectin-1. Size bars represent 5 µm. (B) Hexane, which removes dityrosines and acid-fast lipids, does not affect dectin-1 binding to β-1,3-glucan. Download

Table S1

Unique peptides and percentage of coverage for the 10 most abundant oocyst proteins of Toxoplasma.

Table S2

Triglycerides identified in extracts of oocyst walls of Eimeria, Toxoplasma, and Cryptosporidium.

Table S3

RT-PCR primers for Toxoplasma and Eimeria oocyst mRNAs.

ACKNOWLEDGMENTS

We thank our colleagues at Boston University, including Anirban Chatterjee for help with TEM of Cryptosporidium, Esther Bullitt for help with negative stains, and Rudolf Beiler for help with chicken infections with Eimeria. We thank Ray Fetterer of the USDA for Eimeria oocysts and to Eric Rubin of the Harvard School of Public Health for Mycobacterium smegmatis.

This work was supported in part by grants from the National Institutes of Health (NIH) (AI48082 to J.S., AI07642 [T32] to G.G.B., RR010888, GM104603, and RR015942 to C.E.C., and GM31318 to P.W.R.). Additional support came from the Mizutani Foundation for Glycoscience.

Footnotes

Citation Bushkin GG, Motari E, Carpentieri A, Dubey JP, Costello CE, Robbins PW, Samuelson J. 2013. Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. mBio 4(5):e00387-13. doi:10.1128/mBio.00387-13.

REFERENCES

1. Belli SI, Smith NC, Ferguson DJ. 2006. The coccidian oocyst: a tough nut to crack! Trends Parasitol. 22:416–423. [PubMed]
2. Dubey JP, Lindsay DS, Speer CA. 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11:267–299. [PMC free article] [PubMed]
3. Weiss LM, Dubey JP. 2009. Toxoplasmosis: a history of clinical observations. Int. J. Parasitol. 39:895–901. [PMC free article] [PubMed]
4. Ferguson DJ, Belli SI, Smith NC, Wallach MG. 2003. The development of the macrogamete and oocyst wall in Eimeria maxima: immuno-light and electron microscopy. Int. J. Parasitol. 33:1329–1340. [PubMed]
5. Chapman HD, Jeffers TK, Williams RB. 2010. Forty years of monensin for the control of coccidiosis in poultry. Poult. Sci. 89:1788–1801. [PubMed]
6. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O’Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM. 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382:209–222. [PubMed]
7. Chatterjee A, Banerjee S, Steffen M, O’Connor RM, Ward HD, Robbins PW, Samuelson J. 2010. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryot. Cell 9:84–96. [PMC free article] [PubMed]
8. Bushkin GG, Motari E, Magnelli P, Gubbels MJ, Dubey JP, Miska KB, Bullitt E, Costello CE, Robbins PW, Samuelson J. 2012. β-1,3-Glucan, which can be targeted by drugs, forms a trabecular scaffold in the oocyst walls of Toxoplasma and Eimeria. mBio 3(5):e00258-12. doi: 10.1128/mBio.00258-12. [PMC free article] [PubMed] [Cross Ref]
9. Mai K, Smith NC, Feng ZP, Katrib M, Slapeta J, Slapetova I, Wallach MG, Luxford C, Davies MJ, Zhang X, Norton RS, Belli SI. 2011. Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima. Int. J. Parasitol. 41:1157–1164. [PubMed]
10. Aurrecoechea C, Heiges M, Wang H, Wang Z, Fischer S, Rhodes P, Miller J, Kraemer E, Stoeckert CJ, Jr, Roos DS, Kissinger JC. 2007. ApiDB: integrated resources for the apicomplexan bioinformatics resource center. Nucleic Acids Res. 35:D427–D430. [PubMed]
11. Garcia LS, Bruckner DA, Brewer TC, Shimizu RY. 1983. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J. Clin. Microbiol. 18:185–190. [PMC free article] [PubMed]
12. Layre E, Sweet L, Hong S, Madigan CA, Desjardins D, Young DC, Cheng TY, Annand JW, Kim K, Shamputa IC, McConnell MJ, Debono CA, Behar SM, Minnaard AJ, Murray M, Barry CE, III, Matsunaga I, Moody DB. 2011. A comparative lipidomics platform for chemotaxonomic analysis of Mycobacterium tuberculosis. Chem. Biol. 18:1537–1549. [PMC free article] [PubMed]
13. Yamada H, Bhatt A, Danev R, Fujiwara N, Maeda S, Mitarai S, Chikamatsu K, Aono A, Nitta K, Jacobs WR, Jr, Nagayama K. 2012. Non-acid-fastness in Mycobacterium tuberculosis DeltakasB mutant correlates with the cell envelope electron density. Tuberculosis (Edinb) 92:351–357. [PubMed]
14. Portevin D, De Sousa-D’Auria C, Houssin C, Grimaldi C, Chami M, Daffé M, Guilhot C. 2004. A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc. Natl. Acad. Sci. U. S. A. 101:314–319. [PubMed]
15. Beisson F, Li-Beisson Y, Pollard M. 2012. Solving the puzzles of cutin and suberin polymer biosynthesis. Curr. Opin. Plant Biol. 15:329–337. [PubMed]
16. Hendry C, Dionne K, Hedgepeth A, Carroll K, Parrish N. 2009. Evaluation of a rapid fluorescent staining method for detection of mycobacteria in clinical specimens. J. Clin. Microbiol. 47:1206–1208. [PMC free article] [PubMed]
17. Buda GJ, Isaacson T, Matas AJ, Paolillo DJ, Rose JK. 2009. Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy. Plant J. 60:378–385. [PubMed]
18. Gulder TA, Freeman MF, Piel J. 1 March 2011. The catalytic diversity of multimodular polyketide synthases: natural product biosynthesis beyond textbook assembly rules. Top. Curr. Chem.[Epub ahead of print.] [PubMed]
19. Zhu G, LaGier MJ, Stejskal F, Millership JJ, Cai X, Keithly JS. 2002. Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 298:79–89. [PubMed]
20. Chiang YM, Oakley BR, Keller NP, Wang CC. 2010. Unraveling polyketide synthesis in members of the genus Aspergillus. Appl. Microbiol. Biotechnol. 86:1719–1736. [PMC free article] [PubMed]
21. Mauzy MJ, Enomoto S, Lancto CA, Abrahamsen MS, Rutherford MS. 2012. The Cryptosporidium parvum transcriptome during in vitro development. PLoS One 7:e31715. doi: 10.1371/journal.pone.0031715. [PMC free article] [PubMed] [Cross Ref]
22. Cai X, Herschap D, Zhu G. 2005. Functional characterization of an evolutionarily distinct phosphopantetheinyl transferase in the apicomplexan Cryptosporidium parvum. Eukaryot. Cell 4:1211–1220. [PMC free article] [PubMed]
23. Lesage G, Bussey H. 2006. Cell wall assembly in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 70:317–343. [PMC free article] [PubMed]
24. Drummond RA, Brown GD. 2011. The role of Dectin-1 in the host defence against fungal infections. Curr. Opin. Microbiol. 14:392–399. [PubMed]
25. Possenti A, Cherchi S, Bertuccini L, Pozio E, Dubey JP, Spano F. 2010. Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation. Int. J. Parasitol. 40:1639–1649. [PubMed]
26. Hein EM, Blank LM, Heyland J, Baumbach JI, Schmid A, Hayen H. 2009. Glycerophospholipid profiling by high-performance liquid chromatography/mass spectrometry using exact mass measurements and multi-stage mass spectrometric fragmentation experiments in parallel. Rapid Commun. Mass Spectrom. 23:1636–1646. [PubMed]
27. Quittnat F, Nishikawa Y, Stedman TT, Voelker DR, Choi JY, Zahn MM, Murphy RC, Barkley RM, Pypaert M, Joiner KA, Coppens I. 2004. On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme. Mol. Biochem. Parasitol. 138:107–122. [PubMed]
28. Ramakrishnan S, Docampo MD, Macrae JI, Pujol FM, Brooks CF, van Dooren GG, Hiltunen JK, Kastaniotis AJ, McConville MJ, Striepen B. 2012. Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287:4957–4971. [PubMed]
29. Striepen B, Soldati D. 2007. Genetic manipulation of Toxoplasma gondii, p 391–418 In Kim LM, Kim K (ed), Toxoplasma gondii, the model apicomplexan: perspectives and methods. Academic Press, London, United Kingdom.
30. Johnston WL, Dennis JW. 2012. The eggshell in the C. elegans oocyte-to-embryo transition. Genesis 50:333–349. [PubMed]
31. Samuelson J, Robbins P. 2011. A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia. Trends Parasitol. 27:17–22. [PMC free article] [PubMed]
32. Remmal A, Achahbar S, Bouddine L, Chami N, Chami F. 2011. In vitro destruction of Eimeria oocysts by essential oils. Vet. Parasitol. 182:121–126. [PubMed]
33. Mohanty D, Sankaranarayanan R, Gokhale RS. 2011. Fatty acyl-AMP ligases and polyketide synthases are unique enzymes of lipid biosynthetic machinery in Mycobacterium tuberculosis. Tuberculosis (Edinb) 91:448–455. [PubMed]

Articles from mBio are provided here courtesy of American Society for Microbiology (ASM)