Cryptosporidium andersoni oocyst preparation and excystation
Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows (without other pathogenic microorganisms) according to previous reports with appropriate modifications . Briefly, feces of cows were washed three times with phosphate-buffered saline (PBS), and a preliminary purification using Sheather’s sugar flotation method was used to remove impurities (incompletely digested silage), followed by further purification using cesium chloride gradient centrifugation to obtain pure oocysts. Purified C. andersoni oocysts were counted using a hemocytometer and stored at 4 °C in PBS (pH 7.2) for no longer than 14 days.
For proteomic analysis, a total of 1.8 × 109 C. andersoni oocysts were equally split into non-excysted and excysted groups (9 × 108 oocysts/group). Each group contained three biological replicates (3 × 108 each). Before the experiments, oocysts were subjected to the treatment in 2.5% sodium hypochlorite solution for 10 min at 4 °C and washed three times with PBS. Excystation was performed at 37 °C (~ 3 h and mixing every 10 min) until > 80% excystation was observed by microscopic examination .
The morphology of oocysts and sporozoites was also examined using differential interference contrast (DIC) microscopy (Olympus BL53, Tokyo, Japan) and scanning electron microscopy (SEM). For SEM, specimens were fixed overnight at 4 °C in 2.5% glutaraldehyde in 0.1 M phosphate buffer and then washed twice for 15 min with the same buffer. After dehydration in a graded ethanol series, the ethanol was replaced with isoamyl acetate twice for 20 min, dried using the critical point technique, and coated with gold according to a standard protocol. Specimens were examined under a Hitachi S-3400N scanning electron microscope (Tokyo, Japan).
Protein extraction and SDS-PAGE
Excysted or non-excysted oocysts were suspended in a protein lysis buffer (pH 8.5) containing 7 M urea, 2 M thiourea, 65 mM Tris, 2% dithiothreitol (DTT), 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% IPG buffer (GE Amersham, Boston, USA), and 0.1% v/v protease inhibitor cocktail (Merck, Darmstadt, Germany) . Samples were disrupted by sonication at 80 W for 3 s × 100 at intervals of 10 s. The debris was removed by centrifugation at 12,000×g at 4 °C for 10 min. The supernatants were collected and transferred to new centrifuge tubes. After that, protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China) according to the manufacturer’s instructions. The quality of protein extracts was evaluated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation, followed by Coomassie blue staining.
Trypsin digestion, TMT labeling, and high-performance liquid chromatography fractionation
TMT tagging and analysis were performed as previously described . For protein digestion, the protein extracts were first reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Samples were diluted by adding 100 mM triethylammonium bicarbonate (TEAB) to urea at a concentration less than 2 M. Trypsin was then added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and at a 1:100 trypsin-to-protein mass ratio for a second digestion for 4 h. After digestion, samples were desalted using a Strata-X C18 SPE column (Phenomenex) and vacuum-dried. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit (Thermo Fisher, Waltham, MA, USA). Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted, and dried by vacuum centrifugation. The tryptic peptides were fractionated by high-pH reversed-phase high-performance liquid chromatography (HPLC) using an Agilent 300 Extend C18 column (5-μm particles, 4.6 mm I.D., 250 mm in length). Peptides were separated into 60 fractions with a gradient of 8–32% acetonitrile (pH 9.0) over 60 min, combined into 18 fractions, and dried by vacuum centrifugation.
LC–MS/MS analysis and database search
The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and loaded onto a homemade reversed-phase analytical column (15-cm length, 75 µm I.D.). The gradient comprised an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23%–35% over 8 min, increasing to 80% in 3 min, and then holding at 80% for the final 3 min. All operation procedures occurred at a constant flow rate of 400 nl/min on an EASY-nLC 1000 ultrahigh-performance LC system .
The peptides were subjected to a nanospray ionization (NSI) source, followed by tandem mass spectrometry (MS/MS) in a Q Exactive™ Plus system (Thermo Fisher, Waltham, MA, USA) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350–1800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using the NCE setting of 28. The fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion was used. Automatic gain control (AGC) was set at 5E4. The fixed first mass was set as 100 m/z .
The resulting MS/MS data were processed using the MaxQuant search engine (v.22.214.171.124). Tandem mass spectra were searched against the Cryptosporidium proteome database (28,217 sequences) from the UniProt database (http://beta.uniprot.org/) concatenated with the reverse decoy database. Trypsin/P was specified as a cleavage enzyme, allowing up to two missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and oxidation on Met was specified as a variable modification. The false discovery rate (FDR) was adjusted to < 1.0%, and the minimum score for peptides was set as > 40.
Gene Ontology (GO) annotation of the proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) that classified proteins into three categories: biological process, cellular compartment, and molecular function. The Eukaryotic Orthologous Groups (KOG) database was used for functional classification of differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate metabolic pathways. For GO, KOG, and KEGG enrichment analyses, a two-tailed Fisher’s exact test was applied to test DEPs against all identified proteins, and a corrected p-value < 0.05 was considered significant. Identified protein domain functional descriptions were annotated by InterProScan based on the protein sequence alignment method (http://www.ebi.ac.uk/interpro/). The WoLF PSORT program was used to predict subcellular localization (https://www.genscript.com/wolf-psort.html), using an updated version of PSORT/PSORT II for the prediction of eukaryotic sequences.
Validation of differentially expressed genes by quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify gene expression levels of eight DEPs in non-excysted and excysted C. andersoni oocysts . Total RNA of each sample was extracted from excysted and non-excysted oocysts (three biological replicates) using TRIzol™ Reagent (Thermo Fisher, Waltham, MA, USA). RNA purification and reverse transcription were performed using the Reverse Transcriptase M-MLV Kit with gDNA Eraser (Takara, Japan) according to manufacturer’s instructions. The quantity of RNA was analyzed using a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA). For each sample, 1 µg of total RNA was treated with 1 µl of gDNA Eraser at 42 °C for 2 min. First-strand complementary DNA (cDNA) was synthesized using 1 µl of Oliga (dT) and 1 µl of Random Primers and RNase-free deionized H2O (up to 10 µl) at 70 °C for 10 min and at 4 °C for 2 min. Second-strand cDNA was synthesized using 4 µl of 5× M-MLV buffer, 1 µl of dNTP, 0.5 µl of RI, 0.5 µl of M-MLV, 4 µl RNase-free dH2O, and 10 µl of first-strand cDNA. The resulting products were used as templates for qRT-PCR. Gene-specific qRT-PCR primers were designed with Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). Oligonucleotide sequences of target and reference genes (18S rRNA) for qRT-PCR are listed in Additional file 1: Table S1. Each PCR reaction contained 2 µl of cDNA, 0.4 µM (final concentration) of each primer, and 5 µl of 2× SYBR qPCR mix (Takara, Japan). PCR reactions were performed in duplicate using the qTOWER3G IVD system (Analytik Jena AG, Germany) with the following cycles: one cycle for denaturing at 95 °C for 30 s; 40 cycles for amplification at 95 °C for 5 s, 55 °C for 10 s, and 72 °C for 15 s; and one cycle of melting curve analysis. Relative expression levels were normalized with those of 18S rRNA. The 2−ΔΔCT method was used to determine the fold change. GraphPad Prism version 8.0 software (https://www.graphpad.com/) was used to analyze and plot the data.