Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm’s excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.


Introduction
Parasitic infections caused by blood-feeding hookworms are an ongoing global health threat. Hookworms are nematode roundworms of the Ancylostomatidae family, a part of the Strongylida order [1]. Humans are infected with hookworms through ingestion and/or skin penetration of the infectious third-stage larvae (iL3). Worldwide, Ancylostoma duodenale and Necator americanus were believed to be the most prevalent hookworm species, but more recently, Ancylostoma ceylanicum appeared as an important human hookworm parasite in some countries [2]. More than 500 million people including children are affected [3],

Hamster Gavage Procedure
Hamsters were gavaged using the appropriate technique and were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). Freshly isolated infective A. ceylanicum L3 larvae were counted and distributed in individual Eppendorf tubes, and 80-100 L3 were resuspended per tube in 200 µL of PBS. A curved 18G needle was used to gavage the hamsters. Hamsters were manually restrained by grasping the loose skin at the scruff of the neck with the thumb and forefinger to immobilize the head and torso. Using the needle held next to the animal, the distance from the tip of the nose to the last rib on the left side was measured. Holding the animal with the nose pointing up, the gavage needle was entered into the mouth over the tongue and directed toward the esophagus on the left side of the throat, gently pressing the needle on the back of the mouth and allowing the animal to swallow the needle. The needle was slowly advanced to the measured distance into the stomach. Once the needle was properly positioned, the plunger was gently depressed to dispense~200 µL of resuspended L3 larvae to the hamster. Once the entire inoculum was dispensed, the gavage needle was slowly removed, and the animal was returned to its cage. Gavaged hamsters were observed for any difficulty breathing or for bleeding from the mouth or nose for at least 5 min before returning them to the animal holding room.

Isolation of Adult Ancylostoma Ceylanicum
Twenty-one days post-infection (dpi) with 80 infective A. ceylanicum iL3, three Golden/Syrian hamsters were euthanized using the appropriate technique, equipment, and agents. Compressed CO 2 gas in cylinders was used as a source of carbon dioxide, allowing for the controlled inflow of gas to the induction chamber. Hamsters to be euthanized were placed in a clean transparent chamber 100% CO 2 was introduced at a fill rate of 70% of the chamber volume per minute, to achieve a balanced gas mixture with the existing air in the chamber to fulfill the objective of rapid unconsciousness with minimal distress to the animals. The expected time to unconsciousness was usually within 3-5 min. Each hamster was monitored for lack of respiration and faded eye color. CO 2 was maintained for a minimum of 1 min after observing these signs to avoid unintended recovery upon exposure to natural CO 2 concentrations. Upon completion of the procedure, death was confirmed by ascertaining cardiac and respiratory arrest and noting an animal's fixed and dilated pupils. Following this euthanization procedure, the small intestines were removed and longitudinally cut open. The freshly recovered adult A. ceylanicum were gently washed three times in 1× PBS containing 5× Antibiotics/Antimycotics (Gibco, Waltham, MA, USA). Isolation of adult hookworms from hamsters was performed as previously described [23].

Excretory-Secretory Products (ESPs) Collection
Briefly, adult A. ceylanicum were collected in a 10 mm Petri Dish and placed on the inverted microscope stage with a 20× objective lens (Nikon Eclipse T S 2). The freshly recovered adult A. ceylanicum were sorted with a nail brush under the microscope to group into females and males. Each group was cultured in three biological replicates at a density of 25 worms/well in 24 well plates. The worms were first cultured for 4 h in a humidified incubator at 37 • C, 5% CO 2 , Dulbecco's Modification of Eagle's Medium (DMEM) (Corning, Corning, NY, USA) supplemented with 2% Antibiotic-Antimycotic (Gibco) and 10% Fetal Bovine Serum (Sigma, St. Louis, MI, USA) [24]. The supernatant was discarded, and the worms were gently rinsed twice in 1× PBS containing 5× Antibiotics/Antimycotics. Afterward, the worms were subcultured in 2 mL serum-free DMEM supplemented with 2% Antibiotic-Antimycotic at 37 • C, and 5% CO 2 [24]. Every 24 h, over 3 days, at least 1.8 mL of culture supernatant was aspirated from each well with minimal disturbance to the hookworms. Each well was replaced with fresh serum-free medium, and plates were transferred to the incubator, as described [6]. The drawn culture supernatant was centrifuged at 4000× g at 4 • C for 30 min and transferred (all but the last 1.6 mL) to a new centrifuge tube and stored at −80 • C. Using this method for further media changes prevented voided eggs and remaining hamster or dead worm tissue from contaminating the ESPs [6]. After 72 h, no significant alterations in motility or morphological integrity of the worms were detected, suggesting that the drawn media was comprised of ESPs from the normal physiological activity of healthy adult A. ceylanicum. However, it should be noted that these ESPs are from worms collected from a hamster host and cultured in media to facilitate the collection, but differences may exist in ESPs actually produced in a human intestine.
At the end of 72 h of media collection, the culture supernatant containing ESPs was gradually thawed in ice. To maximize the peptide yield, the three biological replicates were pooled into a 15 mL tube. Approximately, 14 mL of culture supernatant was recovered from 24 to 72 h of media collection per group. The crude ESPs were further cleaned up by centrifugation at 7000× g at 4 • C for 30 min before concentration and transferred (all but the last 13 mL) to a new tube. The culture media containing ESPs was concentrated using 20 mL Pall Macrosep Advance Centrifugal Devices with a defined 3000 Da molecular weight cutoff membrane. Briefly, 13 mL of culture supernatant was placed into the concentrator sample chamber and capped. The concentrator was then placed into the bucket rotor that contains a 20 mL tube. Concentration by 3K Pall membrane was performed at 5000× g at 4 • C until the retention volume reaches 0.5 mL.

Quantification of Protein Concentration
For total proteomic profiling of adult female and male A. ceylanicum proteins, daily ESPs collections of each sex were pooled and quantified. ESPs concentrations were quantified using a Pierce Coomassie Plus Bradford Protein Assay (Thermo Scientific, Waltham, MA, USA). A BSA standard at 1 mg/mL was dissolved in DMEM (Corning) supplemented with 2% Antibiotic-Antimycotic (Gibco). Separately, 5 µL of each standard and ESPs sample were then added to 395 µL of 1× Bradford dye in a 96-well plate. Following 5 min of incubation in the dark at room temperature, the optical density (OD) was measured at 595 nm using Eon Microplate Spectrophotometer (Biotek Instruments, Winooski, VT, USA). A standard curve was generated by plotting the graph of OD (y-axis, nm) versus protein concentration (x-axis, µg/mL). The protein concentrations of both female and male ESPs were then estimated based on linear extrapolation from the standard curve.

SDS-PAGE
Approximately 3 µg of A. ceylanicum adult ESPs was mixed with 2× concentrated Laemmli-SDS buffer (Bio-Rad) with 5% 2-Mercaptoethanol (Sigma-Aldrich). The sample was heated at 95 • C for 5 min and 50 µL of protein sample was loaded into the wells of 12% Mini-PROTEAN TGX gel (Bio-Rad). The precision protein standard (Bio-Rad) was loaded into the first well at 10 µL without dilution with an SDS sample buffer. Electrophoresis was run for 50 min at 150 V using 1× Tris/Glycine buffer (Bio-Rad), [6]. The gel was fixed in (40% ethanol and 10% acetic acid), washed with distilled water, and stained with QC Colloidal Coomassie Stain (Bio-Rad). Gels were imaged in Azure Imaging Systems (C600, Azure Biosystems, Dublin, CA, USA) under LED visible fluorescence detection mode (data not shown).

Sample Preparation for Mass Spectrometry
Concentrated media samples (300 µL of control media, 150 µL of male/female worm media) were precipitated in −20 • C acetone (1 part of the sample to 4 parts cold acetone (vol/vol)) overnight followed by centrifugation at 20,000 rcf at for 10 min. Supernatants were removed and media protein pellets were solubilized in 20 µL lysis buffer for 2 h at 25 • C in a ThermoMixer (Eppendorf) with shaking at 1000 rpm. Trypsin (Promega, Cat. No. V511C) (1 µg) was added and digestion was performed in the ThermoMixer (100 rpm) overnight at 25 • C. The digestion was quenched by the addition of concentrated formic acid (Fluka) to a final concentration of 1% (vol/vol). Peptides were desalted using stage tips [25] and eluted in 50% (vol/vol) MeCN containing 0.1% (vol/vol) FA. An aliquot (1%) was removed for quantification using the Pierce Quantitative Fluorometric Peptide Assay kit (ThermoFisher, Cat. No. 23290). The remaining peptides were transferred to autosampler vials (Sun-Sri, Cat. No. 200046), dried, and stored at −80 • C.

UPLC-MS
The samples were analyzed using ultra-high-performance mass spectrometry [26] using a hybrid quadrupole Orbitrap LC-MS System, Q-Exactive™ PLUS interfaced to an EASY-nanoLC 1000). A 75 µm i.d. × 50 cm Acclaim PepMap 100 C18 RSLC column (Thermo Scientific™) was equilibrated with 100% solvent A (1%FA) on the nano-LC for a total of 11 µL at 700 bar pressure. Samples in 1% FA (vol/vol) were loaded at a constant pressure of 700 bar. Peptide chromatography was initiated with mobile phase A (1% FA) containing 2% solvent B (100%ACN, 1% FA) for 5 min, then increased to 20% B over 100 min, to 32% B over 20 min, to 95% B over 1 min and held at 95% B for 19 min, with a flow rate of 250 nl/min. Data were acquired in data-dependent mode. Full-scan mass spectra were acquired with the Orbitrap mass analyzer using a scan range of m/z = 325 to 1800 and a mass resolving power set to 70,000. Ten data-dependent high-energy collisional dissociations were performed with a mass resolving power at 17,500, a fixed lower value of m/z 110, an isolation width of 2 Da, and a normalized collision energy setting of 27. The maximum injection time was 60 ms for parent-ion analysis and production analysis. Ions that were selected for MS/MS were dynamically excluded for 30 sec. The automatic gain control was set at a target value of 1 × 10 −6 ions for full MS scans and 1 × 10 −5 ions for MS2. Peptide ions with charge states of one were excluded from MS2 acquisition. The MS2 spectra from peptides with +2, +3, and +4 charge states were analyzed using Mascot software [27] (Matrix Science, London, UK; version 2.5.1). The mascot was searched against the improved A. ceylanicum proteome (22,946 entries, described above), using trypsin enzyme specificity, with a maximum of 2 missed cleavages allowed/peptide. The searches were performed with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20 ppm. Carbamidomethylation of cysteine was specified as a fixed modification. Deamidation of asparagine, deamidation of glutamine, acetylation of protein N-terminus, and oxidation of methionine were entered as variable modifications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org, accessed on 1 November 2022) via the iProX partner repository [28] with the dataset identifier PXD037410, under project ID IPX0005214000. The accessions for each sample are IPX0005214001 (Adult_Female_ESPs), IPX0005214002 (Adult_Male_ESPs), and IPX0005214003 (Media-only control). The dataset on iProX includes the .sf3-format Scaffold output file (as described below).
Scaffold (version Scaffold_5.1.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 7.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [46]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Peptide-spectral match (PSM) counts for each peptide sequence are provided in Supplementary Table S2, and the raw scaffold output file with editable threshold values is provided as part of the iProX dataset (PXD037410).

Bioinformatic Analysis of Proteomic Sequence Data
Peptide and peptide-spectral-match (PSM) counts were quantified per protein according to the identifications from the Mascot/Scaffold pipeline described above. Proteins in each sample were intersected to identify proteins only detected in the adult female E/S, the adult male E/S, or both adult female and male E/S. The newly annotated A. ceylanicum proteins were assigned functional annotations using results from InterProScan v5.42 [40] to identify gene ontology [47] classifications and InterPro functional domains [48], and GhostKOALA v2.2 [49] to assign KEGG [50] annotations. Potentially secreted proteins were identified using both SignalP v5.0 [51] for signal peptides and transmembrane domains, and SecretomeP v2.0 [52] to identify proteins with non-classical secretion sequences (where any proteins with 2 or more transmembrane domains were not classified as secreted). Additional protein naming was performed using PANNZER [53] and Sma3s [54]. Significant functional enrichment for GO terms was performed using GOSTATS v2.50 [55] and for Interpro domains and KEGG pathways using WebGestalt v2019 [56] (FDR-adjusted p ≤ 0.05, minimum 3 genes differentially expressed, for all enrichment testing).
Protein conservation data across nematodes and hosts were quantified using BLAST [57] and OrthoFinder [58]. Protein sequence data were downloaded from WormBase Parasite [38] (WBPS16) for A. duodenale (PRJNA72581) and N. brasiliensis (PRJEB511). The N. americanus proteins were based on the improved annotation of the genome (PRJNA72135) presented in Logan et al., 2020 [6], and available for download on Nematode.net [43,44]. The A. caninum protein annotation was based on [59]. In addition, Homo sapiens (Human; GRCh38.103) was downloaded from Ensembl [60]. For BLAST comparisons, the top BLAST [57] hit for each A. ceylanicum protein was identified, including the E value, alignment length, % identity, and whether the top hit was reciprocal (NCBI blastp v2.7.1+, default settings). Orthofinder [58] (v2.4.1) was used to compare protein sequences across all species and define orthologous protein families (OPFs), using default values. E/S proteins identified in N. americanus were retrieved from Logan et al., 2020 [6] and aligned to all A. ceylanicum proteins using the BLAST results.

Improved Genome Annotation Identified~10% More Peptides in Male and Female Hookworms
As a first step in the analysis, genome annotation improvement was performed to ensure the best possible and most complete characterization of the ES proteome. We re-annotated the genome of A. ceylanicum [29] using an improved gene calling pipeline utilizing RNA-seq data [39] and including more extensive up-to-date homologous protein databases ( Table 1). The total number of predicted protein-coding genes decreased by 17,904 from 36,687 to 18,783 with substantial increases in both the mean exon and intron lengths. The mean coding sequence (CDS) length increased by 393 bp with the mean gene length (including introns and UTRs) more than doubling from 3.8 kb to 10 kb. The percentage of BUSCOs [42] detected among the predicted genes increased from 92.5% to 96% (with a 72% reduction in the number of fragmented BUSCOs). Across both samples, 700 of the peptide sequences (7.94% of the 8818 total peptides) detected using the improved annotation were not part of the previous genome/proteome annotation for A. ceylanicum [29]. These peptides represented 10.8% and 9.7% of the total detected spectra in the adult female and male ESPs samples, respectively.

Female and Male Hookworm Secrete Similar Number of Proteins in Culture
Applying ultra-high-performance mass spectrometry methods using a hybrid quadrupole Orbitrap LC-MS system, a total of 795 unique proteins were detected (from 20.79 µg of female and 8.41 µg proteins in the male ESPs), with no peptides identified in the mediaonly control sample. Peptides and spectral counts from A. ceylanicum ESPs proteomics samples were identified using a Mascot/Scaffold pipeline (1% FDR peptide threshold, 95% protein confidence threshold, minimum 2 peptides per protein). Female and male hookworm secrete a similar number of ESPs in culture (670 vs. 684, respectively). Some 29.7% (n = 236) of all detected proteins were uniquely identified in female and male ESPs ( Figure 1A). The relative abundance of all detected proteins in the adult male and adult female is shown for the two samples in Figure 1B, with most proteins having relatively similar abundance in both male and female ESPs. Figure 1C shows the adult-stage normalized mRNA levels (RNA-seq FPKM values) in female and male A. ceylanicum based on results from our previous study [45]. The female-specific ES proteins (orange) also have substantially higher mRNA levels in the female vs. male (below the diagonal). Likewise, many of the male-specific ES proteins (blue) also have substantially higher mRNA levels in the males vs. females (above the diagonal). This analysis shows that the sex-specific ESPs also show sex-specific mRNA levels, which supports their identification using an independent dataset.

Conserved ESPs in Female and Male A. Ceylanicum
A total of 559 proteins were detected in both female and male hookworm (Supplementary Table S1). Table 2 describes the 20 most abundant proteins in both female and male A. ceylanicum. As expected for ESPs, the proteins detected in both females and males were significantly enriched for signal peptides for secretion (63.9% of detected ESPs, vs. 18.6% for all proteins; p < 10 −10 , binomial distribution test, MS Excel; the threshold for significance p ≤ 0.01). They were also enriched for CAP domains (22.2% of proteins vs. 1.9% for all proteins; p < 10 −10 ), which belong to sperm-coating protein (SCP)-like proteins, also known as SCP/Tpx-1/Ag5/PR-1/Sc7 domain-containing proteins (SCP/TAPS). This SCP-like family of proteins is highly expressed by parasitic nematodes and trematodes [61]. The major roles of SCP-like proteins are still unclear, but in hookworms, they have been implicated in larval skin penetration, the transition from the free-living to infectious stages [23,62,63], and host immune response modulation [64,65]. There was significant overlap in detected ESPs with orthologs of the ESPs detected in N. americanus in the previous study [6] (9.3% of detected ESPs, vs. 0.46% of all proteins; p < 10 −10 , binomial distribution test, MS Excel). Signal peptides, CAP domains, and ESPs are indicated for each protein in Table 2 and  Supplementary Tables S1 and S2.
Of the sex-agnostic ESPs, ACEY_07898-1 (Acey_s0073.g740) is the most abundant by far, having more than 1.8 times as many total spectra assigned as the second-most abundant ESPs. Acey_s0073.g740 is a very large protein (3373 amino acids) with conservation only in other hookworm species, so there are no C. elegans or other annotated orthologs that can be used for comparison. The reciprocal best BLAST hit in Necator americanus (NAME_09181) was also by far the most abundant ESPs in the Logan et al. study [6], which provides confidence in its annotation and detection in the ESPs in this study and makes it an interesting candidate for future study. Among the most abundant proteins on the top list are also several implicated in immunomodulation, including ACEY_17598-1: Nematode polyprotein allergen Npa-1 [66], which was more than two-fold more abundant in the male than the female ESPs, and ACEY_09343-1: SCP-like protein [67], and which had very high mRNA levels in the RNA-seq dataset ( Table 2). Npa-1, more commonly known as ABA-1, is a common nematode polyprotein allergen/antigen with a fatty acid binding property [68][69][70]. Ordinarily, nematodes do not have the potential to synthesize fatty acids and must obtain them from the host environment, and Npa-1 is likely used to meet this demand [71]. In the disease model, Npa-1 has been widely used as a serological marker for nematode infection, especially in diseases caused by Ascaris lumbricoides and Ascaris suum [72,73]. Npa-1 is abundant in the somatic tissues of the parasitic nematodes and is also discharged into the tissues of the host by the infective stages of nematode parasites [74].
KEGG pathway, gene ontology (GO) and Interpro domain enrichment among the proteins in both female and male (Table 3) indicated very strong enrichment for peptidases across all three enrichment tests, especially cysteine peptidases, as indicated by Interpro and GO. Cysteine peptidases are known to be particularly important for maintaining parasitic helminth survival in the host, including roles in the invasion of host tissues and in molting and embryogenesis [75]. Very strongly enriched is also the "exosome" KEGG term (p < 10 −10 ) in hookworms; secreted extracellular vesicles are known to interact with host cells and induce immunomodulatory effects, through the action of both proteins and miRNAs [76].
Among the abundant proteins in both female and male A. ceylanicum ESPs, the most differentially abundant protein in female (216 spectra vs. 62 in males) is ACEY_08810-1: Vitellogenin (VIT) ( Figure 1B). Vitellogenins are large glycolipoproteins representing the most conserved family of yolk proteins in oviparous species, where the proteins are naturally synthesized in extraovarian organs, transported to the circulatory system, and absorbed by the ovary [77,78]. Studies looking at the control of germline development and the role of soma-germline interactions in C. elegans revealed that vitellogenins, which serves as a nutrient source for the developing embryo, are made in the intestine, secreted into the somatic tissue, and taken up by oocytes through receptor-mediated endocytosis [79,80]. Vitellogenin, in addition to fatty acid and retinol-binding (FAR) proteins, are the main two lipid transport and storage proteins engaged in cross-membrane transport [81]. According to Wei et al. [81] more than three vitellogenins are fully expressed in the hookworm gut. Vitellogenin comprises four main domains including phosvitin, lipovitellin-1, lipovitellin-2, and a von Willebrand factor type D domain (vWFD) [82,83]. The three-dimensional structure of vitellogenin shows that lipovitellin and phosvitin domains form amphipathic structures of a lipid-binding pocket to accept their lipid cargo [84].  [6]. Y = the top N. americanus BLAST hit is an ES product in that dataset; "-" = the top N. americanus BLAST hit is not an ES product in that dataset; "(N/A)" = there is no significant (E < 10 −5 ) BLAST hit in the N. americanus protein set. Normalized mRNA levels (Fragments per kilobase per million reads, FPKM) based on the RNA-seq dataset from Bernot et al., 2020 [45].

Proteins Detected Only in Female ESPs
Similar to the ESPs detected in both sexes, the 111 female-specific ESPs (14.6% of all female detected proteins) were enriched for signal peptides (49.5% of female-specific ESPs, vs. 18.6% for all proteins; p < 10 −10 , binomial distribution test, MS Excel) and CAP domains (9.0% vs. 1.9%; p = 8.7 × 10 −6 ). This included SCP domain-containing protein (ACEY_06131-2) and Heat shock protein 90 (ACEY_06074-1) ( Table 4). In previous research, many SCP-like proteins that were abundant in the ESPs of the iL3 stage of the canine hookworm A. caninum were considered "Ancylostoma-secreted proteins" [23,63] (ASPs), and many ASP proteins are identified as being only in the female ESPs here, including the second-most abundant protein, asp4. The most abundant female-specific ESPs also include heat shock proteins (Hsp), which act as molecular chaperones essential for regulating the folding and stability of different proteins during periods of increased temperature and other stress [85]. In A. ceylanicum female ES, only two GO terms were enriched among the 111 proteins detected. (Table 5). The represented GO terms related to molecular functions were related to "binding", including pyridoxal phosphate binding (GO: 0030170) and vitamin B6 binding (GO: 0070279) ( Table 5). Hookworm infection can induce vitamin B6 deficiency [86], and another parasitic helminth, Schistosoma mansoni, is known to acquire and metabolize host vitamin B6 during some life cycle stages [87], so these results are suggestive of a similar mechanism in A. ceylanicum adult females.  [6]. Y = the top N. americanus BLAST hit is an ES product in that dataset; "-" = the top N. americanus BLAST hit is not an ES product in that dataset; "(N/A)" = there is no significant (E < 10 −5 ) BLAST hit in the N. americanus protein set. Normalized mRNA levels (Fragments per kilobase per million reads, FPKM) based on RNA-seq dataset from Bernot et al., 2020 [45].

Proteins Detected Only in Male Hookworm
Similarly to the ESPs detected in both sexes and the female-specific ESPs, the 125 male-specific ESPs in A. ceylanicum (18.3% of all male-detected proteins) were significantly enriched for signal peptides (53.6% of male-specific ESPs, vs. 18.6% for all proteins; p < 10 −10 , binomial distribution test, MS Excel) and CAP domains (16.0% vs. 1.9%; p < 10 −10 ). The most abundant male-specific ESPs was ACEY_02196-1, annotated as ttl1: precursor transthyretin-like protein 1 ( Table 6). In a previous study, across 20 nematode secretomes, transthyretin proteins were the only component common across all species in the adult stage [88], highlighting an important yet unknown molecular function for these proteins. In mammals, transthyretin transports the hormones thyroxine and retinol into the circulation [89].
To further understand the functions of proteins identified in the ESPs of adult male A. ceylanicum, GO, KEGG, and Interpro domain functional enrichment was performed. In A. ceylanicum male ES, only 1 KEGG term was enriched among the 125 proteins detected, whereas 7 GO terms and 11 Interpro IDs were enriched (Table 7). Interestingly, the only male-specific KEGG-enriched term, lectin (4091) has been previously characterized for its importance in the reproductive physiology of A. ceylanicum [90]. Lectins are a single or multi-domain large family of calcium-dependent receptors with a wide variety of carbohydrate-recognition domains [91][92][93]. Our Interpro functional analysis predicted three C-type lectin domains-containing proteins including C-type lectin-like/link domain superfamily (IPR016186), C-type lectin fold (IPR016187), and C-type lectin-like (IPR001304) ( Table 7). This lectin domain comprises about 110 to 130 residues. There is increasing evidence that these C-type lectins play an essential role in modulating helminth-mediated immune responses [91]. While these represent a set of male-specific C-type lectins, four others were detected in both males and females, and one was detected only in females. Similarly to the ESPs in both the females and males, the seven enriched GO terms among the male-specific ESPs were all related to peptidase activity (Table 7).  [6]. Y = the top N. americanus BLAST hit is an ES product in that dataset; "-" = the top N. americanus BLAST hit is not an ES product in that dataset; "(N/A)" = there is no significant (E < 10 −5 ) BLAST hit in the N. americanus protein set. Normalized mRNA levels (Fragments per kilobase per million reads, FPKM) based on the RNA-seq dataset from Bernot et al., 2020 [45].

Conclusions
Here we report the exclusive sensitivity of an efficient mass spectrometry strategy combined with improved genome annotation of A. ceylanicum to identify secreted proteins in the ESPs of the adult female and male parasites. This is the first comprehensive profiling of the ESPs proteomes of female and male A. ceylanicum. The protein composition of profiled ESPs in adult A. ceylanicum leads to the conclusion that adult females and males secrete similar numbers of proteins into culture media, many of which are shared between the sexes, and a subset being differentially represented among the sexes, and related to sex-specific biology. This study increases our understanding of A. ceylanicum biology. Our results offer new leads for the development of vaccines and drugs to control the parasites, as well as the identification of new biologics that can be explored for their anti-inflammatory properties. The identified sex-specific secretomes of A. ceylanicum will facilitate other analyses, such as the enrichment of regulatory elements and motifs that mediate the expression of matching genes, information that should facilitate the engineering of transgenic A. ceylanicum to understand functions of specific genes, or to over-express different proteins of interest.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens12010095/s1, Table S1: Gene database including functional annotations (Sma3s, Pannzer2, KEGG, Interpro, Gene Ontology), conservation data, remapped relative mRNA levels (FPKM) for RNA-seq samples spanning the A. ceylanicum life cycle, and peptide and summed peptide spectral match (PSM) counts for all detected proteins in the ESPs samples. Table  S2: Peptide spectral match (PSM) counts per detected peptide, according to output from Scaffold.