2.2. Metabolomics analysis

Serum (25 μL) was precipitated in methanol (1:3 v/v). The soluble fraction was dried under vacuum, reconstituted in 10% acetonitrile (ACN, 75 μL) and analyzed (12 μL) using a TripleTOF 5600 tandem mass spectrometer (Sciex, Concord, ON, Canada) equipped with a Shimadzu Nexera UHPLC and a 150 $\times$ 3 mm 3 μm (130 Å) Scherzo SM-C18 multi-mode HPLC column with water (A) and ACN (B), both containing 0.1% formic acid (FA), at a flow rate of 250 μL/min (40 °C). Liquid chromatography (LC) gradient started at 3% B, held for 3 min, increased linearly to 80% at 17.5 min, held for 2.5 min, increased to 95% at 20.5 min, and held for 3.5 min. Mass spectrometry (MS) analysis was performed in positive (5.5 kV) and negative (−4.5 kV) mode electrospray ionization (ESI) at 450 °C with a declustering potential of 60 V for m/z 80–975. MS/MS spectra were acquired at m/z 50–700 for the 5 most abundant ions using information-dependent acquisition and collision-induced dissociation (collision energy 30 ± 10 eV) for precursor ions >500 intensity units excluded for 20 min after 3 occurrences. Mass tolerances were set to 50 ppm and 50 mDa at the MS and MS/MS levels, and accumulation times were 0.3 s and 0.15 s for MS and MS/MS analyses, respectively. Total cycle time was 0.95 s.

2.3. Metabolite identification and confirmation

Peak finding was performed in MarkerView software (Sciex) for 1.5–18.5 min retention time (RT) with predefined minimum spectral width (0.01 Da) and LC peak width (2 scans). RT tolerance and mass tolerance were 0.2 min and 0.01 Da, respectively. Molecular features with p > 0.01 (Student's t-test) and fold-change (patient/control) < ±1.5 were excluded. Isotopologues, adduct ions, and those resulting from in-source fragmentation of common neutral losses were also excluded. The mass spectra associated with the remaining features were visually inspected in PeakView software (Sciex) to confirm their exact mass and charge state. Confirmed molecular features were searched against the Human Metabolome Database (HMDB) [18], the Metabolite and Chemical Entity Database (METLIN) [19], and the Accurate Mass Metabolite Spectral Library (AMMSL v1.0; Sciex). LC-MS metabolite search was performed with a mass tolerance of 5 ppm for proton transfer adducts. Metabolite identification was performed based on the four following criteria: (1) Only protonated or deprotonated ions were accepted. (2) Features with arguable chromatographic behaviour were rejected; for example, hydrophilic compounds found at a late retention time were dismissed. (3) Physiologically irrelevant compounds such as xenobiotics and odd-chain fatty acids were not retained. (4) Structures with questionable chemical stability such as protonated esters or epoxides were also excluded. SciexOS software was used for the MS/MS spectral library searching using the “candidate search” algorithm with a precursor and fragment mass tolerance of 0.2 Da. Selected metabolites were confirmed by LC-HRMS/MS using RT and MS/MS spectral matching with synthetic standards. Phe-Thr standard was synthesized and purified at the Center of Excellence in Orphan Disease Research–Courtois Foundation (CERMO-FC) at Université du Québec à Montréal (Montreal, QC, Canada). pGlu-Gly and Asn-Gly-Phe-Lys were purchased from CanPeptide (Saint Laurent, QC, Canada). Steroid standards were purchased from Steraloids (Newport, RI, USA) and authorized for import by Health Canada Office of Controlled Substances (authorization # 49322.10.19 & 49323.10.19). Other standards were purchased in analytical grade from Sigma-Aldrich (Saint Louis, MO, USA). Standard solutions (1.0 mg/mL) were made in 50% MS-grade ACN (Sigma-Aldrich). LC-HRMS/MS analysis was conducted at Université du Québec à Montréal. Univariate and multivariate (with respect to metadata confounders) analyses were performed using Microsoft Excel and MetaboAnalyst web application [20], respectively.

2.4. Synthesis and purification of phenylalanyl-threonine

Phe-Thr standard was produced by Fmoc chemistry solid-phase peptide synthesis. Fmoc-L-Thr (tBu)-OH (795 mg, 2 mmol) was linked to 2-chlorotrityl chloride resin (1.176 g, 1 mmol, 100–200 mesh, 1% divinylbenzene, Matrix Innovation) by overnight incubation in N,N-diisopropylethylamine (DIEA, 523 μL, 3 mmol) and dichloromethane (DCM, 20 mL) with agitation by nitrogen sparging. After multiple washes of the resin with DCM, a small aliquot of resin was dried, weighted (10 mg) and the loading efficiency was quantified by measuring dibenzofulvene absorbance at 290 nm after Fmoc removal by 20% piperidine in dimethylformamide (DMF, 5 min), yielding 0.46 mmol/g (54%). The resin was capped (9 mL DCM, 1 mL methanol, 0.5 mL DIEA) for 1 h, washed (DCM twice, then DMF), Fmoc was removed (20% piperidine in DMF, 15 min), and the resin was washed again (DMF, DCM, DMF sequentially, 2 min each). Fmoc-L-Phe-OH (628 mg, 1.62 mmol) was coupled to resin-bound Thr using O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU, 670 mg, 1.62 mmol) and DIEA (280 μL, 1.62 mmol) for 30 min. The resin was washed, the Fmoc group was removed, the resin was washed with diethyl ether and air-dried. The peptide was cleaved off from the resin (20 mL of 95% trifluoroacetic acid/2.5% water/2.5% triisopropylsilane, 2 h) and the resulting solution was concentrated. The crude peptide was precipitated in cold diethyl ether, air-dried, dissolved in deionized water, and lyophilized. The product was then dissolved in 0.5 mL water and purified by size-exclusion chromatography (20 mL packed Sephadex G-10, water elution, 1 mL fractions). Pure fractions were pooled and lyophilized, yielding Phe-Thr (102 mg, 38% total yield). HR-MS (LC-TOF): calcd. for [C₁₃H₁₈N₂O₄+H]⁺ 267.1339; found 267.1338. ¹H NMR (300 MHz, D₂O): δ 7.41–7.26 (m, 5H), 4.40 (d, J = 3.8 Hz, 1H), 4.36 (t, J = 7.0 Hz, 1H), 4.28 (m, 1H), 3.28 (dd, J = 14.2, 6.7 Hz, 1H), 3.18 (dd, J = 14.2, 7.4 Hz, 1H), 2.68 (s, 1H), 1.17 (d, J = 6.5 Hz, 3H). ¹³C NMR (75.44 MHz, D₂O): δ 173.1, 169.4, 133.5, 129.4, 129.1, 128.0, 67.1, 58.4, 54.2, 36.7, 18.8.

2.5. Free fatty acid and cholesterol measurement

Serum (20 μL) was precipitated in acetone (1:10 v/v). The soluble fraction was transferred into a clean tube and evaporated (56 °C, 1 h). Serum lipids were hydrolyzed with potassium hydroxide (1 M in 70% ethanol, 80 °C, 4 h). The solution was then acidified with hydrochloric acid and extracted twice with pentane. Pentane was evaporated (40 °C, 1 h), sample was additionally heated (80 °C, 1 h) to remove moisture and reconstituted in ACN (25 μL). A non-endogenous fatty acid (pentadecanoic acid, 15:0) was added (50 μg/mL) as internal standard. Sample was then derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA, 25 μL, 80 °C, 45 min). Two microliters of the BSTFA derivative was injected into an Agilent 7890-5975C GC-MS instrument (Agilent Technologies, Santa Clara, CA, USA) with a split ratio of 5:1 and inlet temperature of 280 °C. Gas chromatography (GC) separation was done at a starting temperature of 160 °C, then increased to 180 °C (13 °C/min, 0 min), to 225 °C (2 °C/min, 1 min), and to 310 °C (17 °C/min, 2 min) on a HP-5MS 30 m × 250 μm × 0.25 μm capillary column (Agilent) at a carrier gas flow rate of 1.2 mL/min. Selected-ion monitoring (SIM) was conducted in positive ionization mode for m/z 314 (C15:0), 326 (C16:1), 328 (C16:0), 352 (C18:2), 354 (C18:1), 356 (C18:0), 376/150 (C20:4) and 458 (cholesterol) with a dwell time of 0.01 s. To minimize statistical bias, samples were randomized and divided into 5 groups of ~15 samples. Reference standards were purchased from Sigma-Aldrich and used to characterize each analyte based on m/z value and RT. Quantification was performed using the peak area ratio of each analyte to the internal standard. All analytes were detected in all the samples analysed. GC-MS analysis was conducted at Université de Montréal (Montreal, QC, Canada).

2.6. Parasite culturing and transformation

Logarithmic-phase epimastigotes of the Brazil strain [21] were washed three times in EMEM complete medium supplemented with 5% FBS and gentamycin (Wisent, Saint Bruno, QC, Canada) and added to Vero cell monolayers (ATCC CCL-81). Infected cells were washed on the third and fifth days post-infection. Subsequently, the supernatant containing trypomastigotes was collected, centrifuged (1900 g, 20 min), washed in PBS (Wisent) and counted in a Neubauer haemocytometer (Hausser Scientific, Horsham, PA, USA).

2.7. Murine infection and hormone assessment

Six- to eight-week-old CD-1 mice (Charles River Laboratories, Senneville, QC, Canada) were assigned to four study groups of equal number of males and females each (eight subgroups total). Mice were intraperitoneally injected with 10,000 culture-derived T. cruzi trypomastigotes and their weights were monitored weekly. Blood was collected from the lateral saphenous vein weekly in heparinized microcapillaries for immediate detection of parasites in the buffy coat, followed by serum collection and storage at –80 °C. One group was euthanized (isoflurane/CO₂) on day 22 post-infection (PI) (acute phase), underwent cardiac puncture for blood and serum collection. One group was treated with Nfx (100 mg/kg body weight) on day 74 PI (chronic phase) for 20 days q. d. by gavage. The study was terminated on day 110 PI, and blood, serum, testes, and ovaries were collected. Samples were flash frozen in liquid nitrogen immediately after acquisition and stored at –80 °C until analysis. All procedures were carried out in accordance with the guidelines of the Canadian Council on Animal Care, as approved by the Animal Care Committee of McGill University (Animal Use Protocol 7631). The width of the testes was measured weekly with a digital caliper (General Tools, Secaucus, NJ, USA) before, throughout and after the treatment during the chronic phase of infection from day 70 to day 105 PI. Measurements were made to the nearest 0.1 mm. Serum testosterone, progesterone and estradiol levels were assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kits at the Ligand Assay and Analysis Core Facility of the University of Virginia Center for Research in Reproduction (Charlottesville, VA, USA). Nfx was donated by Dr. Eric Chatelain (Drugs for Neglected Diseases initiative, Geneva, Switzerland).

2.8. Statistical analysis

Widths of testes were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Sex hormone levels and weight change were evaluated using two-way ANOVA for the following two factors: sex (male vs female) and disease phase (uninfected vs acute, uninfected vs chronic, uninfected vs treated, acute vs chronic, acute vs treated, chronic vs treated) followed by Bonferroni post-hoc test for significant interactions when appropriate. When no interaction was found, unpaired t-tests were performed on the factor where significance was observed. The results are presented as the mean ± standard error of the mean (SEM). Differences were statistically significant at a p-value of < 0.05. Statistical analyses were performed using GraphPad Prism 5.00 (GraphPad Software, San Diego, CA, USA).

2.9. Proteomics analysis of mouse testes

Testes were thawed on ice, surgically separated from epididymis and ductus deferens, rinsed with PBS and dissected transversely. One section (~25 mg) was used for DNA extraction and T. cruzi PCR. The remainder (25–120 mg) was thoroughly ground in 8 M urea, 0.1 M NaCl, 25 mM Tris HCl pH 8.2 with Pierce Protease Inhibitor (Thermo Scientific, Waltham, MA, USA) to minimize proteolytic degradation. The suspension was further homogenized (10 min, 4 °C) using a 400 W/20 kHz Branson Digital Sonifier (Shanghai, China) for 15 s at 50% maximum power and vigorously mixed for 10 s. The sonication/mixing cycle was repeated three more times. The insoluble fraction was separated by centrifugation (18,000 g, 4 °C, 10 min). The protein concentration of the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). An equivalent of 0.5 mg total protein was then diluted 10-fold in 0.1 M ammonium bicarbonate pH 8.0, reduced with dithiothreitol (10 μL, 0.1 M, 65 °C, 30 min) and alkylated with 2-iodoacetamide (10 μL, 0.25 M, 25 °C, 30 min) in the dark. Protein digestion was performed with porcine pancreas trypsin (Sigma-Aldrich; 5 μL, 2.0 mg/mL) overnight (37 °C, 16 h) at a 1:50 enzyme-to-protein ratio. Digestion was stopped by decreasing pH to 4.0 with 10% acetic acid. Sample was centrifuged (18,000 g, 4 °C, 10 min) and the supernatant was cleaned up using an Oasis HLB solid-phase extraction cartridge (Waters, Milford, MA, USA) following manufacturer's instructions. Peptides were eluted in methanol (0.1% FA), evaporated under vacuum (Labconco, Kansas City, MO, USA; 40 °C, 3 h) and reconstituted in buffer A (120 μL, see below). Strong cation exchange fractionation was performed using a published method [22]. Briefly, the peptide mixture was injected (100 μL) on to a Zorbax 300-SCX 150 mm × 2.1 mm column with 5 μm (300 Å) particles (Agilent) using a Beckman System Gold HPLC (Beckman Coulter, Brea, CA, USA) at a flow rate of 0.25 mL/min with a gradient of 0–50% B in 15 min, up to 100% B at 25 min, then held for an additional 5 min at 100% B, where buffers A and B were 10 mM potassium dihydrogen phosphate in 25% ACN (pH 2.75), and 1 M potassium chloride in buffer A (pH 2.75), respectively. UV absorbance was monitored at 220 and 280 nm. For each sample, 3-min (0.75-mL) fractions were aliquoted into 1.5-ml tubes from 2 to 32 min. Fractions were evaporated to dryness under vacuum and reconstituted (60 μL) in MS-grade water (0.1% FA, Sigma-Aldrich). LC-HRMS/MS analysis was performed (20 μL) on a Maxis II quadrupole-time-of-flight mass spectrometer (Bruker, Billerica, MA, USA) equipped with a Dionex UltiMate 3000 UHPLC system (Thermo Scientific) and an Aeris Peptide XB-C18 1.7 μm 100 Å 150 × 2.1 mm HPLC column (Phenomenex, Torrance, CA, USA) with water (A) and ACN (B), both containing 0.1% FA, at a flow rate of 125 μL/min (50 °C). LC gradient started at 5% B, held for 3 min, was increased to 45% at 25 min, and to 80% at 32 min. Apollo ESI source (Bruker) operated in positive mode with endplate offset and capillary voltage of 0.5 and 4.5 kV, respectively. A continuous flow of 99.5% pure N₂ was provided (Parker, Mayfield Heights, OH, USA) as dry gas (200 °C) at a flow rate of 6 L/min. MS and MS/MS spectra were acquired at m/z 300–2200 using data-dependent acquisition and collision-induced dissociation with a collision energy of 21–55 eV depending on the precursor ion m/z value and charge state. Precursor ions with a charge state of 2–5 were preferred and singly charged ions were excluded. Redundant ions were also excluded for 3 min. Acquisition time was 0.25 s for MS and 0.06–0.25 s for MS/MS scan, depending on precursor ion signal intensity. MS instrument was internally calibrated via post-column infusion of ESI-L Low-Concentration Tuning Mix (Agilent). Samples were analyzed in random order in the sequence to avoid biases due to the injection order. Moreover, at least one blank (5 μL, mobile phase A) was inserted between each two analyses to minimize carryover. Proteomics analysis was conducted at Research Institute of the McGill University Health Centre (Montreal, QC, Canada).

2.10. Proteomics data analysis

LC-HRMS/MS data were imported into the MaxQuant software [23] and searched at 1% false-discovery rate (FDR) using the Andromeda search algorithm against the mouse subset of the UniProt/SwissProt protein database (17,023 sequences, downloaded in January 2020) for tryptic peptides allowing up to 2 missed cleavages. Methionine oxidation and N-terminal acetylation were defined as variable modifications while cysteine carbamidomethylation was set as fixed modification. Mass tolerances were 70 and 6 mDa for the first and main peptide searches, respectively. To account for LC retention shifts, the “match between runs” option was enabled with a match time window of 0.7 min and an alignment time window of 20 min. Only database searches with an FDR-adjusted p < 0.01 were accepted as protein hits. Proteome-wide label-free quantification (LFQ) was performed based on 2 razor unique peptides per protein [24]. Statistical testing was performed by Perseus software [25] at the 2-tailed α level of 0.05 (p < 0.05) to identify significantly differentially abundant proteins (fold-change >1.2) across the two cohorts. Permutation-based FDR [26] was used to correct p-values for multiple hypothesis testing. Protein accession numbers were uploaded to PANTHER [27] for gene ontology classification. Pathway over-representation analysis (ORA) was performed by InnateDB [28] using a hypergeometric search algorithm, Benjamini-Hochberg correction method, and pathway annotations from Reactome, INOH, NETPATH, KEGG, Biocarta and NCI databases. Only over-represented pathways with a corrected p-value < 0.05 were accepted. LC-HRMS/MS raw data from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [29] with the dataset identifier PXD017802.

3.2. Anti-trypanosomal treatment restores serum lipid levels in CD patients

Several major serum fatty acids, including palmitic acid (C16:0), linoleic acid (C18:2), stearic acid (C18:0), arachidonic acid (C20:4), and cholesterol (C27:1) were significantly increased (p < 0.03; ANOVA) in CD patients relative to healthy controls. Palmitoleic acid (C16:1) and oleic acid (C18:1) showed a trend towards increased levels in CD patients compared to healthy controls. They all returned to control levels after treatment, except palmitoleic acid that was unchanged (Figure S3). Several tentative lipid oxidation products, such as oxocaprylic acid (C8:0 + O), nonanedione (C9:0), hydroxycapric acid (C10:0) and hydroxylauric acid (C12:0) had decreased levels in CD patients. Fourteen unconfirmed metabolites were associated with phospholipids (Table S1). Of these, 2 increased and 12 decreased in the patients. All lipid metabolites had levels comparable to CD-negative controls after Nfx treatment.

3.3. T. cruzi infection in mice leads to altered sex steroid metabolism and testes morphology

Our metabolomics analysis of human sera suggested that several steroid metabolites were changing in CD patients. To assess the effects of chronic T. cruzi infection on sex steroid metabolism in vivo, mice were infected with T. cruzi trypomastigotes and their major steroid hormones monitored during both acute and chronic infection phases and following Nfx treatment. Trypomastigotes were detected in the buffy coat of all infected mice until day 25 post-infection (PI), but at day 42, PI parasites were no longer detected, indicating a transition from acute to chronic phase CD [31]. To control for sex-specific differences in the response to infection, male and female groups were analyzed independently. At the experimental endpoint (day 110 PI), the mean serum testosterone level of uninfected male mice was 10.48 ± 2.40 ng/mL, while the testosterone levels of T. cruzi infected animals were drastically reduced (0.64 ± 0.02 ng/mL, 2.25 ± 1.05 ng/mL and 1.15 ± 0.30 ng/mL for acute, chronic and chronic-treated groups, respectively) (Figure 2A). As expected, uninfected female mice had substantially lower levels of testosterone at baseline (0.65 ± 0.07 ng/mL), which did not differ significantly in any of the other experimental groups (Figure 2B). In contrast to our findings with human CD patients, neither progesterone nor estradiol were significantly altered in mice infected with T. cruzi.

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Figure 2

Trypanosoma cruzi infection in mice leads to altered sex steroid metabolism and testes morphology. Serum levels of testosterone (T), progesterone (P) (in ng/mL) and estradiol (O) (in pg/mL) in wild-type CD-1 male (A) and female (B) mice ± acute or chronic T. cruzi infection ±20-day nifurtimox treatment, at day 105 post-infection. Infected male mice had significantly decreased testosterone and increased estradiol levels in both acute and chronic phases whereas female mice had increased estradiol and progesterone levels specifically in acute phase and chronic phase, respectively. Testes width was measured before, during and after the treatment (C) for uninfected (D), chronically infected (E) and treated (F) males. The testes shrank in size in the infected group; however, they presented similar width outcome to those of the uninfected group post-treatment. Significance levels were calculated using ANOVA and F-test: p < 0.05; p < 0.01; p < 0.001. Error bars represent mean ± standard error of mean.

The testes width of male mice was measured during the chronic phase of T. cruzi infection, and during and after treatment (Figure 2C). Prior to treatment, the testes widths were comparable for all experimental groups (13.8 ± 0.3 mm, 13.7 ± 0.4 mm, and 13.6 ± 0.3 mm for uninfected, chronically infected, and treated groups, respectively). As expected, the testes width of uninfected mice gradually increased throughout the experiment. In contrast, the testes width of chronically infected untreated mice showed an initial increase which slowed by day 90 PI and decreased rapidly thereafter. During Nfx treatment, the testes width of treated mice was reduced at day 91 PI, but following treatment completion, this trend reversed and testes width rapidly increased. By the experimental endpoint, the testes of chronically infected Nfx-treated mice were indistinguishable in width and appearance from those of the uninfected mice, while the testes of untreated mice were significantly smaller than the other experimental groups (Figure 2D−F). No significant change in total body weight was observed for either male or female mice during the study.

Funding statement

This work was supported by Drugs for Neglected Diseases initiative (DNDi) (UK Aid (UK), Swiss Agency for Development and Cooperation (SDC; Switzerland), and Médecins Sans Frontières International) [grant number 4759].

Momar Ndao was supported by the Public Health Agency of Canada/National Microbiology Laboratory (contract number 6D063-193271/001), the Foundation of the McGill University Health Centre, the Foundation of the Montreal General Hospital, the Research Institute of the McGill University Health Centre, and the R. Howard Webster Foundation.

Lekha Sleno was supported through the UQAM Strategic Research Chair program. (The LC-MS metabolomics platform at UQAM is supported by the CERMO-FC, through the Courtois Foundation).

Data availability statement

Data associated with this study has been deposited at the ProteomeXchange Consortium via the PRIDE partner repository under the accession number PXD017802.

Declaration of interest's statement

The authors declare no conflict of interest.

We thank Drs. Brian J. Ward, Robert Kiss, Makoto Nagano, Bernard Robaire, Daniel Bernard for helpful discussions; Milli Nath-Chowdhury, Maya Scott-Lourenço, Colton Strong, Elizabeth Ruiz-Lancheros, Amirhossein Daryaei for helping with the animal study and laboratory work; Dr. Kebba Sabally, Patrick Sabourin, Rami Tohme, Wilson Carreiro for technical support; Angela J. Brewer, Louis Cyr, Kelly Marshall, Nathalie Martel for logistics and administrative support; and James I. P. Stewart for reviewing the manuscript. Makan Golizeh thanks Dr. Momar Ndao for his exceptional support during this project.