ABSTRACT
Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis. SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 μM and is found at concentrations between 1 and 10 μM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 μM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 μM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 μM. We interpret these results to suggest that B. dendrobatidis produces an “armamentarium” of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.
INTRODUCTION
According to the best available assessments, amphibians continue to decline in many parts of the world (1, 2) due in part to two closely related species of chytrid fungi, Batrachochytrium dendrobatidis (3) and Batrachochytrium salamandrivorans (4; reviewed in references 5 and 6). Amphibians also have very highly developed and robust immune defense capabilities (reviewed in references 7 and 8). Thus, there is an apparent contradiction. The amphibian immune system should be able to respond to and clear infections due to these skin fungi, yet in many cases, the fungal infections proceed and eventually kill the host. This indicates that Batrachochytrium fungi have evolved mechanisms to evade detection and/or destruction by the amphibian immune system (9).
Previous studies showed that live or dead B. dendrobatidis organisms in coculture with amphibian lymphocytes inhibited their proliferation and induced apoptosis (9), whereas the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza inhibited splenocytes poorly in coculture compared with B. dendrobatidis (9). Inhibition by the fungal cells could also be caused by cell-free supernatants that were resistant to heat and protease treatments (9). The B. dendrobatidis cell-free supernatants also inhibited a delayed-type hypersensitivity reaction in frogs injected with a plant lectin (10). These cell-free supernatants contained two inhibitory metabolites, methylthioadenosine (MTA) and kynurenine (KYN) (11), but their immunosuppressive activity seemed unlikely to explain all of the inhibition due to the fungus. Thus, we sought to further detect and characterize additional immunomodulatory factors produced by the fungus.
The previous report on the abundance and activity of MTA in the supernatants of B. dendrobatidis led us to hypothesize that additional small molecules might help the fungus evade the immune system. In particular, MTA is generated during the biosynthesis of polyamines, which are low-molecular-weight organic compounds that are positively charged at neutral pH. The polyamines spermidine (SPD) and spermine are derived from putrescine (PUT), a metabolite generated from either the amino acid ornithine or arginine (reviewed in references 12 and 13). They are abundant in nature and have a wide variety of reported functions (reviewed in reference 14). They are critical for proliferation of eukaryotic and prokaryotic cells (reviewed in reference 14), but at high concentrations they are also associated with inhibition of proliferation of lymphocytes and induction of apoptosis (15, 16). We hypothesized that B. dendrobatidis releases significant amounts of spermidine in the local environment of the skin cells, resulting in inhibition of the functions of lymphocytes or other immune cells responding to the foreign presence. If polyamines play a role in immune evasion by B. dendrobatidis, we predicted that they would be produced by B. dendrobatidis, be required for growth, be detected in active cell-free supernatants, and be released at concentrations sufficient to inhibit lymphocyte functions.
RESULTS
Effects of B. dendrobatidis zoosporangia and supernatants on proliferation of lymphocytes.We have shown previously that coculture of B. dendrobatidis zoosporangia, but not zoospores, inhibited proliferation of frog splenocytes (primarily lymphocytes and antigen-presenting cells) (9). For this study, we examined how few zoosporangia would be necessary to inhibit splenic lymphocytes. Coculture of very low numbers of B. dendrobatidis zoosporangia with phytohemagglutinin (PHA)-stimulated lymphocytes resulted in significant inhibition of proliferation. As few as one zoosporangium in culture with about 62 lymphocytes resulted in consistent inhibition of proliferation, and the inhibition was increased with greater numbers of zoosporangia (Fig. 1A and B). This suggests that local interactions of the zoosporangia with spleen cells result in the release of an inhibitory substance or substances.
Effects of B. dendrobatidis zoosporangia and supernatants on proliferation of lymphocytes. (A) Splenocytes (Spl) (105/well) from X. laevis were cultured alone or with PHA. The PHA-stimulated Spl were cultured alone or with increasing numbers of B. dendrobatidis zoosporangia (one representative experiment of four is shown). Significantly reduced [3H]thymidine uptake was detected as counts per minute (CPM) compared to control PHA-stimulated lymphocytes; **, P ≤ 0.01 by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. (B) Summary of four experiments reported as percent growth of cells with added B. dendrobatidis in comparison with [3H]thymidine uptake by PHA-stimulated control splenocytes (normalized to 100). Shown are means ± standard errors (SE) of the results of four independent experiments. Significant inhibition in comparison with the PHA-stimulated control cells; *, P ≤ 0.05, and **, P ≤ 0.01 by one-way ANOVA with Tukey’s post hoc test. (C) Spl were cultured alone or with PHA. The PHA-stimulated Spl were cultured alone or with increasing concentrations of B. dendrobatidis supernatant (Sup) (one representative experiment of three is shown). Significantly reduced [3H]thymidine uptake was detected as counts per minute compared to control PHA-stimulated splenocytes; **, P ≤ 0.01 by one-way ANOVA with Tukey’s post hoc test. (D) Summary of the results of three independent experiments reported as percent [3H]thymidine uptake by splenocytes cocultured with B. dendrobatidis supernatants in comparison with the PHA-stimulated cells (normalized to 100). Means ± SE of three experiments are shown. Significant growth reduction in comparison with the PHA-stimulated cells; **, P ≤ 0.01 by one-way ANOVA with Tukey’s post hoc test.
To demonstrate that this inhibition was not contact dependent (due to direct cell-to-cell interactions) and that supernatants can replace live cells, we examined the effects of cell-free supernatant factors on lymphocyte proliferation. We produced B. dendrobatidis supernatants by inoculating 107 zoosporangia/ml in distilled water for 24 h. The supernatant also inhibited PHA-stimulated lymphocytes when concentrated 2.5-fold or more (Fig. 1C and D) (9).
Isolation and identification of spermidine from growing cultures of B. dendrobatidis.To examine the nature of inhibitory factors produced by B. dendrobatidis, a large bulk culture of the fungus was grown at 19°C with constant shaking for 10 days. Because we had recently identified MTA using reverse-phase chromatography and we hypothesized that polyamines might be present in the spent B. dendrobatidis culture, we employed an alternative fractionation strategy to broaden our search for bioactive molecules that might be implicated in pathogenesis. By using weak cation-exchange chromatography on a heat-stable, ethanol-precipitated fraction of B. dendrobatidis cells and supernatant, we observed that positively charged molecules possessed significant cytotoxic activity against Jurkat T cells at concentrations of 1 and 0.1 mg/ml (Fig. 2A). Specifically, the formic acid eluate demonstrated nearly complete inhibition of Jurkat T cell growth at a concentration of 1 mg/ml and ∼75% inhibition at 0.1 mg/ml in vitro (Fig. 2A).
Isolation and identification of spermidine from growing cultures of B. dendrobatidis. (A) Jurkat T cell viability upon treatment with crude B. dendrobatidis-derived fractions (1 mg/ml) resulting from cation-exchange chromatography. Viability is expressed as percent viability (mean ± SE) relative to the positive control (no additions). Column FT, column flow-through; MeOH, methanol eluate; Formic Acid, 0.5% formic acid/water eluate; Formic Acid (1:10), 10-fold dilution of formic acid treatment (0.1 mg/ml). Each column represents testing of duplicate samples. (B) Derivatization and detection of putrescine and spermidine using dansyl chloride. The decreased polarity of these derivatized polyamines allows them to be separated using reverse-phase HPLC, and the addition of the dansyl chromophore (λmax = 340 nm) allows the polyamines to be detected using a UV-Vis photo diode array detector. (C to G) LC-MS analysis of derivatized polyamines in B. dendrobatidis supernatant. (C) Total ion chromatogram. (D) Extracted ion chromatogram (m/z = 555 to 556) of derivatized supernatant. (E) Extracted ion chromatogram (m/z = 845 to 846) of derivatized B. dendrobatidis supernatant. (F) Mass spectrometry identification of compound eluting at 20.9 min in panel D, assigned as didansylputrescine (expected [M + H]+, 555.2). (G) Mass spectrometry identification of compound eluting at 23.4 min in panel E, assigned as tridansylspermidine (expected [M + H]+, 845.3).
Identification of spermidine and putrescine as components of B. dendrobatidis cell-free supernatants.The enrichment of inhibitory activity from a B. dendrobatidis-derived sample containing positively charged molecules merited further investigation. Polyamines are positively charged small molecules that are both essential and potentially toxic to eukaryotic cells if their levels are not well regulated (reviewed in reference 17). Although these polyamines cannot be directly visualized by UV-visible (Vis) spectrometric analysis following high-performance liquid chromatography (HPLC) separation, they can be detected after derivatization using dansyl chloride (Fig. 2B) (18). Accordingly, after dansylation of B. dendrobatidis supernatants, a didansylated putrescine derivative ion and a tridansylated spermidine derivative ion were detected by liquid chromatography-mass spectrometry (LC-MS) with mass to charge ratio (m/z) values of 555.1 and 845.4, respectively (Fig. 2C to G); the UV-Vis characteristics and HPLC retention times matched standards of pure polyamines that were experimentally derivatized using dansyl chloride. Using these derivatives as standards, we estimated that the concentration of spermidine in the B. dendrobatidis supernatants was between 1 and 10 μM. As is the case for most other fungi, the polyamine spermine was not detected in B. dendrobatidis supernatants (19).
Effects of spermidine and other polyamines on lymphocyte proliferation and viability.To determine the effects of pure spermidine on lymphocyte activity, we induced splenocytes of Xenopus laevis to proliferate using PHA and examined the effects of increasing concentrations of spermidine on proliferation. Lymphocyte proliferation was significantly inhibited at concentrations of 10 μM or more (Fig. 3A and B). Thus, spermidine is active at the approximate concentrations found in cell-free supernatants of B. dendrobatidis zoosporangia. To determine how the spermidine sensitivity of amphibian lymphocytes compares with that of mammalian lymphocytes, we also examined the effects of spermidine on the viability of proliferating Jurkat T cells. The Jurkat cells were somewhat more sensitive, showing significant inhibition at concentrations as low as 1 μM (Fig. 3C and D). To determine whether inhibition of lymphocytes was a unique property of spermidine or was shared with other polyamines, we tested the activities of four other related polyamines on proliferation of X. laevis lymphocytes. The polyamines differed in size and chemical structure (Fig. 4A). In addition to spermidine, norspermidine and spermine also inhibited lymphocyte proliferation in a dose-dependent manner, whereas the smaller polyamines, putrescine and cadaverine, did not (Fig. 4B and C). Only putrescine and spermidine were detected in B. dendrobatidis supernatants.
Spermidine inhibits proliferation of frog (X. laevis) lymphocytes and a human T cell line (Jurkat). (A and B) Inhibition of PHA-stimulated X. laevis splenocytes by spermidine. (A) Data from one experiment representative of ten similar experiments. X. laevis splenocytes were cultured alone or with PHA. The PHA-stimulated cells were incubated with increasing concentrations of spermidine, as shown. (B) Percent inhibition of proliferation at each concentration (n = 5 to 10 experiments at each concentration). (C and D) Inhibition of Jurkat T cells by spermidine. (C) One experiment representative of 11 independent experiments showing inhibition of growth measured as the reduction of MTT at 570 nm. The positive control (Pos.) was cells cultured in medium alone, and the negative control (Neg.) was cells treated with etoposide (12.5 μg/ml). (D) Summary of data from 11 experiments reported as percent inhibition of growth. The error bars show standard errors, and the indicated treatments were significantly different from the positive control by one-way ANOVA with Tukey’s post hoc test. **, P ≤ 0.01, except for percent inhibition of Jurkat T cells at 1 μM; *, P ≤ 0.05.
Effects of other polyamines on frog lymphocyte proliferation. (A) Two biosynthetic pathways for spermidine biosynthesis. The pathway inhibitors DFMA and DFMO are highlighted in red, and the previously reported MTA pathway by-product is shown in blue. Additional polyamines that could not be detected in B. dendrobatidis supernatant but that were tested in this study are shown in the inset. (B and C) Comparison of the lymphotoxic activities of other polyamines with that of spermidine. (B) One representative experiment with five or more replicate wells per polyamine and dose tested. Significantly different from the PHA-stimulated control cells by one-way ANOVA with Tukey’s post hoc test; *, P < 0.05; **, P < 0.01. (C) Average percent inhibition. Each polyamine was tested in three to five independent experiments. SPD was tested in 12 independent experiments. PUT and cadaverine (CAD) were only slightly inhibitory even at concentrations as high as 100 μM. Norspermidine (NSP) and spermine (SPM) were inhibitory in a dose-dependent fashion, similar to SPD. Inhibition by NSP, SPD, and SPM was significantly greater than for the positive control (set at zero); **, P < 0.01 by one-way ANOVA with Tukey’s post hoc test.
Effects of inhibitors of ornithine decarboxylase and arginine decarboxylase on growth of B. dendrobatidis.In order to determine the role of spermidine in survival and growth of B. dendrobatidis and the pathway of biosynthesis of the molecule, we used irreversible inhibitors of the enzymes ornithine decarboxylase and arginine decarboxylase. Ornithine decarboxylase is inhibited by α-difluoromethylornithine (DFMO) (20), and arginine decarboxylase is inhibited by α-difluoromethylarginine (DFMA) (21). At a concentration of 5 mM DFMO, growth of B. dendrobatidis was inhibited by about 53%, and at 10 mM DFMO, growth was inhibited by about 84% (Fig. 5A and B). This demonstrates that ornithine decarboxylation is critical for B. dendrobatidis growth. Some bacteria, plants, and fungi also synthesize spermidine by way of an alternative pathway from arginine via arginine decarboxylase through the intermediate agmatine (22; reviewed in reference 23). Inhibition of arginine decarboxylase by DFMA also resulted in dose-dependent inhibition of the growth of B. dendrobatidis by as much as 80% at 10 mM (Fig. 5C), indicating that arginine decarboxylation is also important for B. dendrobatidis growth. We then tested inhibitors of each enzyme in combination to determine the predominant pathway of spermidine synthesis in the fungus. Both DFMO and DFMA inhibited B. dendrobatidis growth in a dose-dependent fashion, with maximal inhibition at 10 mM (Fig. 5A to C). The addition of both at 10 mM showed greater inhibition than that due to DFMA alone, suggesting that the ornithine decarboxylase pathway is dominant (Fig. 5D). To determine if blocking the ornithine decarboxylase pathway would allow compensatory growth through the arginine decarboxylase pathway, we maximally inhibited both pathways with DFMO and DFMA and then relieved the arginine decarboxylase inhibition by decreasing the amount of DFMA in some of the cultures. The results showed that allowing arginine decarboxylation to recover did not reverse the growth inhibition due to DFMO (Fig. 5E). To demonstrate that the ornithine decarboxylase pathway is the dominant pathway and the arginine-through-agmatine pathway is not present, we blocked each pathway and provided either putrescine or agmatine to restore growth. Putrescine restored some growth regardless of the inhibitor (Fig. 5F and G), but agmatine was not able to restore growth in DFMA-blocked cultures (Fig. 5G). This suggests that one or more enzymes necessary to convert agmatine to putrescine, leading to spermidine, are lacking in B. dendrobatidis. These studies support the hypothesis that spermidine is necessary for B. dendrobatidis growth and that its production from ornithine is the major synthesis pathway in the fungus.
Spermidine synthesized from ornithine is essential for growth, and B. dendrobatidis appears to lack an alternative pathway of synthesis from arginine. (A and B) Inhibition of B. dendrobatidis growth by DFMO (one of two experiments) measured as a change in optical density (OD490) (A) and measured as average percent inhibition compared to the positive control (B) (each concentration was tested more than five times, except 5 mM, which was tested twice). (C) Inhibition of growth by DFMA (one of two experiments). For panels A to C, B. dendrobatidis growth was significantly different from the positive control; *, P ≤ 0.01 by one-way ANOVA with Tukey’s post hoc test. (D) Percent inhibition (average of the results of three experiments) by DFMO alone, DFMA alone, or DFMO plus DFMA. The effects of DFMO alone and DFMO plus DFMA were significantly different from the effects of DFMA alone by one-way ANOVA with Tukey’s post hoc text; *, P ≤ 0.01. (E) Reducing DFMA did not rescue cells inhibited by DFMO. Ornithine decarboxylase was blocked by DFMO. Addition of 10 mM DFMA also reduced growth, but decreasing DFMA in the presence of DFMO did not allow for recovery due to a hypothesized arginine pathway of synthesis of spermidine (shown is percent growth in comparison with the positive control in three experiments). (F and G) Addition of putrescine (PUT), but not agmatine (AG), rescued B. dendrobatidis cells inhibited by DFMO and DFMA. (F) 10 mM DFMO inhibited growth, which was rescued by the addition of PUT. Significantly different from DFMO alone; *, P ≤ 0.01 by one-way ANOVA with Tukey’s post hoc test. (G) 10 mM DFMA inhibited growth of B. dendrobatidis, which was rescued by addition of PUT, but not by AG. DFMA-PUT was significantly different from DFMA alone or DFMA-AG by one-way ANOVA with Tukey’s post hoc test; *, P ≤ 0.01. n.s., the difference between growth in 10 mM DFMA and growth in 10 mM DFMA-AG was not significant. The positive control (Pos.) for all experiments was cells in broth only, and the negative control (Neg.) was heat-killed cells (10 min; 60°C). The error bars indicate standard error of the mean (SE).
MTA enhances the inhibitory effects of spermidine at low concentrations.Because MTA is another immunomodulatory metabolite that is present in B. dendrobatidis cell-free supernatants and is a by-product of spermidine biosynthesis (11, 12), we tested whether MTA would synergize with spermidine to inhibit lymphocytes. Alone at a suboptimal concentration (10 μM), MTA was only slightly inhibitory (Fig. 6A). However, in the absence of serum factors, 10 μM MTA enhanced the inhibition of spermidine at low concentrations (0.1 and 1 μM), concentrations at which spermidine alone was not inhibitory. Thus, at a suboptimal concentration of the two metabolites together, they were able to inhibit to a greater extent than either one alone (Fig. 6B).
A suboptimal concentration of MTA enhanced inhibition induced by spermidine. (A) Inhibition of PHA-stimulated X. laevis spleen cells by SPD or SPD plus 10 μM MTA (one representative experiment of four similar experiments). MTA alone did not significantly inhibit PHA-induced proliferation, but addition of MTA with SPD significantly inhibited the response at both 1 and 10 μM SPD over that of SPD alone by two-tailed Student's t test; *, P ≤ 0.035. (B) Percent inhibition of proliferation at each concentration (n = 5 or 6 experiments at each concentration). Significantly different from spermidine alone by one-way ANOVA with Tukey post hoc test; P = 0.009.
DISCUSSION
Spermidine and stress.Batrachochytrium dendrobatidis is an unusual pathogen because it may be able to persist in the environment without amphibian hosts and grow in culture on limited sources of nutrients (3, 24–26), but it is also able to parasitize amphibian skin. Within amphibian skin, it must cope with the adaptive immune system. Epithelial cells and resident immune cells (innate lymphocytes, Langerhans cells, etc.) would be expected to recognize B. dendrobatidis by pathogen- or damage-associated patterns and subsequently to induce an inflammatory response to clear the infection. However, the immune response to the pathogen is often ineffective, characterized by little or no inflammatory response or lymphocyte infiltration (27, 28). Studies to examine immune-related genes that are activated following infection of vulnerable species, such as Atelopus zeteki and Litoria verreauxii alpina, suggest an immune response that is highly dysregulated and ineffective (29, 30). Thus, B. dendrobatidis appears to have mechanisms to evade immune clearance. Among the natural immunomodulatory metabolites produced by the fungus are MTA and KYN (11). Here, we show that another metabolite, SPD, but not its biosynthetic precursor PUT, is also a potent inhibitor of amphibian lymphocyte function. We hypothesize that fungal cells within the skin environment experience stress, including possible oxidative stress, perhaps due to mediators released by macrophages or neutrophils. Under stress, they may synthesize increased amounts of spermidine as protection. Saccharomyces cerevisiae cells deficient in the ability to synthesize polyamines are especially sensitive to oxygen (31; reviewed in references 32 and 33). Saccharomyces cerevisiae cells respond to oxidative stress by exporting spermidine and spermine (34). Thus, it seems possible that macrophages and neutrophils may release reactive oxygen species when they encounter B. dendrobatidis pathogens. In turn, the zoosporangia may release SPD to readjust the level of polyamines in the cell in response to this stressor. It might be possible, using imaging mass spectrometry, to find increased amounts of spermidine in skin tissue with a high intensity of B. dendrobatidis infection (35, 36). Future studies will examine whether B. dendrobatidis in the presence of reactive oxygen species, such as hydrogen peroxide, is induced to produce spermidine and whether the pathogen is more sensitive to oxidative stress in the presence of DFMO. Another possible mechanism of inhibition is that macrophage encounters with B. dendrobatidis could alter their functional phenotype towards a less phagocytic and more immunosuppressive phenotype and release of cytokines, such as interleukin 10 (IL-10), that suppress lymphocytes responding to the infection (37). Thus, an ancient stress response common to many fungi may enable B. dendrobatidis to evade a protective immune response.
Biosynthesis of spermidine and MTA.Our results strongly suggest that the dominant pathway of spermidine biosynthesis in B. dendrobatidis is via ornithine to putrescine to spermidine, with MTA produced as a by-product (reviewed in reference 12). A preliminary search of the B. dendrobatidis genome (Broad Institute bioproject PRJNA13653) revealed the presence of a number of the putative enzymes needed for synthesis of spermidine and production of MTA, including ornithine decarboxylase, arginine decarboxylase, and spermidine synthase. Biosynthesis of spermidine from arginine requires at least four enzymes, arginine decarboxylase converting arginine to agmatine, agmatine iminohydrolase converting agmatine to N-carbamoylputrescine, N-carbamoylputrescine amidohydrolase converting N-carbamoylputrescine to putrescine, and spermidine synthase converting putrescine to spermidine (22). Using polypeptide sequences from the plant Arabidopsis thaliana to search the B. dendrobatidis genome for a homolog to agmatine iminohydrolase, we found a poor match. This suggests that the enzyme necessary to convert agmatine to a spermidine precursor is missing in B. dendrobatidis. Thus, a pathway from ornithine to spermidine is indicated or may be dominant in B. dendrobatidis. We speculate that rapidly proliferating B. dendrobatidis zoosporangia within amphibian skin produce inhibitory concentrations of both SPD and MTA. When B. dendrobatidis is stressed, exported SPD can directly inhibit lymphocyte proliferation, and excess MTA may enhance this effect.
MATERIALS AND METHODS
Culture of B. dendrobatidis and enrichment of zoospores.Batrachochytrium dendrobatidis isolate JEL 197 was cultured in 1% tryptone broth (T-broth) at 21°C and subcultured twice weekly (9, 38). Zoospores were purified as previously described by washing the agar surfaces of 5- to 7-day-old cultures of B. dendrobatidis growing on 1% tryptone agar three times using 3 to 5 ml of sterile 1% T-broth (9, 38). The combined broth containing zoospores was passed over sterile nylon spectra/mesh filters (BioDesign Inc. of New York, Carmel, NY, USA) with a 20-μm mesh opening size to remove mature zoosporangia.
Frog lymphocyte culture.Splenic lymphocytes from X. laevis were enriched over a Ficoll gradient as previously described (9). Briefly, splenocytes were cultured in complete Leibovitz's L-15 medium adjusted to amphibian tonicity and supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 12.5 mM sodium bicarbonate, 50 μM 2-mercaptoethanol, 2 mM l-glutamine, and 1% heat-inactivated fetal calf serum. The spleen cells were cultured at a density of 5 × 105/ml (105 per well), and T lymphocytes were induced to proliferate by addition of PHA at a final concentration of 2 μg/ml. The cells were incubated at 26°C in the presence of 5% CO2-95% air for 3 days and were pulsed with 0.5 μCi [3H]thymidine (5 μCi/ml; specific activity, 2 Ci/mmol) (Perkin Elmer, Waltham, MA, USA) during the last 24 h prior to harvesting. Proliferation was measured by uptake of [3H]thymidine in the last 24 h of a 3-day culture and recorded as counts per minute.
Coculture of lymphocytes with B. dendrobatidis zoosporangia or supernatant factors.To determine the effects of coculture of living B. dendrobatidis zoosporangia or cell-free supernatants of B. dendrobatidis on frog lymphocyte proliferation, splenic lymphocytes were cultured at a density of 5 × 105/ml (105 per well) and stimulated with PHA (2 μg/ml) and with the addition of varying numbers of zoosporangia (all cells larger than zoospores) ranging from 100 to 105 per well or with B. dendrobatidis supernatant at a 1.25- to 10-fold concentration of the supernatant. Supernatants were prepared as previously described (9). Control wells contained splenocytes only with no addition of PHA (negative) or splenocytes with PHA but no added zoosporangia (positive). Proliferation was measured by uptake of [3H]thymidine in the last 24 h of a 3-day culture and recorded as counts per minute.
To examine the effects of addition of purified polyamines (HCl salts of spermidine, cadaverine, spermine, and putrescine; norspermidine as a liquid [Sigma-Aldrich, St. Louis, MO, USA]) on lymphocyte proliferation, lymphocytes were cultured with or without PHA (2 μg/ml) and increasing concentrations of each polyamine dissolved in complete L-15 medium. The cultures developed for 3 days, and [3H]thymidine was added in the last 24 h of culture.
To determine the effects of addition of MTA and spermidine to frog lymphocytes stimulated with PHA, a suboptimal concentration of MTA (10 μM) was added, along with spermidine, at the beginning of the 3-day culture period. For these experiments examining the interactive effects of MTA and spermidine, fetal calf serum was replaced by 1% (wt/vol) bovine serum albumen (BSA) (Fraction V powder; Fisher Scientific) to eliminate possible serum effects on spermidine activity (39, 40).
To examine the effects of purified polyamines on growth of mammalian lymphocytes, the Jurkat T cell line was cultured in RPMI medium supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2-95% air as described previously (11). The Jurkat cells (104/well) in 50 μl were cultured for 3 days with or without increasing concentrations of purified polyamines or 25 μg/ml etoposide (negative control for growth; final concentration in culture, 12.5 μg/ml) in 50 μl of RPMI medium so that the total volume of cells and inhibitors was 100 μl. Viability of the Jurkat cells was determined by the addition of 100 μl of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide at 500 μg/ml, as previously described (11)]. Percent inhibition of PHA-induced splenocyte proliferation and percent inhibition of Jurkat growth were determined as previously described (11).
Effects of enzyme inhibitors on B. dendrobatidis growth.To examine the effects of DFMO and DFMA on growth (Sigma-Aldrich, St. Louis, MO, USA), T-broth was supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin to reduce the chance of bacterial contamination, and cultures were enriched for zoospores as previously described. The zoospores were counted using a hemocytometer slide and cultured with or without DFMO or DFMA or both at concentrations ranging from 0.1 to 10 mM at a density of 5 × 105/ml (5 × 104/well) in a total volume of 100 μl. Growth was monitored as a change in optical density at 490 nm (OD490) on day 7. Positive controls for growth were cells cultured in T-broth only, and negative controls were heat-killed cells (10 min at 60°C). In order to determine whether putrescine or agmatine could rescue B. dendrobatidis cells inhibited by DFMO or DFMA, the blocking reagents (10 mM DFMO or 10 mM DFMA) were added on day 0, and the cultures were supplemented with 1 mM putrescine or 1 mM agmatine on day 4. The supplemented cultures were observed again on day 7, and growth was recorded as the OD490.
Isolation and characterization of spermidine from B. dendrobatidis.A 1-liter culture of B. dendrobatidis (isolate JEL197) was grown in a 4-liter Erlenmeyer flask with constant shaking at 100 rpm at 19°C. The culture, including cells and supernatant, was transferred to a round-bottom flask and heated for 6 h at 95°C. Following the heat treatment, the sample was centrifuged and then concentrated under vacuum to 200 ml using a rotary evaporator. The concentrated supernatant was precipitated through the addition of 4 volumes of cold ethanol (final concentration, 80% [vol/vol]) and stirred for 16 h at 4°C. The ethanol precipitate was collected by centrifugation and resuspended in water before attempting to further enrich for bioactive molecules using a Strata-X-CW weak cation-exchange column (Phenomenex, Golden, CO, USA) using the manufacturer’s recommended protocol. Briefly, the supernatant was loaded onto a 5-g column, and the flowthrough was pooled with a 30-ml water wash. Next, the bound fraction was eluted with a 30-ml volume of methanol and then an equal volume of 0.5% formic acid in water. The fractions were collected in glass vials and dried using a speed vacuum system (Savant SPD2010; Thermo Fisher) overnight at room temperature. The vials were weighed, and fractions were resuspended in water at a concentration of 10 mg/ml. A modified version of the Jurkat T cell viability assay described above was used to evaluate the bioactivity of the fractions. In a 96-well plate, 20 μl of the Strata X-CW fractions was added to 180 μl of fresh medium containing 104 Jurkat T cells. The Jurkat T cell line was cultured in RPMI medium supplemented with 10% fetal calf serum, 2 mM l-glutamine at 37°C with 5% CO2-95% air; however, no antibiotics were added. After 24 h, 50 μl of a 500-mg/ml MTT solution was added to the treated or untreated (water-treated control) cells before reincubation under the same conditions for 4 h. After 4 h, the 96-well plate was centrifuged, and the insoluble formazan was resuspended in 100 μl of dimethyl sulfoxide (DMSO) for quantification as previously described. The normalized cell viability is presented relative to an untreated control sample.
Demonstration of the presence of polyamines in cell-free supernatants of B. dendrobatidis.Cell-free supernatants prepared in the Vanderbilt laboratory were lyophilized and sent to the Villanova and Gwynedd Mercy laboratories for identification of potentially immunomodulatory components. Polyamines present in the B. dendrobatidis cell-free supernatant were derivatized using a variation of a previously published method (18). In a scintillation vial, 100 μl of supernatant was mixed with 200 μl saturated sodium carbonate and 400 μl of a dansyl chloride solution (7.5 mg/ml; 28 mM) in acetone. The reaction mixtures were stirred at room temperature in the dark for 1 to 2 h for a complete reaction. After the reaction was completed, 400 μl of 28 mM aqueous proline was added to quench any unreacted dansyl chloride. Dansylated products were extracted with dichloromethane, the dichloromethane was evaporated under a gentle air stream, and the residue was reconstituted in 1 ml of methanol for LC-MS analysis. All samples were analyzed using a Shimadzu LC-20 liquid chromatograph equipped with an ACE C18 column (3 μm; 150 by 4.6 mm) and a Shimadzu SPD-M20A diode array detector. For MS analysis, an identical system with an Applied Biosystems Sciex API 2000 triple-quadrupole mass spectrometer operating in positive electrospray ionization (ESI+) mode was used. Masses of compounds were obtained as follows: [M + H]+. Compounds were separated with a binary mobile phase flowing at 0.5 ml min−1 consisting of acidified water (0.1% [vol/vol] formic acid; solvent A) and acidified acetonitrile (0.1% [vol/vol] formic acid; solvent B). The gradient was as follows: 10% B (2-min hold) ramped to a final mobile-phase concentration of 100% B over 20 min (5-min hold). The gradient was then dropped back down to 10% B for the remainder of the run. Each run lasted 30 min, and the compounds were detected and quantified at 340 nm. The HPLC and MS control and spectra were obtained using the software LC Lab Solutions 5.82 (Shimadzu) and Analyst 1.6.2 (Applied Biosystems), respectively.
ACKNOWLEDGMENTS
Work in the Rollins-Smith, Umile, and Minbiole laboratories was supported by National Science Foundation grants IOS-1121758, IOS-1557634, and IOS-1557592. Work in the Clardy laboratory was funded by National Institutes of Health grant R01 AT009874. Antonio Ruzzini is currently funded by the Saskatchewan Health Research Foundation. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
FOOTNOTES
- Received 14 January 2019.
- Returned for modification 18 February 2019.
- Accepted 1 March 2019.
- Accepted manuscript posted online 4 March 2019.
- Copyright © 2019 American Society for Microbiology.
