Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine

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.

Keywords: Batrachochytrium dendrobatidis; amphibian declines; difluoromethylarginine (DFMA); difluoromethylornithine (DFMO); immunomodulation; methylthioadenosine; polyamine; putrescine; spermidine; splenocytes.

FIG 1

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.

FIG 2

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 (λ_max = 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).

FIG 3

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.

FIG 4

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.

FIG 5

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).

FIG 6

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.