Lymphocyte Inhibition by the Salamander-Killing Chytrid Fungus, Batrachochytrium salamandrivorans.

Amphibian populations have been declining around the world for more than five decades, and the losses continue. Although causes are complex, major contributors to these declines are two chytrid fungi, Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, which both cause the disease termed chytridiomycosis. Previously, we showed that B. dendrobatidis impedes amphibian defenses by directly inhibiting lymphocytes in vitro and in vivo by release of soluble metabolites, including kynurenine (KYN), methylthioadenosine (MTA), and spermidine (SPD). Here, we show that B. salamandrivorans cells and cell-free supernatants also inhibit amphibian lymphocytes as well as a human T cell line. As we have shown for B. dendrobatidis, high-performance liquid chromatography (HPLC) and mass spectrometry revealed that KYN, MTA, and SPD are key metabolites found in the B. salamandrivorans supernatants. Production of inhibitory factors by B. salamandrivorans is limited to mature zoosporangia and can occur over a range of temperatures between 16°C and 26°C. Taken together, these results suggest that both pathogenic Batrachochytrium fungi have evolved similar mechanisms to inhibit lymphocytes in order to evade clearance by the amphibian immune system.

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

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.