Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis

Abstract

cAmphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.

Immunomodulatory Metabolites Released by the Frog-Killing Fungus Batrachochytrium dendrobatidis

“…Alternatively, it may be a natural product of rapidly growing B. dendrobatidis cells that accumulates when fungal cells grow at a high cell density, as occurs in bacterial biofilms (26). Its role in B. dendrobatidis metabolism awaits further study, but its presence in inhibitory supernatants suggests that it may be one factor that contributes to the immune evasion of B. dendrobatidis in vitro and in vivo (11,12). Although the mechanism of action of MTA in the inhibition of lymphocytes is not well understood, this small molecule can inhibit both T lymphocytes and B lymphocytes in mammalian model systems, and it has been proposed for use in the treatment of melanoma and autoimmune disorders (18,27).…”

“…Further studies demonstrated that B. dendrobatidis cells release molecules that inhibit proliferation of lymphocytes and transformed cell lines and induce apoptosis of lymphocytes (11). Enriched fungal supernatants also interfere with the development of a delayed-type hypersensitivity (DTH)-like response to injected phytohemagglutinin (PHA) in X. laevis (12). Thus, the inhibitory factors can act locally in the skin environment.…”

“…Heavily infected frogs show lethargy, loss of appetite, and behavioral changes (1,3,7,8), and they die due to a failure to maintain a balance of essential ions across the damaged skin (9). Although amphibians have robust and complex immune defenses (10), immune responses against this pathogen are impaired (11)(12)(13). Our previous studies showed that sublethal X irradiation of South African clawed frogs (Xenopus laevis) resulted in decreased lymphocyte numbers in the spleen and increased fungal burdens, demonstrating that lymphocyte-mediated adaptive immune responses are important for control of the infection (14).…”

Amphibian Alliance for Zero Extinction Sites in Chiapas and Oaxaca

“…More recently, there is evidence that Bd inhibits lymphocyte-mediated swelling in vitro (Fites et al 2013) and that genes which might lead to an immune response to Bd can be activated and subsequently suppressed (Ellison et al 2014). In vivo, experiments indicate that Bd inhibits immunity Fites et al 2014). Thus, it is likely that some species may be better at defending themselves against Bd not because they inherently have more robust immune systems, but because the immune suppressive factors emitted by Bd are less effective on them.…”

“…Ellison et al (2014) have shown 33 Lowered immune response at higher elevation has been put forward as a contributing factor, but if this were the case one would expect to find amphibians succumbing to a combination of diseases. Amphibian necropsies instead show that chytridiomycosis alone often kills (Berger et al 1998(Berger et al , 2004 Fites et al 2014). The new focus on Bd immunosuppression might help to explain why some amphibian species are less susceptible than others to chytridiomycosis.…”