Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation

Abstract

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Keywords: conservation; disease resistance; emerging infectious disease; major histocompatibility complex; peptide-binding domain; pocket residue.

Figure 1.

Alignment of the β1 domain of the MHC class II in amphibians illustrating peptide-binding residues. Each sequence in the alignment represents a consensus sequence grouping alleles isolated in a species, or in a subgroup within species. Susceptibility of each was determined by experimental infections or field observation of Bd-associated population declines. Positions encoding amino acids similar to Bd-resistant Bufo gargarizans are represented by a dot. Variable positions in consensus sequences are represented by the most frequent amino acid in the group (lower case letters). Dashes indicate missing data. Peptide-binding residues are highlighted in colours, as denoted, to indicate their association with pockets of the MHC peptide-binding groove. Bd-resistant Litoria verreauxii alpina (Livea-R), Bd-susceptible L. v. alpina (Livea-S), Bufo gargarizans (Buga), Bombina orientalis (Boor), Bombina bombina (Bobo), Bombina variegata (Bova), Bombina pachypus (Bopa), Bufo bufo (Bubu), Bufo [Epidalea] calamita (Buca), Rana [Lithobates] yavapaiensis (Raya), Rana [Lithobates] catesbeiana (Raca), susceptible Rana spp. (sRana) including Rana [Lithobates] clamitans, R. pipiens, R. [Lithobates] sylvatica, R. [Lithobates] warszewitschii and Rana temporaria, Alytes obstetricans (Alob), Xenopus laevis (Xela), Andrias davidianus (Anda), Ambystoma mexicanum and A. tigrinum (Anme/Amti). Raya-Q, allele from Rana [Lithobates] yavapaiensis associated with Bd resistance. The complete alignment with all MHC-II β1 included in the study is available in the electronic supplementary material.

Figure 2.

Experimental infection of L. v. alpina frogs from populations with varied Bd infection histories. (a) Survival curves for captive-reared frogs from two historically infected sites (A and B) and an uninfected site (C) in Kosciuszko National Park, Australia, experimentally infected with a virulent Bd culture. (b) Mean infection intensity as a function of time since Bd inoculation for those subjects that survived five weeks or less, eight weeks, or through the duration of the 12-week experiment. These results include the weekly swabs from up to 30 frogs from each site, selected by stratified random sampling based on days survived. Infection intensity is given in log-transformed ZSE. Error bars represent 95% confidence intervals.

Figure 3.

Association of the MHC class II β1 domain with survival to Bd in L. v. alpina. (a) Frequency of MHC class II β1 alleles (Livea) in subjects that survived (red) and succumbed (blue) to experimental infection by Bd, indicating alleles encoding the P9 and P6 pocket compositions most frequent in Bd-resistant amphibians; frequency of individuals with two (2/2), one (1/2) or no (0/2) MHC-II β1 alleles with the specific P9 residue composition. (b) Survival curves for individuals during the course of the Bd infection experiment with respect to the presence of the specific P9 pocket residue composition in two (2/2), one (1/2) or none (0/2) of their two MHC-II β1 domains.