Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens

Abstract

The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes.

Introduction

Molecular variation is the raw material for evolution. Species need molecular variation in order to respond to environmental changes, including new infectious diseases and altered ecosystems. Traditionally, intraspecies variation is studied at either the macroscopic or the genetic level, and there is less emphasis on describing differences in expressed protein families. However, an understanding of functional differences among organisms at the protein level is essential for a complete picture of adaptive variation that connects genes to phenotypes.

Many amphibian species are experiencing steep population declines (reviewed in [1], [2], [3], [4], [5]), and emerging infectious diseases are thought to be a major cause (reviewed in [6], [7], [8]). It is important to assess and conserve genetic variation at immune system loci if amphibian species are to recover from these declines [9]. Leopard frogs have long been model amphibians for studying genetic variation at the molecular level [10]. A recent taxonomic revision proposed by Frost et al. [11] places leopard frogs and related species (previously genus Rana) in the genus Lithobates. However, Hillis [12] argued persuasively that the newer classification scheme that replaces widely used species names is unnecessary. Because this issue is not resolved, we have chosen to retain the genus designation Rana throughout this paper which allows our work to be more directly connected to previous literature on this species. The northern leopard frog, Rana pipiens, has experienced population losses in the last several decades, especially in the western portions of its range. Some of the losses are associated with emerging infectious disease pathogens ([13], [14], [15], [16], [17], reviewed in [6], [18]). Although the species is still abundant in many locations, its populations have experienced significant losses in comparison with historical numbers (reviewed in [18]). The status of the pickerel frog, R. palustris (or Lithobates palustris), with respect to emerging infectious disease is less clear, although it does not appear to be in serious decline [19].

Antimicrobial peptides (AMPs) are cationic, amphipathic molecules that kill pathogens by disrupting the cellular membrane or disrupting nucleic acids or protein synthesis within the cell (reviewed in [20], [21], [22], [23], [24], [25], [26]). Amphibian AMPs are produced in specialized granular glands (also called poison glands) in the skin. They are functionally diverse and thought to be an important component of innate defenses against pathogens that would enter by way of the skin ([27], [28], [29], [30], [31], reviewed in [32], [33], [34]). AMPs from many amphibian species have been isolated, sequenced, and tested for antimicrobial activity (reviewed in [25], [32], [34]), but very few studies have investigated AMP differences among individuals of the same species. Furthermore, the genetic basis for AMP diversity is not well-understood. Both R. pipiens and R. palustris are known to express complex mixtures of AMPs in their skin [35], [36], [37], [38] (Table 1). In these species, a genetic locus encoding peptides of the brevinin-1 family, Brevinin1.1, shows a highly unusual pattern of genetic variation [39]. In R. pipiens, several divergent allelic lineages are segregating at Brevinin1.1, each encoding a different peptide. The most common peptides encoded by these alleles are brevinin-1Pa, brevinin-1Pb, brevinin-1Pg, and brevinin-1PLa (Table 1). These divergent alleles may have arisen through simple point mutations, through gene conversion from a paralogous locus, or through introgression from a different species. Regardless of the origin of the genetic diversity at this locus, multiple tests of selective neutrality performed on the nucleotide sequences encoding these peptides indicate that the diversity is non-neutral and maintained by balancing natural selection [39]. Geographic variation in the frequencies of these alleles is shown in Fig. 1. Other loci in R. pipiens also encode brevinin-1Pa and brevinin-1Pb, as well as other peptides [40]. In contrast to this genetic diversity, the allele encoding brevinin-1PLa appears to be fixed at the Brevinin1.1 locus in R. palustris, possibly owing to a selective sweep [39]. As with R. pipiens, R. palustris also expresses other AMPs encoded by other loci [38], [41]. The peptides brevinin-1Pa and brevinin-1Pb have previously been shown to be active against bacterial and fungal microbes infecting humans [36]. Other Rana peptides are known to be active against amphibian pathogens [27], [28], [29], [42], [43], [44]. Nothing is known about the antimicrobial activities of brevinin-1Pg, brevinin-1PLa, or many of the other peptides known to be produced by these frogs.

Given that the process of balancing selection appears to maintain divergent alleles for AMPs in R. pipiens, we hypothesized that peptides which differ among individuals would show substantial differences in antimicrobial activity. Presumably there are fitness trade-offs associated with the peptides encoded by the Brevinin1.1 locus, resulting in their continued preservation by natural selection. One possibility is that individual peptides are specialized to inhibit a different class of pathogen (e.g. fungi, gram-positive bacteria, or gram-negative bacteria). A second possibility is that families of peptides target the same class of pathogen, but some individual peptides within a family are specialized to target a unique species or a unique genetic strain of a single species, perhaps a species which is coevolving with the Brevinin1.1 locus. A third possibility is that some peptides are universally more active than others at killing a broad spectrum of microbes, but there is a cost of production in the absence of deadly pathogens. The more effective peptide(s) may also damage host cells or beneficial skin bacteria [45], [46], [47], [48], [49], [50] that would inhibit growth of skin pathogens.

We tested the activities of R. pipiens peptides against three amphibian pathogens: the fungus B. dendrobatidis, the gram-negative bacterium Aeromonas hydrophila, and the gram-positive bacterium Staphylococcus epidermidis. The most serious emerging infectious disease affecting amphibians at the present time is chytridiomycosis caused by B. dendrobatidis [51], [52], [53], reviewed in [6], [7], [8], [32], [33], [34]. This chytrid fungus is transmitted by a swimming zoospore that attaches to amphibian skin and enters living skin cells of the epidermis [51], [52], [53], [54]. It does not become systemic, but replicates within the skin. Infectious zoospores emerge and re-enter the skin of the same individual or a new host [51], [52], [53], [54]. Amphibian AMPs vary remarkably in their ability to kill B. dendrobatidis [32], [34], and current evidence suggests that AMPs in the mucus are an important component of innate defenses against this pathogen [28], [29], [30], [31], [43], [55]. Populations of R. pipiens suffered some of the earliest recorded epizootics of B. dendrobatidis [6], although most populations today appear to be relatively resistant [56]. A. hydrophila can cause dermatosepticemia, commonly called “red-leg” disease in frogs [57], [58]. This species has been isolated from both R. palustris and R. pipiens in the wild [59], [60]. Although it occurs on the skin and in the digestive tracts of healthy animals, it can induce fatal disease in some anuran species when the animals are stressed [61], [62]. Most amphibian AMPs tested thus far are not effective against A. hydrophila, so it represents the most challenging test of the brevinin-1 peptides [27]. S. epidermidis has been isolated from the skin of R. pipiens [63] and R. catesbeiana [64]. Although not thought to be a major epizootic agent, it represents the gram-positive bacteria, which often respond differently to AMPs than gram-negative bacteria [22].

Here we report on the patterns of expression of AMPs of the brevinin-1 family and other AMP families within three geographically separate populations of R. pipiens. By mass spectrometry, we show that each population (Michigan, Minnesota, and Vermont) expresses a unique suite of AMPs. The differences in expressed brevinin-1 peptides reflect differences in the distribution of alleles for the Brevinin-1.1 locus [39]. Furthermore, enriched peptide mixtures from Vermont and Minnesota frogs were significantly more effective in growth inhibition of B. dendrobatidis than those of Michigan frogs. When the most abundantly expressed peptides (brevinin-1Pa, brevinin-1Pb, brevinin-1Pg, and brevinin-1PLa) were tested individually for antimicrobial activity against B. dendrobatidis, A. hydrophila, and S. epidermidis, some additional differences were observed. Taken together, the differences in antimicrobial activity of peptides from different populations suggest possible differences in resistance to pathogens that may reflect past and ongoing encounters with disease organisms.