The adaptive microbiome hypothesis and immune interactions in amphibian mucus

Abstract

The microbiome is known to provide benefits to hosts, including extension of immune function. Amphibians are a powerful immunological model for examining mucosal defenses because of an accessible epithelial mucosome throughout their developmental trajectory, their responsiveness to experimental treatments, and direct interactions with emerging infectious pathogens. We review amphibian skin mucus components and describe the adaptive microbiome as a novel process of disease resilience where competitive microbial interactions couple with host immune responses to select for functions beneficial to the host. We demonstrate microbiome diversity, specificity of function, and mechanisms for memory characteristic of an adaptive immune response. At a time when industrialization has been linked to losses in microbiota important for host health, applications of microbial therapies such as probiotics may contribute to immunotherapeutics and to conservation efforts for species currently threatened by emerging diseases.

Keywords: Antimicrobial peptides; Chytridiomycosis; Disease ecology; Homeostasis; Microbiota; Resilience; Stress physiology; Symbiosis.

Figure 1.. Characteristics of host microbiomes subsequent to pathogen exposure are conceptualized by the Adaptive Microbiome Hypothesis.

The hypothesis posits that the microbiome of hosts shifts in response to pathogen exposure with the following characteristics: beta-diversity shifts and microbiome dispersion decreases as the diversity of microbes on individual hosts decreases but taxa functioning in anti-pathogen competition increase (A). The microbiome of eight individual hosts is shown changing through time (circles on day 0 and squares on day 5 changing upon pathogen exposure), with alternative states depicted at day 20 post-exposure (panel C). Stability of the microbial rescue effect may depend on continued selection pressure from pathogen exposure. Clearance of infection may lead to microbiome resilience and host recovery (B), or tolerance of infection leading to microbial rescue of the host population (C). Disease is thought to lead to Anna Karenina Principle (AKP) effects (C) in which increased dispersion and dysbiosis results from homeostatic overload, or the threshold beyond which the holobiont can no longer adapt. An alternative to the indicated trajectories is a depauperate initial microbial diversity that leads to homeostatic failure, such that the microbiome is unable to mediate the stress of infection (see Bletz et al., 2018; Greenspan et al., 2022).

Figure 2.. Bacteria isolated from amphibians in Panama indicate shifts in microbiome function against the fungal pathogen (Batrachochytrium dendrobatidis, Bd) depending on host community disease state.

Upon recovery from the chytridiomycosis epizootic, amphibian assemblages in a Bd endemic state have higher proportions of individuals with at least one anti-Bd bacterium isolated from their skin (A; Campana X²₁ = 18.958, p < 0.0001; El Cope X²₁ = 7.289, p = 0.0069). Individuals with anti-Bd skin bacteria may be considered “protected” from Bd infection to some extent and show reduced infection prevalence (B; X ²₁ = 20.027, p < 0.0001). Populations of some species persisting after the epizootic show increased prevalence of the anti-Bd bacterium Pseudomonas fluorescens (C; Fisher’s exact test, p = 0.0232), a bacterium more common on the skin of tropical than on temperate amphibians (Kueneman et al., 2019). At the same time, these amphibians showed a significant reduction in prevalence of Serratia marcescens and Pseudomonas mosselii post-epidemic. Amphibians in rainforests of Panama exhibited culturable isolate diversity ranging from 3-21 unique isolates as indicated in this “living histogram” (D) showing morphologically distinct isolates stacked by individual amphibian. (Methods and data collection described in Woodhams et al., 2015; Voyles et al., 2018).

Figure 3.. The acute phase response or Reactive Homeostasis (Romero et al., 2009) to infection in amphibians involves several interrelated components that mediate homeostasis.

(A) Activation of the hypothalamic-pituitary-interrenal axis stimulates an immediate corticosterone response, here accumulating in water rinses within the first hour of exposure to the fungal pathogen Batrachochytrium salamandrivorans (Bsal) as quantified from spotted salamanders, Ambystoma maculatum (data from Barnhart et al., 2020). Not shown is the activation of acute phase proteins including complement proteins from hepatocytes (reviewed in Jain et al., 2011; Rodriguez and Voyles, 2020). Catecholamines may also be stimulated by infection (Rollins-Smith, 2017). (B) Homeostatic circuit modified from Medzhitov (2021) inflammatory circuit model. The adaptive microbiome is hypothesized to be a novel homeostatic variable as well as an effector in the negative feedback loop regulating homeostasis. Tolerance, or adaptation of the microbiome to a new stable state that does not influence homeostatic variables is an alternative outcome. (C) Skin bacterial communities of Eastern red-spotted newts, Notophthalmus viridescens, depicted in a principal coordinates analysis showing samples colored by Bd infection load (white = 0 to red = 3.1x10⁶ zoospores max), and points scaled by predicted anti-Bd function of the microbiome of each individual (data from Carter et al., 2021). Shifts in amphibian skin microbiome communities are a typical response to infection (Jani and Briggs, 2014), although the mechanism and timing of response remains under investigation. Baseline corticosterone, immune defenses, and microbiome can change seasonally and with circadian rythms in amphibians and in other vertebrates (Martinez-Bakker and Helm 2015; Le Sage et al., 2021). (D) Sickness behaviors in N. viridescens in response to B. salamandrivorans infection include inappetence, unusual shedding patterns (reviewed in Grogan et al., 2018) and body posturing perhaps functioning to dry the skin or inducing movement away from the water potentially inhabited by conspecifics (photo credit: Julia McCartney). (E) Sickness behaviors in N. viridescens may also include thermal regulation (behavioral fever, reviewed in Lopes et al., 2021).

Figure 4.. Compounds including antimicrobial peptides and bufadienolides secreted from skin granular glands into the mucosome may differentially regulate microbial growth on amphibian skin.

(A) Natural mixtures of antimicrobial peptides purified from skin secretions of midwife toads, Alytes obstetricans, from Switzerland have differential activity on growth of bacteria cultured from toad skin (Davis, 2013; parentheses indicate the phyla). Ideal probiotics, as conceptually modeled in the figure, for amphibian skin are not inhibited by antimicrobial skin defense peptides or other immune defenses, but rather are facilitated in growth or anti-pathogen function, and function over a range of host temperatures in ectotherms, and are not inhibited by the endogenous microbial community or by the target pathogen (Woodhams et al., 2014). In addition to the promicrobial function of some peptides (Woodhams et al., 2020b), (B) bufadienolides have microbe-specific activities that may help regulate the host microbiome. Shown are the bioactivities of gamabufatalin described from boreal toads, Anaxyrus boreas. Data from Barnhart et al. (2017), photo credit Timothy Korpita.

Figure 5.. The microbiome interacts with multiple aspects of the amphibian skin landscape.

Microbes present on amphibian skin can interact with the host directly and indirectly. The immune system influences microbes present on the skin through avenues such as mucosal antibodies and pattern recognition receptors inducing innate immune responses (upper left panel). Other microbes present on the skin may influence the overall community composition through competitive and cooperative behaviors (upper right panel). Mucus is known to interact with microbes, acting as a food source and an anchor point for both microbes and bacteriophage (Barr et al., 2013), and mucus turnover rates (caused by shedding, ciliated cells in larvae, etc.) may help remove microbes attached to the matrix (lower left panel). Small molecules released by the host, such as antimicrobial peptides (shown: Subasinghagea et al. 2008; PBD ID: 2K10), can directly influence the survival and growth of microbes present on the skin, changing the community following granular gland release (lower right panel). Created with BioRender.com.

Figure 6.. Bacteria commonly isolated from amphibian skin (x-axis) also tend to dominate in culture-independent targeted amplicon sequencing (y-axis) and metagenomic studies, with some exceptions.

For example, Pseudomonas is a commonly isolated bacterial genus that is dominant in both culture and culture-independent assessments of amphibian skin bacteria. In contrast, Microbacterium is under-represented in culture-independent assessments perhaps due to difficulty in lysing Gram-positive cells during DNA extraction. The addition of a lysozyme incubation step during DNA extraction may help to reduce this bias (Teng et al., 2018). (Data from cultured isolates from Woodhams et al. (2015); data from amplicon sequencing studies from Kueneman et al. (2019).

Figure B1.

The adaptive microbiome hypothesis as represented by panarchy, or nested adaptive cycles after Gunderson and Holling (2002). In the top panel, adaptive cycles are represented at the scales of microbial population, individual amphibian host, and amphibian population or community. Connections across scale are indicated by processes of remember and revolt. Lower panels are representative figures of experiments in which newts were exposed to infectious Batrachochytrium salamandrivorans (Bsal) zoospores obtained from sterile culture (lower right panel), or exposed to Bsal and co-transmitted adaptive microbiota from direct contact of naïve to infected newts (lower left panel). Upon infection, newts with undetermined co-transmission may have gained a head-start at adapting to infection by assembling a protective microbiota.