Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Heat stress and amphibian immunity in a time of climate change.

As a class of vertebrates, amphibians, are at greater risk for declines or extinctions than any other vertebrate group, including birds and mammals. There are many threats, including habitat destruction, invasive species, overuse by humans, toxic chemicals and emerging diseases. Climate change which brings unpredictable temperature changes and rainfall constitutes an additional threat. Survival of amphibians depends on immune defences functioning well under these combined threats. Here, we review the current state of knowledge of how amphibians respond to some natural stressors, including heat and desiccation stress, and the limited studies of the immune defences under these stressful conditions. In general, the current studies suggest that desiccation and heat stress can activate the hypothalamus pituitary-interrenal axis, with possible suppression of some innate and lymphocyte-mediated responses. Elevated temperatures can alter microbial communities in amphibian skin and gut, resulting in possible dysbiosis that fosters reduced resistance to pathogens. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

The importance of antimicrobial peptides (AMPs) in amphibian skin defense.

Antimicrobial peptides (AMPs) are produced for defense in nearly all taxa from simple bacteria to complex mammalian species. Some amphibian families have developed this defensive strategy to a high level of sophistication by loading the AMPs into specialized granular glands within the dermis. Enervated by the sympathetic nervous system, the granular glands are poised to deliver an array of AMPs to cleanse the wound and facilitate healing. There have been a number of excellent review publications in recent years that describe amphibian AMPs with an emphasis on their possible uses for human medicine. Instead, my aim here is to review what is known about the nature of amphibian AMPs, the diversity of amphibian AMPs, regulation of their production, and to provide the accumulated evidence that they do, indeed, play an important role in the protection of amphibian skin, vital for survival. While much has been learned about amphibian AMPs, there are still important gaps in our understanding of peptide synthesis, storage, and functions.

Disease state associated with chronic toe lesions in hellbenders may alter anti-chytrid skin defenses.

Hellbenders (Cryptobranchus alleganiensis) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans (Bsal) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal. Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd. MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects of disease associated with toe lesions.

Once a reservoir, always a reservoir? Seasonality affects the pathogen maintenance potential of amphibian hosts.

Host species that can independently maintain a pathogen in a host community and contribute to infection in other species are important targets for disease management. However, the potential of host species to maintain a pathogen is not fixed over time, and an important challenge is understanding how within- and across-season variability in host maintenance potential affects pathogen persistence over longer time scales relevant for disease management (e.g., years). Here, we sought to understand the causes and consequences of seasonal infection dynamics in leopard frogs (Rana sphenocephala and Rana pipiens) infected with the fungal pathogen Batrachochytrium dendrobatidis (Bd). We addressed three questions broadly applicable to seasonal host-parasite systems. First, to what degree are observed seasonal patterns in infection driven by temperature-dependent infection processes compared to seasonal host demographic processes? Second, how does seasonal variation in maintenance potential affect long-term pathogen persistence in multi-host communities? Third, does high deterministic maintenance potential relate to the long-term stochastic persistence of pathogens in host populations with seasonal infection dynamics? To answer these questions, we used field data collected over 3 years on >1400 amphibians across four geographic locations, laboratory and mesocosm experiments, and a novel mathematical model. We found that the mechanisms that drive seasonal prevalence were different from those driving seasonal infection intensity. Seasonal variation in Bd prevalence was driven primarily by changes in host contact rates associated with breeding migrations to and from aquatic habitat. In contrast, seasonal changes in infection intensity were driven by temperature-induced changes in Bd growth rate. Using our model, we found that the maintenance potential of leopard frogs varied significantly throughout the year and that seasonal troughs in infection prevalence made it unlikely that leopard frogs were responsible for long-term Bd persistence in these seasonal amphibian communities, highlighting the importance of alternative pathogen reservoirs for Bd persistence. Our results have broad implications for management in seasonal host-pathogen systems, showing that seasonal changes in host and pathogen vital rates, rather than the depletion of susceptible hosts, can lead to troughs in pathogen prevalence and stochastic pathogen extirpation.

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.

Batrachochytrium fungi: stealth invaders in amphibian skin.

Amphibian populations around the world have been affected by two pathogenic fungi within the phylum Chytridiomycota. Batrachochytrium dendrobatidis (Bd) has infected hundreds of species and led to widespread declines and some species extinctions. Batrachochytrium salamandrivorans (Bsal) has devastated some native European salamanders, especially the iconic fire salamanders (Salamandra salamandra). Comparative genomic studies show that Bd is more diverse and widespread than previously thought, and global lineages occur together allowing for the development of hybrid lineages. New studies raise the concern of greater pathogenesis if both Bd and Bsal infect the same host. Although amphibians possess robust immune defenses, co-infected and many single-infected hosts seem unable to mount effective immune responses. A strong defense may actually be harmful. Analysis of Bd and Bsal secretions documents small metabolites that signal high density to limit their growth and to suppress adaptive immune defenses, thus enabling a stealth presence in the skin compartment.

Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Preparatory immunity: Seasonality of mucosal skin defences and Batrachochytrium infections in Southern leopard frogs.

Accurately predicting the impacts of climate change on wildlife health requires a deeper understanding of seasonal rhythms in host-pathogen interactions. The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), exhibits seasonality in incidence; however, the role that biological rhythms in host defences play in defining this pattern remains largely unknown. The aim of this study was to examine whether host immune and microbiome defences against Bd correspond with infection risk and seasonal fluctuations in temperature and humidity. Over the course of a year, five populations of Southern leopard frogs (Rana [Lithobates] sphenocephala) in Tennessee, United States, were surveyed for host immunity, microbiome and pathogen dynamics. Frogs were swabbed for pathogen load and skin bacterial diversity and stimulated to release stored antimicrobial peptides (AMPs). Secretions were analysed to estimate total hydrophobic peptide concentrations, presence of known AMPs and effectiveness of Bd growth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibit Bd growth in vitro were used as an estimate of predicted anti-Bd function of the skin microbiome. Batrachochytrium dendrobatidis dynamics followed the expected seasonal fluctuations-peaks in cooler months-which coincided with when host mucosal defences were most potent against Bd. Specifically, the concentration and expression of stored AMPs cycled synchronously with Bd dynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibit Bd growth was greatest when risk of Bd infection was highest. We interpret the increase in peptide storage in the fall and the shift to a more anti-Bd microbiome over winter as a preparatory response for subsequent infection risk during the colder periods when AMP synthesis and bacterial growth is slow and pathogen pressure from this cool-adapted fungus is high. Given that a decrease in stored AMP concentrations as temperatures warm in spring likely means greater secretion rates, the subsequent decrease in prevalence suggests seasonality of Bd in this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.

Antimicrobial Secretions of Toads (Anura, Bufonidae): Bioactive Extracts and Isolated Compounds against Human Pathogens.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

Caerin 1 Antimicrobial Peptides That Inhibit HIV and Neisseria May Spare Protective Lactobacilli.

Although acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is a manageable disease for many, it is still a source of significant morbidity and economic hardship for many others. The predominant mode of transmission of HIV/AIDS is sexual intercourse, and measures to reduce transmission are needed. Previously, we showed that caerin 1 antimicrobial peptides (AMPs) originally derived from Australian amphibians inhibited in vitro transmission of HIV at relatively low concentrations and had low toxicity for T cells and an endocervical cell line. The use of AMPs as part of microbicidal formulations would expose the vaginal microbiome to these agents and cause potential harm to protective lactobacilli. Here, we tested the effects of caerin 1 peptides and their analogs on the viability of two species of common vaginal lactobacilli (Lactobacillus rhamnosus and Lactobacillus crispatus). Several candidate peptides had limited toxicity for the lactobacilli at a range of concentrations that would inhibit HIV. Three AMPs were also tested for their ability to inhibit growth of Neisseria lactamica, a close relative of the sexually transmissible Neisseria gonorrhoeae. Neisseria lactamica was significantly more sensitive to the AMPs than the lactobacilli. Thus, several candidate AMPs have the capacity to inhibit HIV and possible N. gonorrhoeae transmission at concentrations that are significantly less harmful to the resident lactobacilli.

Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Scheele et al (Reports, 29 March 2019, p. 1459) bring needed attention to the effects of amphibian infectious disease. However, the data and methods implicating the disease chytridiomycosis in 501 amphibian species declines are deficient. Which species are affected, and how many, remains a critical unanswered question. Amphibians are imperiled; protective actions require public support and robust science.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Out in the cold and sick: low temperatures and fungal infections impair a frog's skin defenses.

Amphibians worldwide continue to battle an emerging infectious disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Southern leopard frogs, Rana sphenocephala, are known to become infected with this pathogen, yet they are considered 'of least concern' for declines due to chytridiomycosis. Previous studies have shown that R. sphenocephala secretes four antimicrobial peptides (AMPs) onto their skin which may play an important role in limiting susceptibility to chytridiomycosis. Here, we examined (1) the effects of temperature and AMP depletion on infections with Bd and (2) the effects of temperature and Bd infection on the capacity to secrete AMPs in juvenile leopard frogs. Pathogen burden and mortality were greater in frogs exposed to Bd at low temperature but did not increase following monthly AMP depletion. Both low temperature and Bd exposure reduced the capacity of juvenile frogs to restore peptides after monthly depletions. Frogs held at 14°C were poorly able to restore peptides in comparison with those at 26°C. Frogs held at 26°C were better able to restore their peptides, but when exposed to Bd, this capacity was significantly reduced. These results strongly support the hypothesis that both colder temperatures and Bd infection impair the capacity of juvenile frogs to produce and secrete AMPs, an important component of their innate defense against chytrid fungi and other pathogens. Thus, in the face of unpredictable climate changes and enzootic pathogens, assessments of disease risk should consider the potential for effects of environmental variation and pathogen exposure on the quality of host defenses.

Disease and the Drying Pond: Examining Possible Links among Drought, Immune Function, and Disease Development in Amphibians.

Drought can heavily impact aquatic ecosystems. For amphibian species that rely on water availability for larval development, drought can have direct and indirect effects on larval survival and postmetamorphic fitness. Some amphibian species can accelerate the timing of metamorphosis to escape drying habitats through developmental plasticity. However, trade-offs associated with premature metamorphosis, such as reduced body size and altered immune function in the recently metamorphosed individual, may have downstream effects on susceptibility to disease. Here, we review the physiological mechanisms driving patterns in larval amphibian development under low water conditions. Specifically, we discuss drought-induced accelerated metamorphosis and how it may alter immune function, predisposing juvenile amphibians to infectious disease. In addition, we consider how these physiological and immunological adjustments could play out in a lethal disease system, amphibian chytridiomycosis. Last, we propose avenues for future research that adopt an ecoimmunological approach to evaluate the combined threats of drought and disease for amphibian populations.

Viscosin-like lipopeptides from frog skin bacteria inhibit Aspergillus fumigatus and Batrachochytrium dendrobatidis detected by imaging mass spectrometry and molecular networking.

Amphibian populations worldwide have declined and in some cases become extinct due to chytridiomycosis, a pandemic disease caused by the fungus Batrachochytrium dendrobatidis; however, some species have survived these fungal epidemics. Previous studies have suggested that the resistance of these species is due to the presence of cutaneous bacteria producing antifungal metabolites. As our understanding of these metabolites is still limited, we assessed the potential of such compounds against human-relevant fungi such as Aspergillus. In this work we isolated 201 bacterial strains from fifteen samples belonging to seven frog species collected in the highlands of Panama and tested them against Aspergillus fumigatus. Among the 29 bacterial isolates that exhibited antifungal activity, Pseudomonas cichorii showed the greatest inhibition. To visualize the distribution of compounds and identify them in the inhibition zone produced by P. cichorii, we employed MALDI imaging mass spectrometry (MALDI IMS) and MS/MS molecular networking. We identified viscosin and massetolides A, F, G and H in the inhibition zone. Furthermore, viscosin was isolated and evaluated in vitro against A. fumigatus and B. dendrobatidis showing MIC values of 62.50 µg/mL and 31.25 µg/mL, respectively. This is the first report of cyclic depsipeptides with antifungal activity isolated from frog cutaneous bacteria.

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.

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Lymphocyte Deficiency Induced by Sublethal Irradiation in Xenopus.

In many studies of diseases affecting amphibians, it is important to determine to what extent lymphocyte-mediated defenses are involved. For example, in studies of the nature of the immune response of Xenopus laevis to the amphibian chytrid fungus, Batrachochytrium dendrobatidis, it was essential to determine if mucosal antimicrobial peptides or lymphocyte-mediated immunity was most important for resistance to this skin pathogen. In this protocol, we describe a method for sublethal irradiation to reduce lymphocyte numbers. Briefly, X. laevis adults or tadpoles are exposed to 9 Gy (900 rads) of irradiation applied by exposure to a cesium source or gamma irradiator to reduce lymphocyte populations in the spleen.