We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.
Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.
Amphibians are currently experiencing one the greatest losses of global biodiversity of any vertebrate taxa1. A major cause of these declines is the fatal skin disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd2. The pathogen superficially infects the epidermis, which can lead to the disruption of skin function resulting in severe electrolyte loss, cardiac arrest, and death3. Various potential host immune mechanisms against Bd are currently being studied, such as antimicrobial peptides4,5, cutaneous bacterial flora6, immune cell receptors7,8, and lymphocyte activity9,10. However, few studies explore whether epidermal apoptosis and cell death is an immune mechanism against this deadly pathogen.
Cell death, either through apoptosis (programmed cell death) or necrosis (unprogrammed death), in the epidermis may be a pathology of Bd infection. Previous research suggests that Bd infection may induce apoptosis because disruption of intracellular junctions is observed when skin explants are exposed to zoospore supernatants in vitro11. Additionally, degenerative epidermal changes in Bd-infected frogs are observed using electron microscopy12,13. Transcriptomic analyses indicate that apoptosis pathways are upregulated in infected skin14, and amphibian splenocytes undergo apoptosis when they are exposed to Bd supernatants in vitro15. Despite the growing volume of evidence suggesting that Bd can induce apoptosis and host cell death in vitro, in vivo studies that explore or quantify apoptosis mechanisms through the progression of infection are lacking. Further, it is unknown if the host uses apoptosis as a defensive immune strategy to combat Bd infection, or if apoptosis is a pathology of disease.
In this study, we aimed to detect epidermal cell death and apoptosis in infected animals in vivo using two methods: caspase 3/7 protein assay, and terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ assay. As each assay detects different aspects of cell death16, together these methods provide a full understanding of the mechanisms involved in cell death, and ensure an accurate measure of the effect. The caspase 3/7 assay quantifies the activity of effector caspases 3 and 7, which enables quantification of both the intrinsic and extrinsic apoptosis pathways. In contrast, the TUNEL assay detects DNA fragmentation, which is caused by cell death mechanisms including apoptosis, necrosis and pyroptosis17. We use the TUNEL assay to investigate the location of cell death within the epidermis of both clinically infected and uninfected animals using three different skin sections: the dorsum, the venter and the thigh of Pseudophryne corroboree. This method identifies the anatomical site of cell death, as well as distinguishing its location within specific epidermal layers. We then use the caspase 3/7 assay to conduct a time series quantification of apoptosis throughout an 8-week infection in Litoria verreauxii alpina. We take toe tip samples fortnightly from the same animals and are able to correlate pathogen infection load with caspase 3/7 activity.
James Cook University approved animal ethics in applications A1875 for P. corroboree and A1897 and A2171 for L. v. alpina.
1. Animal Husbandry and Monitoring
2. Testing for Bd Infection
3. Inoculation
4. TUNEL Assay
5. Caspase 3/7 Assay
TUNEL Assay
There were more TUNEL positive cells in the infected animals than in the uninfected control animals. The in situ location of TUNEL positive cells differed in infected and control animals. In control animals, there was an even distribution of TUNEL positive cells throughout the dermal and epidermal skin layers at low levels (See Figure 1A), but in the infected animals, the TUNEL positive cells were more frequent in the epidermis (Figure 1B). The sites of infection (as observed on the H&E stained slides) were observed as clumped Bd sporangia scattered through the thigh and ventral skin sections with none in the dorsum sections. While the TUNEL positive cells were more concentrated at and directly adjacent to sites of infected cells (Figure 1C), there was also more widespread TUNEL positive cells over the epidermis and in epidermal layers that were deeper than where Bd resided. In Bd infected animals there were more TUNEL positive cells in all three skin types analyzed, but a particularly high level in the thigh and venter skin (12.01 times higher (95% CI: 4.92 – 26.30) in the thigh skin and 22.31 times higher (95% CI 5.25 – 94.82) in the venter skin) (Figure 2).
Caspase 3/7 Assay
Over the course of infection there was a difference in caspase activity. In Bd infected animals, infection intensity was positively correlated with caspase 3/7 activity (Figure 3). There was also a difference between infected and uninfected animals through time, post inoculation. Caspase 3/7 activity decreased within the first few weeks after inoculation (with 48.36% less activity in infected animals), and then increased toward the end of week 7 (Figure 4) in Bd infected animals, but remained constant in uninfected individuals.
Figure 1: Terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ assay of infected and uninfected animals. A) Bd– control thigh skin section of Pseudophryne corroboree, and B) Bd+ thigh skin section of P. corroboree stained by in situ TUNEL assay. The blue is DAPI staining indicating nuclei of the cells, and the red is the rhodamine stain, which indicates DNA fragmentation characteristically caused by apoptosis. The yellow arrow indicates the position of the Bd cluster seen in panel C. C) P. corroboree section of thigh skin stained with H&E. The H&E section is serial to panel B. There is a cluster of empty Bd sporangia (arrow) and a few dark immature sporangia near the skin surface. For all three panels the epidermis is at the top of the photo. Comparing panels B and C shows that the rhodamine stained epidermal cells are concentrated around and below the cluster of Bd and where skin damage is visible, such as micro-vesicle formation between basal epidermal cells. 400X magnification and the scale bar indicates 0.03 mm. Adapted from Figure 2 in Brannelly et al. 2017 Peer J22. Please click here to view a larger version of this figure.
Figure 2: The proportion of TUNEL positive (TUNEL+) cells per skin type. The proportion of TUNEL positive apoptotic cells per skin type in P. corroboree, with infected animals indicating animals that succumbed to disease (n = 9) and uninfected control animals (n = 10). Error bars indicate 95% confidence intervals of a proportion and * indicates a significant increase in TUNEL+ cell proportions. In the dorsal skin, the infected animals had 14.38 (95% CI 3.32 – 62.24) times more TUNEL positive cells than control animals (Odds Ratio: Z = 3.57, p <0.01). In the thigh skin, infected animals had 12.01 (95% CI: 4.92 – 26.30; Odds Ratio: Z = 5.46, p <0.01) times more TUNEL positive cells than control animals (Pearson's Chi Squared: χ21 = 44.30, p <0.01). In the venter skin, infected animals had 22.31 (95% CI 5.25 – 94.82) times more TUNEL positive cells than control animals (Odds Ratio: Z = 4.21, p <0.01). Adapted from Figure 3 in Brannelly et al. 2017 Peer J22. Please click here to view a larger version of this figure.
Figure 3: The correlation between infection intensity, Log10(zoospore equivalents), and caspase 3/7, Log10(Caspase) of inoculated Litoria verreauxii alpina over the course of the experiment. The correlation between infection intensity and caspase activity is 0.463, and the trend line has an equation of y = (0.229)x + 0.939. Adapted from Figure 4 in Brannelly et al. 2017 Peer J22. Please click here to view a larger version of this figure.
Figure 4: Caspase 3/7 activity through week 7 for each group of Litoria verreauxii alpina: Bd-infected animals that succumbed to disease (n = 4) and uninfected controls (n = 8). Caspase activity is defined as the luminescence reading controlled for by protein concentration per sample and then log base 10 transformed. The caspase activity (Log10 transformed) for each group per week. Error bars indicate standard error. * indicates that the infected animals differed from the uninfected controls at that week. (Linear mixed effects model: week, F4 = 11.974, p <0.01; week*status, F8 = 2.139, p = 0.037; Week 3 ANOVA, F2,18 = 5.512, p = 0.014), Adapted from Figure 5 in Brannelly et al. 2017 Peer J22. Please click here to view a larger version of this figure.
We explored epidermal apoptosis and cell death as a potential mechanism of pathology of the deadly disease chytridiomycosis or a mechanism of disease resistance in Bd susceptible species. We used two methods of assessing cell death in the epidermis, TUNEL assay for in situ epidermal cell death analysis, and caspase 3/7 assay for monitoring epidermal cell death throughout the progress of infection. We found that cell death and apoptosis are correlated with infection load and cell death is significantly higher at the site of infection. In early infection, there is a decrease in apoptosis in the epidermis, but apoptosis increases as pathogen burden increases within the infected tissue.
Apoptosis in cells can be tested using numerous different approaches, each with benefits and limitations. It is important to study apoptosis and cell death using multiple assays in order to confirm findings. In this study, we explored two different assays to investigate epidermal cell death and apoptosis in frogs with chytridiomycosis. First, we trialled the TUNEL assay to assess cell death in situ. This test is time consuming, and expensive per sample, and slides must be read immediately as the fluorescent signal quickly fades. Despite these limitations, the results of this in situ experiment are important to further understanding the mechanisms of host-pathogen interactions. Information regarding localization can be determined using this method, and in our study apoptosis was present in high levels at the site of fungal infection and more diffusely at other sites. Additionally it must be noted that the TUNEL assay measures DNA damage, which can be caused by a number of different cell death mechanisms including apoptosis, necrosis and pyroptosis17. While there are signatures of apoptosis-associated cell death (such as single cells with membranes still intact, see protocol line 4.6.3, the classification relies on the interpretation of the researcher. Because the mechanism of cell death is not determined by the TUNEL assay, it is possible that another non-apoptosis cell death pathway may have caused the increase in TUNEL positive cells at morbidity of infected animals. The difference in what each assay measures might explain pattern differences in the two assays trialled here.
The caspase 3/7 assay can be used to quantify the effect of infection through time. However, as frog toe samples are small, there is limited sample for analysis and standardization. A common method for sample standardization is to determine the total protein concentration of each extract. As the Bradford protein quantification assay consumes sample, it is not an effective method for small extracts. In addition, we found that pigments within the frog skin precluded analysis by nano UV-Vis spectrometry. Small sample sizes also prevented standardization by dry weight. We suggest estimating skin surface area of the toe using photography, as the toe tip samples (these frogs were less than 3 g in body size) were too small to allow assays for both protein concentration and caspase concentration. We found that photographing the toes and using a skin surface area estimate is as effective as traditional protein concentration analyses.
In this study, we used two standard techniques for quantifying cell death and apoptosis, but adapted the methods for amphibians and small tissue samples. For the TUNEL assay, we euthanized the animal to ensure enough tissue was available for the assay, and allowed for the comparison of different skin locations. It is possible to adapt this method to smaller tissue samples, such as a toe webbing biopsy, which would not require euthanasia of the animal. Depending on the size of the animal, biopsies could allow for a time series test similar to our approach for the caspase 3/7 assay.
In future studies, we suggest the use of additional assays for exploring cell death and apoptosis over the course of chytridiomycosis infection because there is still much to learn of the causal mechanisms of chytridiomycosis pathogenesis. These results suggest that apoptosis and epidermal cell death may be important in the pathogenesis of Bd; however, more research is needed in order to determine the influence of apoptosis on disease outcomes, particularly in hosts that do not succumb to infection.
The authors have nothing to disclose.
We thank the following people who assisted with husbandry and data collection: D. Tegtmeier, C. De Jong, J. Hawkes, K. Fossen, S. Percival, M. McWilliams, L. Bertola, M. Stewart, N. Harney, and T. Knavel; and M. Merces for assistance with dissections. We would also like to thank M. McFadden, P. Harlow and Taronga Zoo for raising the L. v. alpina, and G. Marantelli for raising the P. corroboree. We thank F. Pasmans, A. Martel for advice on apoptosis assays, C. Constantine, A. Kladnik and R. Webb for assistance with TUNEL assay, and T. Emeto and W. Weßels for help with protocol and kit for caspase 3/7 assay. This manuscript and protocol is adapted from Brannelly et al 2017 Peer J22.
POLARstar Omega | BMG Labtech | Luminescent plate reader | |
384 well flat clear bottom plate | Corning | 3707 | |
384 well low flange white flat bottom plate | Corning | 3570 | |
Agar Bacteriological (Oxoid) | Fisher | OXLP0011B | |
Formal-Fixx 10% Neutral Buffered Formalin | Fisher | 6764254 | |
Lactose Broth (Oxoid) | Fisher | OXCM0137B | |
Sodium Bicarbonate | Fisher | BP328-500 | |
Tricane-S (MS-222) | Fisher | NC0872873 | |
Tryptone | Fisher | BP1421-500 | |
Bovine Serum Albumin | Invitrogen | 15561020 | |
Sterile rayon swab | Medical Wire & Equipment | MW-113 | |
ApopTag Red In Situ Apoptosis Detection Kit | Merck Millipore | S7165 | |
Coomassie Bradford reagent | Pierce | 23200 | |
Caspase Glo 3/7 | Promega | G8090 | |
HEPES buffer | Sigma Aldrich | H0887-20ML | |
Magnesium chloride | Sigma Aldrich | 1374248-1G | |
Gelatin hydrolysate Enzymatic | Sigma-Aldrich | G0262 | |
PBS (Phosphate Buffered Saline), pH 7.2 (1X) | Thermo/Life | 20-012-043 | |
Prepman | Thermo/Life | 4318930 | |
TaqMan Fast Advanced Master Mix | ThermoFisher | 4444556 | |
Parafilm | Bemis | PM996 | |
Clorox bleach | Clorox | ||
Ethanol, 200 Proof, Molecular Grade | Fisher | BP2818500 | |
ZEISS Axio Scan florescent miscroscope | Carl Zeiss | Florescent microscope | |
3.2mm stainless steel beads | BioSpec | 11079132SS | |
Primer ITSI-3 Chytr (5′-CCTTGATATAATACAGTGTGCCATATGTC-3′) | Taqman | Individual design for primers and probe | |
Primer 5.8S Chytr (5′-TCGGTTCTCTAGGCAACAGTTT-3′) | Taqman | Individual design for primers and probe | |
Minor groove binder probe Chytr MGB2(5′-CGAGTCGAAC-3′) | Taqman | Individual design for primers and probe | |
Rotor-Gene qPCR Instruments | Qiagen | qPCR machine | |
Microcentrifuge tubes 1.5ml | Fisher | 02-681-372 | |
Cell culture petri plates | Nunc | 263991 | |
Mini-beadBeater Zircornia-Silicate Beads, 0.5mm | BioSpec | 11079105Z |