Cathelicidin gene characterization

Six cathelicidins were identified in the Tasmanian devil genome. Cathelicidin names were assigned at random and do not imply their position on the chromosome or orthology with other species. All six cathelicidins are located on chromosome two29, however their relative positions on the chromosome are unknown due to the fragmented nature of the genome assembly.

Multiple sequence alignment revealed that Tasmanian devil peptides contained all the characteristic features of cathelicidins, including peptide domains and motifs (Supplementary Figure S1). All six genes contained four exons, similar to other cathelicidins. The prepropeptides ranged from 154 to 172 amino acids in length and contained a signal peptide, conserved cathelin domain and variable antimicrobial domain.

The signal peptide sequence ranged from 21–27 amino acids in length with a high proportion of leucine residues. The cathelin domain ranged from 89 to 95 amino acid residues in length and contained a number of conserved residues. The characteristic four cysteine motif, which is an identifying feature of cathelicidins, was present and is shown in Supplementary Figure S1. The antimicrobial domain was highly variable in length and composition, ranging from 20 to 37 amino acids with a high proportion of charged residues. This imparts a cationic charge on the mature peptide, ranging from 3.9 to 8.1 at pH7 (Table 1). Few conserved residues were evident, with only 17 to 47% similarity amongst Tasmanian devil mature peptide sequences. Significant sequence heterogeneity was also observed within marsupial mature peptides, as Tasmanian devil sequences were only 15–40% similar to tammar wallaby and 3–30% to opossum.

Table 1 The amino acid sequence of six Tasmanian devil cathelicidins, their molecular weight, charge at pH7 and hydrophobic percentage. Full size table

As expected, Tasmanian devil cathelicidins cluster with those of other marsupials in the phylogenetic tree (Fig. 1), indicating that they are more similar to marsupial cathelicidins than to those of monotremes or eutherians. Saha-CATH1 and 2 cluster with opossum and tammar wallaby cathelicidins, suggesting that these genes arose prior to the divergence of these species around 70 million years ago30. In comparison, Saha-CATH3, 5 and 6 form a species-specific clade, which suggests that they have arisen through more recent gene duplications.

Figure 1 Neighbour joining phylogenetic tree showing the evolutionary relationships among Tasmanian devil Saha-CATH1 to 6, tammar wallaby MaeuCath 1 to 8, opossum Modo-CATH1 to 7 and 9 to 12, platypus Oran-CATH1 to 7, human CAMP, mouse CAMP, pig Protegrin-1 and PMAP-37, cow Bac5, BMAP-27 and BMAP-28, sheep SMAP-29 and chicken fowlicidin-1. Full size image

Expression profile

Expression level of Tasmanian devil cathelicidins were assessed in a wide range of immune, reproductive, secretory, respiratory and gastrointestinal tissues using relative qPCR (Fig. 2). Saha-CATH1, 2, 4, 5 and 6 were expressed in all tissues tested, whilst Saha-CATH3 was not expressed in the pouch, lung or heart. As expected, all six cathelicidins were present in blood, spleen and lymph node as they are stored within neutrophil granules. Saha-CATH1 and 4 were most highly expressed in the blood, whilst Saha-CATH3, 5 and 6 were most highly expressed in the spleen. Conversely, Saha-CATH2 was present at the lowest levels within the spleen and was most highly expressed in the pouch. Saha-CATH1, 4, 5 and 6 were also expressed in the pouch at lower levels compared to their expression in other tissues. Additionally, transcripts of Saha-CATH1, 2, 5 and 6 were also detected in a milk transcriptome31. Cathelicidin expression within the mouth mucosa, skin, pouch, uterus and milk supports their functional role in protecting naïve young during development.

Figure 2 Relative fold expression of Tasmanian devil cathelicidin genes in different tissues. For each cathelicidin, relative expression was calculated in comparison to the tissue with the lowest expression (i.e. the tissue with fold 1 expression in each graph). Three tissues (pouch, lung, and heart) that showed no detectable Saha-CATH3 expression were not included in the CATH3 graph. Full size image

Change of pouch microbiome during lactation

To test the hypothesis that the microbial community in devil’s pouch undergoes compositional changes in response to lactation, we examined the pouch microbiota of three non-lactating and three lactating devils by sequencing PCR amplicons of the bacterial 16S rRNA gene V3-V4 region (around 465 bp) on the Illumina MiSeq System. A total of 468,268 sequences were obtained after quality filtering, with the smallest number of sequences obtained for one sample being 44,880 and the largest being 116,364. These sequences were grouped into 4,410 operational taxonomic units (OTUs), which were categorised to 26 bacterial phyla, 79 classes, 138 orders, 261 families, and 487 genera. Consistent with previous observation32, the pouch flora of non-lactating devils consisted mainly of bacteria from five phyla: Proteobacteria (35.3%), Firmicutes (28.4%), Fusobacteria (27.5%), Bacteroidetes (5.7%), and Actinobacteria (1.9%) (Fig. 3).

Figure 3 Comparison of pouch microbiome between non-lactating (NL) and lactating (L) Tasmanian devils. (A) Relative abundance of bacterial families. (B) PCoA analysis of unweighted UniFrac distances. (C) Bacterial phylotype richness inferred using Chao1 metric and 40,000 sequences for rarefaction. Full size image

Several differences were detected between the pouch of non-lactating and lactating devils (Fig. 3). Firstly, the unweighted UniFrac distance within the non-lactating group (0.58 ± 0.03) was significantly lower than the distance between lactating and non-lactating samples (0.68 ± 0.07), suggesting a high degree of compositional dissimilarity between the pouch flora of non-lactating and lactating devils. This can be seen in the principal coordinates analysis (PCoA) plot (Fig. 3) where non-lactating samples grouped together but were separate from the lactating samples. Thirty-four OTUs exhibited significantly lower relative abundance in the lactating pouch than in non-lactating samples (Wilcoxon rank sum test p < 0.05), with the most pronounced changes occurring in the level of Leptotrichiaceae (20.9% in non-lactating samples vs. 0.4% in lactating), Porphyromonas (4.5% vs. 0.3%), Pasteurellaceae (1.7% vs. 0.1%), and Parvimonas (1.0% vs. 0.2%). Despite the decrease in these bacteria, the overall level of bacterial diversity in the pouch of lactating and non-lactating devils was similar (Fig. 3), which may be a result of faecal contamination from the pouch young. Indeed, the prevalence of Cetobacterium (1.9% in non-lactating samples vs. 10.9% in lactating) and Clostridium (1.0% vs. 4.3%), two genera that are main components of devil gut microbiota, significantly increased in the pouch that had pouch young present.

Antimicrobial activity

To assess the antimicrobial activity of devil cathelicidins, we tested synthesized mature peptides on 25 bacterial and 6 fungal strains (Table 2). As there were no Tasmanian devil specific isolates available, human and veterinary strains were used. The antimicrobial activity of Tasmanian devil cathelicidin mature peptides Saha-CATH1 to 6 against bacteria and fungi is summarised in Table 2. Saha-CATH1, 2 and 4 did not show antimicrobial activity against the bacteria and fungi tested, with a minimum inhibitory concentration (MIC) of greater than 64 μg/mL. As such, these values are not included in Table 2. Saha-CATH3 had highly specific activity, as it was unable to kill any bacteria, but killed Cryptococcus neoformans. Saha-CATH6 killed a number of Streptococcus species and vancomycin-resistant Enterococcus faecalis (VREF), however was ineffective against other bacteria. On the other hand, Saha-CATH5 had broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria and killed the drug resistant pathogens VREF and methicillin-resistant Staphylococcus aureus (MRSA) (Table 2). The antifungal activity of Saha-CATH5 and 6 was more restricted, but both peptides showed activity against Candida krusei, Cryptococcus neoformans and Cryptococcus gattii. The MIC values for ampicillin, tetracycline and fluconazole were within the acceptable limits for ATCC strains according to clinical and laboratory standards institute (CLSI) guidelines, and are included in Table 2.

Table 2 Antimicrobial activity of Tasmanian devil cathelicidins Saha-CATH3, 5 and 6 against bacteria and fungi, expressed as the minimum inhibitory concentration (MIC). Full size table

Cytotoxicity and haemolysis

Further experiments were carried out to assess cytotoxic and haemolytic potencies of devil cathelicidin mature peptides. Tasmanian devil cathelicidin mature peptides Saha-CATH1, 2, 3 and 4 were not toxic to the human cell line A549 as seen in Fig. 4a. Cell survival of Saha-CATH1 treated cells remained close to the control at all concentrations. Survival of Saha-CATH2 treated cells also remained close to the control over dilutions ranging between 1.9 μg/mL to 250 μg/mL, however survival of cells at 500 μg/mL was significantly higher than the growth control (p < 0.05). The same pattern was observed for Saha-CATH3 at 1.9, 7.9, 15.6 and 250 μg/mL and all dilutions of Saha-CATH4 ranging between 15.6 to 500 μg/mL (p < 0.05). The significant increase in cell survival above the level of the untreated growth control, as seen in Fig. 4a, does not necessarily indicate cathelicidins are inducing proliferation of A549 cells, rather it is more likely a reflection of cell growth throughout the assay. Alamar blue is an indicator of mitochondrial activity, hence cell growth during the assay results in an increase in the reduction of alamar blue and an elevated percentage cell survival relative to the growth control. Cell survival of Saha-CATH5 and 6 treated cells was significantly higher than the growth control over the concentrations of 1.9 to 250 μg/mL, and 3.9 to 250 μg/mL respectively (p < 0.05). As such, Saha-CATH5 and 6 were non toxic up to a concentration of 250 μg/mL. Saha-CATH5 was the most toxic as treatment of cells with 500 μg/mL resulted in 42% cell survival compared to the untreated growth control (p < 0.05). Similarly, treatment with Saha-CATH6 at the same concentration resulted in 59% cell survival (p < 0.05).

Figure 4 Toxicity of Tasmanian devil cathelicidins Saha-CATH1 to 6 to human cells. (a) cytotoxic activity against the human A549 cell line expressed as percent cell survival compared to untreated control cells. (b) human haemolytic activity expressed as percent red blood cell lysis compared to the 1% Triton X-100 positive control. The mean values ± SD (bars) of two independent experiments performed in duplicate are reported. Full size image

The majority of Tasmanian devil cathelicidin mature peptides did not lyse human red blood cells as shown in Fig. 4b. Saha-CATH1, 2, 3 and 4 caused very low levels of haemolysis, below 7% at all concentrations compared with the positive control. Despite this, mean absorbance values for red blood cells treated with Saha-CATH2 at 500 μg/mL, Saha-CATH3 at 250 and 500 μg/mL and Saha-CATH4 from 125 to 500 μg/mL were significantly different from the negative control (p < 0.05). Similarly, mean absorbance values of Saha-CATH5 and 6 from 31.25 to 125 μg/mL were significantly different from the negative control (p < 0.05), however lysis remained below 15% at these concentrations. Saha-CATH6 was moderately haemolytic at 250 and 500 μg/mL, however lysis remained below 26% (p < 0.05). In support of the cytotoxicity data above, Saha-CATH5 was the most toxic as it caused 37% haemolysis at 500 μg/mL (p < 0.05).