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provided by Sydney eScholarship Immunogenetics (2017) 69:133–143 DOI 10.1007/s00251-016-0959-1

ORIGINAL ARTICLE

Characterization of the antimicrobial family in the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii)

Elizabeth A. Jones1 & Yuanyuan Cheng1 & Denis O’Meally1,2 & Katherine Belov1

Received: 19 August 2016 /Accepted: 5 November 2016 /Published online: 12 November 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Defensins comprise a family of -rich anti- Keywords Tasmanian devil . Koala . Tammar wallaby . microbial with important roles in innate and adaptive . Evolution immune defense in vertebrates. We characterized alpha and genes in three Australian marsupials: the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos Introduction cinereus), and tammar wallaby (Macropus eugenii) and iden- tified 48, 34, and 39 defensins, respectively. One hundred and Defensins are a family of cysteine-rich polypeptides that play twelve have the classical antimicrobial peptides characteristics critical roles in innate and adaptive immune defense (Yang et al. required for pathogen membrane targeting, including cationic 1999) in vertebrates (Lehrer and Ganz 2002;Ganz2004), in- charge (between 1+ and 15+) and a high proportion of hydro- vertebrates (Hoffmann and Hetru 1992;LehrerandGanz1999; phobic residues (>30%). Phylogenetic analysis shows that de la Vega and Possani 2005), and plants (Broekaert et al. gene duplication has driven unique and species-specific ex- 1995). These include defense against bacterial (Lehrer et al. pansions of devil, koala, and tammar wallaby beta defensins 1989; Schibli et al. 2002), fungal (Edgerton et al. 2000;Feng and devil alpha defensins. Defensin genes are arranged in et al. 2005) and viral pathogens (Daher et al. 1986), immuno- three genomic clusters in marsupials, whereas further duplica- modulatory functions (Bowdish et al. 2006;Grigatetal.2007; tions and translocations have occurred in eutherians resulting Steinstraesser et al. 2011), roles in reproduction and fertility in four and five gene clusters in mice and humans, respective- (Zhou et al. 2004; Patil et al. 2005; Narciandi et al. 2011; ly. Marsupial defensins are generally under purifying selec- Tollner et al. 2011), and natural flora control (Salzman et al. tion, particularly residues essential for defensin structural sta- 2007). Defensin characterization is critical in defining a species’ bility. Certain hydrophobic or positively charged sites, pre- host-defense peptide repertoire and understanding its immune dominantly found in the defensin loop, are positively selected, system. Defensins are also ideal for studying adaptive molecu- which may have functional significance in defensin-target in- lar evolution because of their intrinsic link with rapidly evolv- teraction and membrane insertion. ing pathogens (Hughes 1999;Sempleetal.2003; Tennessen 2005; Cheng et al. 2015). The antimicrobial affinity of defensins makes them promising templates for future classes Electronic supplementary material The online version of this article of novel antibiotics and in vivo gene therapy (Huang et al. (doi:10.1007/s00251-016-0959-1) contains supplementary material, 2002; Thevissen et al. 2007;Eastonetal.2009), which are which is available to authorized users. urgently required to combat multidrug-resistant pathogens. α β * Katherine Belov Three defensin subfamilies, alpha ( ), beta ( ), and theta [email protected] (θ), have been described in mammals, with alpha and beta genes found in all lineages (Ganz 2004). Defensins typically 1 Faculty of Veterinary Science, School of Life and Environmental consist of a conserved signal sequence and a propeptide se- Sciences, University of Sydney, Camperdown, NSW 2006, Australia quence encoded by the first one to two exons, and a mature 2 Centre for Animal Health Innovation, University of the Sunshine peptide domain encoded by the terminal exon (Ouellette and Coast, Sippy Downs, QLD 4556, Australia Selsted 1996;Ghoshetal.2002). They are initially synthesized 134 Immunogenetics (2017) 69:133–143 as precursor molecules which are post translationally processed defensin sequences were used to create the initial HMMs, to form an active mature peptide (Valore and Ganz 1992). whichwerethenupdatedtoincludenovelAustralianmarsupial Intramolecular bridges form between three cysteine defensin sequences with HMMER searches repeated. All pairs in mature peptides, defining the subfamilies: disulfide HMMER searches were performed on a six frame translation bond bridge 1–6, 2–4, and 3–5 in alpha defensins of each marsupial genome. Based on previously defined and cysteines 1–5, 2–4, and 3–6inbetadefensins(Ganzetal. defensin intron lengths (Patil et al. 2005), signal sequences 1985; Selsted et al. 1985). within 15 Kbp upstream of a mature peptide motif were pre- The gray short-tailed opossum (Monodelphis domestica)is dicted to be the matching signal sequence. Defensins were the only marsupial in which defensins have been characterized named sequentially as they were identified using an abbreviated (Belov et al. 2007). Australian marsupials diverged from species name, a subfamily suffix, and a gene number. American marsupials 80 million years ago (MYA) (Meredith et al. 2008). The Tasmanian devil (Sarcophilus harrisii), koala Phylogenetic analysis (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii) last shared a common ancestor 60 MYA,representing Alpha and beta defensin peptide sequences were each aligned three distinct lineages within the Australasian radiation using CLUSTALW. A beta defensin tree was constructed (Phillips et al. 2006;Meredithetal.2008). The Tasmanian using 41 devil, 32 koala, 36 wallaby, 46 opossum, 36 human, devil and koala have both been severely affected by conta- and 46 mouse sequences, and the tree consisted gious diseases in recent years (McCallum et al. 2007; of seven devil, two koala, three wallaby, two opossum, five Rhodes et al. 2011). The devil faces extinction due to devil human, six chimpanzee, 13 mouse, and 11 rat sequences facial tumor disease (Lachish et al. 2007), while the koala (Supplementary Tables 2 & 3). Phylogenetic trees were con- faces an ongoing battle with chlamydiosis and the koala ret- structed using the neighbor-joining method based on p- rovirus (KoRV) (Polkinghorne et al. 2013). Characterization distance of aligned amino acids in MEGA 5 (Tamura et al. of defensins in these species is the first step in defining their 2011). A thousand bootstrap replicates were used to test phy- role in host health and will inform our understanding of the logeny reliability (Felsenstein 1985). evolution of this important peptide family. Here, we decribe alpha and beta defensins in the Tasmanian devil, tammar wal- Synteny analysis laby, and koala and discuss their evolution. Synteny groups were based on genomic organization and phy- logenetic analysis of orthologs between species. The Methods ENSEMBL genome browser (Release 84) (Cunningham et al. 2015) was used to determine the position and orientation Computational search for defensin genes of human and mouse defensin genes according to the latest human (Genbank assembly ID GCA_000001405.20 released Known opossum, platypus, human, and mouse defensin se- 2014) and mouse (GenBank Assembly ID GCA_000 quences were used to perform BLASTN and BLASTP 001635.6 released 2012) genome assemblies. Human and (Altschul et al. 1997) searches against the devil (Murchison mouse defensin cluster flanking genes were used as query et al. 2012), tammar wallaby (Renfree et al. 2011), and koala sequences to BLAST the Tasmanian devil, koala, wallaby, (KGC, unpublished data) genomes. For evidence of transcrip- and opossum genomes to determine their location and orien- tion and to refine genome annotations, BLASTP and tation (Supplementary Table 2). Wallaby was excluded from TBLASTN searches were performed on published tran- synteny analysis due to the fragmented nature of the genome scriptome data from devil (Murchison et al. 2012) assembly. (Hewavisenti et al. 2016), tammar wallaby (Wong et al. 2011), and koala (Hobbs et al. 2014) (Supplementary Selection tests Table 1). Significant BLAST hits (e-value <10−5; identity over 60%) were retrieved and aligned with CLUSTALW The data monkey web server (Pond and Frost 2005a; Delport (Thompson et al. 1994) and examined for the defensin cysteine et al. 2010) was used to assess individual residues under pos- motif or the conserved signal sequence in Bioedit 7.0.0 (Hall itive and negative selection. Fixed Effect Likelihood (FEL) 1999). Novel defensin sequences were then used as the query (Pond and Frost 2005b) and Fast Unconstrained Bayesian sequences for additional BLASTN and BLASTP searches. Approximation for Inferring Selection (FUBAR) (Murrell Seven hidden Markov models (HMMs) were generated using et al. 2013) were used to detect both negatively and positively HMMER 3.0 (Finn et al. 2011): four targeted the six cysteine selected sites. Mixed Effects Models of Evolution (MEME) mature peptide motif and three targeted the first exon signal (Murrell et al. 2012) was also used to test positively selected sequence. Representative opossum, human, mouse, and dog sites. Significance value for FEL and MEME were set at Immunogenetics (2017) 69:133–143 135 p < 0.05, and posterior probability for FUBAR was >0.9. defensins may also have prosegment pieces encoded by third Overall nucleotide substitution rates in the first and second exons, which will require transcript data to identify. The ma- exon were calculated using the Kumar method (Nei and ture peptide length varied between 40 and 140 amino acids in Kumar 2000) in MEGA 5. Due to defensin cleavage site var- beta defensins and between 30 and 35 amino acids in alpha iability, N-terminus of the putative mature beta defensin was defensins. Mature defensins are usually shorter than 50 amino trimmed at four amino acids prior to the first cysteine of the 6- acids (Ganz 2003). Large beta defensins with elongated C- cysteine motif; alpha defensin mature peptide start site was terminal tails, like the ones we found in marsupials, have also predicted as the first trypsin cleavage site (arginine: R or ly- been described in mice, yet the biological function or potential sine: K) prior to the first cysteine. cleavage of these tails is unknown (Schutte et al. 2002). Classically, mammalian defensins have a conserved 6- Defensin sequence characterization cysteine mature peptide motif. The majority of marsupial defensins identified exhibit this motif, though we also observed The overall hydrophobicity percentage of each defensin was a few defensin-like variants, including three 8-cysteine alpha calculated using an antimicrobial peptide database (Wang and variants (SahaDefA7, MaeuDefA3, PhciDefA2), three 5- Wang 2004). Hydrophobic residues are defined according to cysteine beta variants (SahaDefB41, PhciDefB23, the Kyte-Doolittle scale (Kyte and Doolittle 1982). Peptide MaeuDefB29), and three 7-cysteine variants (SahaDefA4, sequence logos were created for each defensin subfamily SahaDefB31, PhciDefA1). The 8-cysteine variant in the devil using WebLogo (Crooks et al. 2004) and were based on align- (SahaDefA7) was transcribed in the devil facial tumor and the ments of all Tasmanian devil, koala, and tammar wallaby one in koala (PhciDefA2) was transcribed in multiple tissues defensin sequences identified. The absolute site variability (Supplementary Table 2). Disulfide bridges in defensins assist using the Wu-Kabat variability coefficient (Kabat 1969)was in peptide stabilization; facilitate target membrane interaction, used to generate a variability plot (Garcia-Boronat et al. 2008) binding, and pore formation; and are associated with chemotaxis using the same sequence alignments used for the selection properties (White et al. 1995;Wuetal.2003; Klüver et al. 2006). tests and web logo generation. Peptide charge was calculated Changes in disulfide bonding numbers and topology can influ- using pKa values of negatively and positively charged resi- ence the three dimensional structure of defensins, affecting their dues taking into account disulfide bonds, as previously de- antimicrobial potency, chemotaxis, and the efficiency of CCR6 scribed (Cheng et al. 2015). and monocyte binding (Mangoni et al. 1996;Wuetal.2003; Klüver et al. 2005; Varkey and Nagaraj 2005). However, alter- ations to the 6-cysteine motif can also be beneficial and increase Results and discussion the antimicrobial repertoire, as observed in rat and mice with defensin variants containing 5, 9, or 11 cysteines (Campopiano Defensin gene annotation et al. 2004;Anderssonetal.2012; Patil et al. 2013). Hydrophobicity is an important factor governing antimi- Forty-one beta defensins and seven alpha defensins were iden- crobial potency of peptides (Klüver et al. 2005;Klüveretal. tified in the genome of the Tasmanian devil; 33 beta defensins 2006). The alpha and beta defensins we identified have a high and two alpha defensins were identified in the koala genome; proportion of hydrophobic residues >30% (Figs. 1 and 2; and 36 beta defensins and three alpha defensins were identi- Supplementary Table 4). Hydrophobic residues cluster in the fied in the tammar wallaby genome. Due to the fragmented of both subfamilies, which facilitates defensin nature of these genome assemblies, 70 of the 121 putative transmembrane transport. Hydrophobic residues are common- defensins identified were not assigned a first exon signal se- ly located in close proximity to positively charged amino quence and therefore may include some pseudogenes acids, which is a noted structural feature of cytolytic peptides (Supplementary Table 2). (Kini and Evans 1989) including previously characterized defensins (Yang et al. 2000). Characteristics of marsupial defensins Due to proportionally high arginine and lysine content, 112 of the defensins identified were positively charged Intron length varied considerably in the annotated defensin (Supplementary Table 4). Beta defensins had a charge ranging genes, with the shortest spanning 99 bp (SahaDefB26) and from −5.4 to 15.4 (average 4.6), while alpha defensins were all the longest 14,844 bp (PhciDefB29); the mean intron length cationic with charge ranging from 2.7 to 10.7. Peptide charge was 2 Kbp. Based on transcriptome data available, 10 devil plays a crucial role in antimicrobial activities and immunomod- defensins and nine koala defensins were transcribed ulatory functions of defensins (Klüver et al. 2005). We identi- (Supplementary Table 2). One defensin had a third exon fied eight beta defensin mature peptides with negative or neutral encoding a short prosegment piece. As our HMMs targeted charges (Supplementary Table 4). While this is not widely re- signal sequences and mature peptides only, some other ported for defensins (Yenugu et al. 2004; Wei et al. 2015), 136 Immunogenetics (2017) 69:133–143

Signal pepde Prosegment Mature defensin

- + - + + + + - - + - - - + - - + ++ - - - - - + - - - - - + + - - - + + +

Fig. 1 A peptide sequence logo for marsupial alpha defensins showing positions of the β-strands within the mature peptide are shown beneath full-length gene variability and amino acid sites under selection (p <0.05) the logo. Basic residues are blue, acidic residues are red, hydrophobic according to FEL, MEME, and FUBAR tests. Negatively selected sites residues are black, polar residues are green, and neutral residues are shown by a dash (−) (FEL green, FUBAR black) and positively selected purple site shown by a plus (+) (FEL blue,FUBARyellow,MEMEred). The anionic antimicrobial peptides such as dermcidin from the hu- between 9.7 and 10.7 (mean 10.1) and an anionic signal/ man (Steffen et al. 2006) and maximin H5 from the toad prosegment peptide between −8.1 and −13.8 (mean −11.0), Bombina maxima (Lai et al. 2002) possess innate immune func- which conform to the charge balance theory. However, this tions and antimicrobial properties, indicating charge alone does charge balance was not observed for the 8-cysteine alpha vari- not fully account for antimicrobial potential. Alpha defensins ants, which had prosegments with a slightly negative charge characteristically have a long anionic propiece, which acts to ranging from −0.1 to −0.9. balance the cationic charge in the mature peptide domain to One hundred and twelve defensins identified in this study reduce autocytotoxicity prior to secretion (Michaelson et al. have the characteristic hallmarks of cationic antimicrobial 1992; Hughes and Yeager 1997). The classical 6-cysteine alpha defensins. Cationic antimicrobial peptides are being actively defensins that we annotated had a mature peptide charged investigated as source for novel antibiotics (Aoki and Ueda

Prosegment Mature defensin

------+ -- - + - - + - + - + + + - + --++ + -- + + + + - + - + - + + + - + --+ -- + + + +

Fig. 2 A peptide sequence logo for marsupial beta defensins showing site shown by a plus (+) (FEL blue,FUBARyellow,MEMEred). The full-length gene variability and amino acid sites under selection (p <0.05) positions of the α-helix, β-strands, signal/prosegment and mature peptide according to FEL, MEME, and FUBAR tests. Negatively selected sites are shown beneath the logo. Basic residues are blue, acidic residues are shown by a dash (−)(FELgreen,FUBARblack) and positively selected red, hydrophobic residues are black and neutral residues are purple Immunogenetics (2017) 69:133–143 137

2013). Mimetics are currently in clinical trial stages and show Human, mouse, and opossum have 39, 52, and 37 beta defensin promising results against skin infections and Staphylococcus genes in three, four, and five clusters, respectively (Patil et al. aureus, as well as broad spectrum in vitro activity against 2004; Patil et al. 2005)(Fig.3). The Tasmanian devil, koala, multidrug-resistant gram positive and negative (Tew and wallaby have a similar number of beta defensins (48, 34, et al. 2009; Mensa et al. 2014). The unique marsupial cysteine and 39). The Tasmanian devil defensins are located on 13 variants identified, and those defensins with high charge and supercontigs that map to chromosome 1 and chromosome 4. hydrophobic properties, may be good targets for novel thera- Koala genes are yet to be mapped to chromosmes, but due to peutic development. the high quality of the genome assembly, all defensins except DefB25 can be located on three scaffolds. Synteny analysis and phylogenetics Tasmanian devil and koala defensins belong to three syntenic clusters (cluster A, C, D) as in the opossum Vertebrate defensins are arranged in chromosomal clusters that (Fig. 3). These clusters share synteny with eutherian gene each span less than 1.2 Mb (Patil et al. 2004; Patil et al. 2005). clusters A, C, and D (Fig. 3). Beta defensin expansions have

A B

1 6 4 XKR5 Mouse DEFA23 46 ANGPTD2 50 DEFA24 DEFA17 13 AGPAT5 3 35 15 9 10 2 DEFA20 DEFA21 11 7 14 DEFA26 DEFA2 33 5 40 54 11a 8 DEFA5 52 39 38 37 42 DEFA26 DEFA23 DEFA25 11b NEIL2 FDFT1 CTSB 30 43 47 48 51 34 12 DEFA4 Mouse 8qA1, 3.A2 Chr.14C3, Cen Tel (1235kb) (71kb)

Human DEFA1/2 137 CSMD1 ANGPTD2 AGPAT5 1 DEFA6 DEFA3 DEFA8 DEFA9 DEF10 DEFA3 DEFA11 DEFA7 DEFA5 109P1 103 4 108 11e 11c 104 106 105 107 XKR5 DEFA1/2 DEFT1 DEFT1

NEIL2 FDFT1 CTSB 136 135 134 131 130 108P3 109P1 Human 8p23.1 Tel 8p23.1 (639kb) Tel (184kb)

NEIL2 Opossum CSMD1 ANGPTD2 AGPAT5 XKR5 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 DEFA1 DEFA2 20 21 22 23 24 25 26 27 28 CTSB FDFT1 9 Chr.1 (1750kb)

Koala 27 9 ANGPTD2 AGPAT5 XKR5 15 2 3 1 10 8 20P 18 13 14 5 A1 A2 17 6 16 19 22 28 11 26 Chr.Un Scaffold 337 (825kb) DEFA7 34 35 CTSB FDFT1 26 31 22 27 NEIL2 DEFA4 25 28 32 16 30 29 5 15 DEFA2 DEFA5 DEFA1 DEFA6 41 13 40 6 4 36 9 11 3 DEFA3 1 33 XKR5 10 7 2 14 8 12

Devil Devil Devil Devil Devil Devil Devil Devil Devil Chr. 1 Chr. 1 Chr 1 Chr.1 Chr. 1 Chr. 1 Chr. 1 Chr. 1 Chr. 1 SC 836 SC 1042 SC 854 SC 771 SC 845 SC 924 SC 794 SC 571 SC 289 (200kb) (21kb) (12kb) (84kb) (100kb) (46kb) (12kb) (586kb)

C D

Mouse TFAP2D 18 PKHD1 TFAP2B 17 41 49 CRISP4 Mouse C20ORF96 19 45 Rem1 Id1 28 23 SOX12 ZCCHC3 26 29 21 36 25 H13 Tel Chr.1qA3, 20 22 (56kb) Chr.2qH1 Cen (164kb)

PKHD1 PKHD1 TFAP2B TFAP2D 112 110 113 114 133 CRISP1 Human 6p12.3 Human SOX12 ZCCHC3 C20ORF96 132 129 128 127 126 125 Cen 115 116 117P 118 119 121 122P 123 124 REM1 HM13 ID1 (103kb) Cen Cen 20q11.21 (214kb)

Opossum

PKHD1 PKHD1 TFAP2B TFAP2D 33 32 31 30 CRISP1 Chr. 2

SOX12 ZCCHC3 C20ORF96 34 35 36 37 REM1 HM13 ID1 Opossum (266kb) Chr1, (310kb)

Koala Chr. Un. 4 24 7 30 21P Koala Chr. Un. SOX12 ZCCHC3 C20ORF96 33 32 23 12 29 Scaffold 818 REM1 HM13 ID1 Scaffold 328 (149kb) (114kb) TFAPD2 TFAP2B 23 18 17 CRISP1 SOX12 ZCCHC3 C20ORF96 Devil Chr 1 20 21 37 24 REM1 HM13 ID1 Devil Chr 1 SC 218 SC 217 Devil Chr4 , Devil Chr4 , (69 kb) Scaffold 175 Scaffold (41kb) 174 Fig. 3 Genomic organization of alpha and beta defensins in eutherians between >60 and 79% and blue lines indicate bootstrap value >80%. and marsupials. The position and orientation of genes are represented by Size indicated under chromosome number is the defensin cluster size triangles. Genes with labels containing solely numbers are beta-defensins. excluding flanking genes. White filled arrows indicate pseudogenes; Solid lines link orthologous genes across species and dotted lines colored boxes around genes indicate species-specific duplications which represent paralogs. Orthologs and paralogs were determined based on are also highlighted in the phylogenetic trees the phylogenetic analysis where red lines indicate bootstrap support 138 Immunogenetics (2017) 69:133–143 occurred in the Tasmanian devil, koala, tammar wallaby opos- peptide gene expansions in marsupials are associated with sum, and mouse lineages (Fig. 4). Genes within these expan- their reproductive physiology (Wang et al. 2011;Peeletal. sions have no clear orthologs, and in the koala, Tasmanian 2016). Marsupials give birth to immunologically naïve young devil, and mouse, they tend to cluster together in the genome. that develop in a non-sterile pouch. It is possible that defensin This suggests that the expansions are the result of recent gene duplications have evolved in marsupials to protect the immu- duplications and, as has been proposed in mice, are likely an nologically naïve young as broad-spectrum natural antibiotics adaptive response to species-specific pathogenic challenges (Belov et al. 2007). As mice also have short gestation periods (Patil et al. 2004). It has been proposed that antimicrobial and are born with less mature immune systems compared to

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Fig. 4 Phylogenetic tree showing relationship between human, mouse, respectively. Species-specific expansions are highlighted: mouse opossum, Tasmanian devil, koala, and tammar wallaby beta defensins. (maroon), koala (blue), devil (pink), tammar wallaby (green), opossum Bootstrap values are shown next to the nodes; nodes with bootstrap (purple) support of 60–79 and >80% are highlighted in red and blue, Immunogenetics (2017) 69:133–143 139 humans (Chappuis 1998), mouse beta defensin duplications share a common ancestral beta defensin (Liu et al. 1997; Patil may also be associated with reproductive traits. et al. 2004). In contrast to beta defensins, there are no clear Beta defensin gene expansions in the Tasmanian devil, ko- orthologs between eutherian and marsupial alpha defensins ala, opossum, and mouse are all located within the largest (Fig. 5). Alpha defensins in mice and rats have evolved in a syntenic cluster A (Fig. 3), which also contains the alpha lineage-specific manner, whereas orthologs are shared between defensin family in all species. Alpha defensins are thought to humans and primates, and within the marsupials (Fig. 5).

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3 0.93 ModoDefA2 0.97

0.7 PatrDefA 2 0.9 aeuDefA1 1 M 4

1 5 S ahaDefA HosaDefA5 3 1 0.81 SahaDefA 0.43 PatrDefA6 1 1 1 0.54 0.71 SahaDefA5 0.81 HosaDefA6 1 0.87 SahaDefA6 0.81

RanoNP3b MumuDefA3 0.55 0.99 0 MumuDefA1 0.74 0.59 RanoDefA1 1 7 1 0.73 MumuDefA23 0.99 RanoNP4 1 1 1 0.82 MumuDefArs7 0.28

RanoDefA11 1 1 0.75 MumuDefA1

0.85 0.37 RanoNP MumuDefA2

1 0.79 MumuDefA2

0.6

RanoDefA5 1 MumuDefA26 4 0.67 7

RanoNP2 0. MumuDefA5

1

1 MumuDefA4 5

MumuDefA2

RanoDefA7 MumuDefA20 MumuDefA2 1

2

RanoDefA9

RanoDefA8

RanoDefA6

MumuDefars 1

MumuDefA2

1.0 Fig. 5 Phylogenetic tree showing relationship between human, mouse, support of 60–79 and >80% are highlighted in red and blue, opossum, Tasmanian devil, koala, and tammar wallaby alpha defensins. respectively. The Tasmanian devil-specific expansions are highlighted Bootstrap values are shown next to the nodes; nodes with bootstrap in pink 140 Immunogenetics (2017) 69:133–143

Tasmanian devils are the only marsupial to have an expansion and hydrophobic sites is likely due to the essential func- in the alpha defensin family, possessing seven alpha defensins tion of these residues, which are required to maintain compared to other marsupials that have two. Three devil alpha defensin structural integrity, transport, and the biological defensins were transcribed in the facial tumor and one was functions discussed earlier. transcribed in the testis. As alpha defensin expression is gener- Overall, there were nine sites under positive selection in ally limited to myeloid cells, intestinal paneth cells, and repro- marsupial alpha defensins and 16 sites in beta defensins. ductive epithelial cells (Selsted and Ouellette 2005), further Like the distribution of negatively selected sites, positive- characterization of the expression and function of these genes ly selected sites were also concentrated in the mature pep- will help determine the significance of this expansion in the tide in both subfamilies, with five sites under positive se- devil. Considering the carrion-scavenging diet of devils lectioninthealphamaturepeptideand11inthebetama- (Pemberton et al. 2008), a heightened defense against food- ture peptide. These sites mostly contain hydrophobic or borne pathogens may be a possible explanation to this devil- charged residues and are located mainly within the loop specific alpha defensin expansion. regions between the antiparallel beta sheets, or within N- The conserved synteny of defensin gene regions across terminal or C-terminal positively charged clusters. The species is supported by the order of genes that flank the defensin loops and terminal charge clusters have previous- clusters. Interestingly, the flanking genes CTSB, FDFT1, ly been shown to protrude from the defensin core and have and NEIL2 of Tasmanian devil and opossum cluster A are functions associated with membrane docking and micro- found downstream of mouse and human cluster B bial membrane integration (Romestand et al. 2003). The defensins (Fig. 3). This suggests that the mouse and hu- close proximity of sites under negative and positive selec- man cluster B defensins were likely translocated from tion in the mature peptide suggests coordinated selection cluster A to a different chromosomal region during euthe- acts to maintain this variable loop region. Positive selec- rian evolution. No clear orthologous relationship between tion favoring charge alterations has been previously shown eutherians and marsupials was inferred in cluster B and C to be a driver of mammalian defensin gene divergence (Fig. 3), indicating that cluster B and C defensins may (Hughes 1999;Sempleetal.2003). This charge-favoring have evolved independently in each lineage after the mar- selection is a hallmark of the evolutionary arm race be- supial eutherian divergence. tween cationic defensins and their anionic microbial tar- gets (Semple et al. 2003). Natural selection on marsupial defensin genes

Acomparisonofsynonymous(dS) and nonsynonymous (dN) substitution rates revealed that an overall purifying selection Conclusion

(dN < dS significance 0.05) was acting across both exons of marsupial beta defensins and across the second exon of alpha In this study, we characterized defensin genes of the defensins (Table 1). Tasmanian devil, koala, and tammar wallaby and performed Thirteen codon sites in alpha defensins (Fig. 1) and 18 the first evolutionary comparison of defensins across multiple sites in beta defensins (Fig. 2) were under purifying se- marsupial species. This work provides an important first step lection. The majority of negatively selected sites in both in characterizing the function of these peptides in marsupial subfamilies was concentrated in the mature peptide do- immunity. main and involved mainly polar residues, including the AMP, antimicrobial peptide; DefA, alpha defensin; DefB, six conserved cysteines. In the alpha defensin signal pep- beta defensin; HMM, hidden Markov model; Hosa, human; tide, five sites were under purifying selection, most of Maeu, tammar wallaby; Modo, opossum; Mumu, mouse; which are hydrophobic. Negative selection of these polar Phci, koala; Saha, Tasmanian devil.

Table 1 Mean rates of synonymous (dS +/− S.E) and nonsynonymous (dN +/− S.E) nucleotide substitutions and test for overall selection in alpha and beta defensins

Defensin subfamily Signal peptide and propiece Mature peptide

a a a a dS dN Stat P dS dN Stat P

Beta defensins 1.005 ± 0.074 0.585 ± 0.088 3.934 <0.001 1.335 ± 0.058 0.966 ± 0.136 2.096 0.019 Alpha defensins 0.643 ± 0.085 0.508 ± 0.062 1.533 0.064 0.800 ± 0.185 0.395 ± 0.180 2.066 0.020 a Z test statistics for purifying selection (dS > dN) Immunogenetics (2017) 69:133–143 141

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