WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young

Developmental and Comparative Immunology 36 (2012) 584–590

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WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young Ashalyn P. Watt ⇑, Julie A. Sharp, Christophe Lefevre, Kevin R. Nicholas Institute for Technology Research and Innovation, Deakin University, Waurn Ponds, Victoria 3217, Australia

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Article history: Received 4 August 2011 Revised 13 September 2011 Accepted 2 October 2011 Available online 15 October 2011 Keywords: WFDC2 Mammary gland Antibacterial Lactation Immune protection

a b s t r a c t WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction WFDC2 is also known as human epididymis 4 (HE4) and is part of a large family of WAP four disulfide core (WFDC) proteins (Bingle et al., 2002). Proteins that belong to this family all contain four-disulfide core (4-DSC) domains that consist of eight cysteine residues in a conserved arrangement (Topcic et al., 2009; Hennighausen and Sippel, 1982; Campbell et al., 1984). Tammar wallaby (Macropus eugenii) WFDC2 has two 4-DSC domains that have previously been annotated domain III on the amino terminal end and domain II at the carboxyl terminal end based on similarities to the annotated whey acidic protein (WAP) domains (Sharp et al., 2007). WFDC2 was first identified in epididymis but was later found to have high expression in the oral cavity (Kirchhoff et al., 1991; Bingle et al., 2002; Idoji et al., 2008) and more recently the WFDC2 gene has been identified in the mammary gland of the tammar wallaby during lactation (Sharp et al., 2007). In the tammar wallaby milk composition changes throughout lactation in order to meet the developmental requirements of the growing young (Trott et al., 2003; Green et al., 1988). The lactation cycle in the tammar wallaby has been divided into four phases (1, 2A, 2B and 3) which have been defined by changes in milk composition and growth and sucking patterns of the young (Tyndale -Biscoe and Janssens, 1988; Nicholas et al., 1997; Green et al., ⇑ Corresponding author. E-mail address: [email protected] (A.P. Watt).

1988). Phase 1 consists of 26 days of pregnancy and parturition, this precedes the start of lactation (Nicholas et al., 1997). Phase 2A consists of the first 100–120 days of lactation when the young is permanently attached to the teat and in the pouch (Lefevre et al., 2007; Trott et al., 2003; Simpson et al., 2000). During this phase the volume of milk produced is low and dilute containing high levels of complex carbohydrates and low levels of protein and fat (Nicholas et al., 1997). In phase 2B the young is no longer permanently attached to the teat but remains in the pouch and sucking is less frequent. This phase usually lasts for the next 100 days (Lefevre et al., 2007; Nicholas et al., 1997; Trott et al., 2003) and the levels of protein and fat in milk remain low and carbohydrate levels remain high. Growth of the young is slow in these two phases but there is considerable development of a competent immune system (Simpson et al., 2000; Tyndale-Biscoe and Janssens, 1988). The onset of phase 3 is correlated with an increase in growth of the mammary gland and larger volumes of milk being produced that corresponds to an increased growth rate of the young (Nicholas et al., 1997). This phase begins at approximately 200 days after parturition and represents the time when the young begins to leave the pouch, and ends approximately at 350 days (Nicholas et al., 1997). In this phase there is a dramatic change in milk composition, including reduced carbohydrate concentration and increased fat and protein concentrations (Trott et al., 2003; Nicholas et al., 1997; Tyndale-Biscoe and Janssens, 1988). The composition of the major milk proteins change progressively throughout the lactation cycle (Nicholas et al., 1997). However,

0145-305X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.10.001

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not all milk proteins have been characterized for expression pattern in the tammar lactation cycle and their function in either the mammary gland or neonate remains unknown. Our previous studies have shown that the WFDC2 gene is expressed in the mammary gland of the tammar wallaby and appeared to be differentially regulated during lactation. The expression profile of the WFDC2 gene in the tammar wallaby mammary gland was shown to have high expression during phase 1 (pregnancy), a lower level of expression during phase 2A (early lactation) and no expression in Phase 2B to 3 (mid to late lactation) (Sharp et al., 2007). However, a more extensive expression profile is needed to show precise changes in WFDC2 gene expression levels during the lactation cycle. In addition, WFDC2 was not investigated in the mammary gland during involution, a time when the mammary gland undergoes significant remodeling to return to a mammary morphology that resembles a mature virgin-like structure (Li et al., 1997). There is limited information on the biological activity of the WFDC proteins, with the exception of SLPI (Zhu et al., 2002; Hiemstra et al., 1996), Elafin (Tomee et al., 1997) and Eppin (Yenugu et al., 2004) which have been shown to have antibacterial activity as well as other functions such as anti-inflammatory, anti-viral and anti-proteinase (Moreau et al., 2008; Williams et al., 2006). Whey acidic protein (WAP) is another WFDC family member that is found in milk in some species including the tammar wallaby (Simpson et al., 2000; Topcic et al., 2009), and recently rat WAP was shown to have antibacterial activity against Staphylococcus aureus but not Escherichia coli, suggesting selectivity of antibacterial activity (Iwamori et al., 2010). There is very limited information about the WFDC2 gene in the tammar wallaby and WFDC2 has not been identified in the milk of other species except for the platypus and echidna (Sharp et al., 2007). The role of WFDC2 is unknown, but it has been suggested that WFDC2 in the human may play a role in innate immune defense in the lung and oral cavity (Bingle et al., 2006). Here, we have presented an extensive expression profile of the WFDC2 gene in the mammary gland throughout the lactation cycle and in involution of the tammar wallaby. We also present functional studies which suggest that WFDC2 has a role in innate immunity by providing antibacterial activity against S. aureus, Salmonella enterica and Pseudomonas aeruginosa and that this activity is found exclusively in domain II of the two 4-DSC domains.

2.2. Animals Tammar wallabies (M. eugenii) were maintained in open enclosures at The University of Melbourne Macropod Research Facility (Wantirna, VIC, Australia), with stock originally from Kangaroo Island, South Australia. Mammary glands were collected from non-pregnant, pregnant (days 9, 14, 15, 17, 20, 22, 23, 24), lactation (days 1, 3, 5, 40, 76, 80, 87, 135, 168, 190, 240, 293) and involution (days 1, 3, 5, 10 and 45) animals. In total mammary tissue was collected from two non-pregnant virgin animals, eight pregnant animals, seven animals at phase 2A of lactation, three animals at phase 2B of lactation, two animals at phase 3 of lactation, and five animals at involution. The mammary glands of tammar wallabies were excised under sterile conditions after animals were euthanized. The day of gestation was scheduled as described previously (Shaw et al., 1996). The day of lactation was determined using head length measurements of the pouch young to estimate their age (Poole et al., 1991). All experiments were approved by the Institutional Animal Care and Ethics Committee. 2.3. Expression of the WFDC2 gene Total RNA was extracted from tammar wallaby mammary gland tissue that had been stored at 80 °C. A total of 26 tissue samples from pregnancy, lactation and involution were used to extract RNA using the TriPure Isolation Reagent from Roche Diagnostics following manufacturer’s protocol. RNA concentrations were quantitated using a Nanodrop1000 spectrophotometer (Thermo scientific) with absorbance at 260 and 280 nm. Reverse transcription of total RNA (1 lg) was carried out using SuperScript III (Invitrogen) following manufacturer’s protocol. The cDNA sample concentrations were quantitated using a Nanodrop1000 spectrophotometer (Thermo scientific) and cDNA samples were stored at 20 °C until needed. Reverse transcriptase polymerase chain-reaction (RT-PCR) was used to determine the expression profile of the WFDC2 gene using gene-specific primers (Table 1) and 0.1 lg cDNA. PCR was performed using GoTaqGreen master mix (Promega). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a house keeping gene to normalize expression of the WFDC2 gene. PCR conditions used were initial denaturation 94 °C for 3 min, followed by 27 cycles for GAPDH and 28 cycles for WFDC2 of 94 °C 30 s, 60 °C 30 s and 72 °C 30 s followed by a final extension of 72 °C for 5 m. The size of the PCR products in Table 1 was confirmed using a 1 kb plus DNA marker (Promega). The levels of gene expression relative to GAPDH were quantitated using ImageJ software (http://rsb.info.nih.gov/ij/).

2. Materials and methods 2.4. WFDC2-FLAG tag gene constructs 2.1. Phylogenetic analysis of WFDC2 and its 4-DSC domains Phylogenetic relationships of WFDC2 proteins between species were performed using either the whole protein sequence or a single 4-DSC domain sequence. Sequences were aligned using the ClustalW program (Thompson et al., 1994) to determine any similarities between the WFDC2 proteins and between the 4-DSC domains of WFDC2 protein. One hundred bootstrap replicates were performed on the aligned protein sequences using seqboot followed by Protdist, a program to measure distances of protein sequences (Felsenstein, 1989). Subsequently the program FITCH was used and CONSENSE for majority-rule consensus tree, (BioManager (ANGIS). Phylograms were viewed using Tree View v1.6. Accession number for sequences were; human Q14508, mouse Q9DAU7, rat Q8CHN3, echidna A7J9L4, platypus A7J9L5, opossum XP_001379667, tammar wallaby A7J9L6, Cow Q3T0Z0, Pig Q8M169, Chimpanzee XP_514678, Rhesus monkey XP_001107499, Dog Q28894, Rabbit Q28631, Beetle Xp_969824.

Primers were designed to amplify the full tammar wallaby WFDC2 cDNA. The reverse primer was designed at the stop codon and included a sequence for FLAG-Tag which was added for subsequent purification of the protein with a FLAG affinity purification column. The cDNA from day 23 pregnant was used for PCR with WFDC2 cloning primers (Table 2). The PCR conditions comprised an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C

Table 1 WFDC2 expression profile primers. Primer

Sequence

GAPDH F GAPDH R tWFDC2 F tWFDC2 R

50 -gac tca tga cta cag tcc atg cca t-30 50 -gga cat gta gac cat gag gtc cac-30 50 -cca tcg gtc aga acc aca c-30 50 -gca aat ccc cag cag aga-30

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1.5 min, 60 °C 1.5 min and 72 °C 1.5 min followed by a final extension of 72 °C for 5 min. The amplified DNA was run on a 1% agarose gel using electrophoresis and DNA bands were visualized using SYBR-Safe staining under UV light, excised and purified using QIAquick Gel Extraction Kit (Promega) following the manufacturers protocol then ligated into pTarget expression vector (Promega) using T4 DNA ligase (Promega) following manufacturer’s instructions. Construction of individual WFDC2 4-DSC domain constructs used the protocol by (Topcic et al., 2009) with the following modifications. The WFDC2 full length cDNA in pTarget vector was used as the DNA template to produce the cDNA for single 4-DSC domains (DII and DIII) together with the leader, FLAG-tag and stop codon. The PCR reaction comprised a final concentration of 1  phusion GC buffer, 200 lM dNTPs each, 0.5 lM forward and reverse primers (Table 2) 10 ng template plasmid DNA, 3% DMSO and 0.02 U/ll Phusion DNA polymerase (Finnzymes Phusion High-fidelity DNA Polymerase Kit). The PCR conditions used comprised an initial denaturation 94 °C for 2 min, followed by 30 cycles of 94 °C 30 s, (65 °C for WFDC2 domain II and 58 °C for WFDC2 domain III) 30 s and 72 °C 6 min followed by a final extension of 72 °C for 5 min. Resultant DNA bands were excised and purified as mentioned previously. Purified plasmid with insert was phosphorylated before ligation by incubating plasmid (1 lg), 1  T4 polynucleotide kinase reaction buffer and nuclease free water in a total volume of 41 ll and incubation at 70 °C for 10 min. Subsequently 1 ll T4 polynucleotide kinase (New England Biolabs) and 5 ll 10 mM ATP was added and the reaction mix incubated at 37 °C for 30 min. The kinase was inactivated by heating to 65 °C for 20 min and the plasmid purified using QIAquick PCR Purification Kit (Promega) following the manufacturers protocol. The plasmids were subsequently self-ligated using T4-DNA ligase (Promega) following manufacturer’s instructions; clones were selected and verified by sequencing (Applied Genetic Diagnostics Division, Departments of Pathology, The University of Melbourne). 2.5. Transfection of HEK-293T cells for generation of conditioned media HEK293T cells (human embryonic kidney cell line) were maintained in DMEM high glucose media with 10% FBS and 1% L-glutamine and incubated at 37 °C (5% CO2). Transfections were carried out using Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. The HEK-293T cells (106 cells/35 mm well) were seeded and incubated overnight in 2% FBS/Opti-MEM and the following day media removed and 2 ml serum free Opti-MEM was added. Cells were transfected with the constructs WFDC2 full length gene cloned into pTarget, WFDC2 domain II cloned into pTarget, WFDC2 domain III cloned into pTarget, pTarget without insert (empty vector) and GFP (Green fluorescent protein) as a fluorescent marker for transfection efficiency. Cells transfected with pGFP were photographed at 24 h after transfection to confirm successful transfection. Conditioned media was collected 48 h after

Table 2 Primers for generation of WFDC2 constructs. Primer

Sequence

WFDC2-Fwd WFDC2-Rev with flag tag WFDC2-DII Fwd WFDC2-DII Rev WFDC2-DIII Fwd WFDC2-DIII Rev

50 -gag cgc cca atg gcc agc-30 50 -tca ctt gtc atc gtc atc ctt gta atc atg gtg agg tgt gag gca g-30 50 -aac gag aag aag ggc aaa tgc-30 50 -gtg gtt ctg acc gat ggc a-30 50 -cac cat gat tac aag gat gac-30 50 -ggg gat tgt gca gac cga-30

transfection and centrifuged at 5000g for 5 min to remove cell debris and conditioned media was stored at 80 °C until needed. 2.6. Protein purification using FLAG affinity chromatography Media containing WFDC2 protein, WFDC2 domain II and WFDC2 domain III was subjected to FLAG affinity purification chromatography (Sigma; following manufacturers conditions), and the concentration of eluted proteins was measured using nanodrop1000 spectrophotometer (Thermo scientific) and examined by SDS–PAGE (15% gels) followed by silver staining (Pierce scientific). The size of the protein bands was determined using See Blue Plus 2 Pre-stained standard (Invitrogen). 2.7. Antibacterial assay Antibacterial assays were carried out using AlamarBlue cell viability reagent (Invitrogen) following manufacturer’s protocol. Overnight cultures of ATCC25923 S. aureus, ATCC27853 P. aeruginosa, ATCC43971 S. enterica and ATCC10100 Enterococcus faecalis were prepared in Iso Sensitest broth (Oxoid) and incubated overnight at 37 °C on a shaker. An aliquot (200 ll) was added to 1.8 ml ISA broth and incubated at 37 °C on a shaker for 3hours when the bacteria reached log phase. Cell number was determined by measuring the absorbance on a plate reader at 600 nm (Bio-Rad, xMark microplate spectrophotometer). Conditioned media (40 ll of WFDC2 intact protein, WFDC2 domain II, WFDC2 domain III and pTarget empty vector control) and 10 ll AlamarBlue (Invitrogen) along with 1  104 cells/ml to a total of 100 ll was used for measuring antibacterial activity of WFDC2 and the individual domains. Plates were incubated at 37 °C on a shaker and fluorescence measured each hour at 544 nm excitation and 590 nm emission. All experiments were conducted in triplicate and performed at least three times. 2.8. Statistical analysis Statistical analyses were carried out using a two-tailed t-test assuming unequal variances between means. Differences between mean values were considered statistically significant at P < 0.05. 3. Results 3.1. Phylogenetic analysis of the WFDC2 protein To determine the evolutionary relationships between WFDC2 orthologues a phylogenetic analysis of WFDC2 proteins from different taxa was performed (Fig. 1A). This analysis showed WFDC2 proteins from marsupial (tammar wallaby and short-tailed opossum) clustered together within the same clade while WFDC2 protein from monotremes (platypus and echidna) formed a sister clade. Classification of the type of 4-DSC domain (DIII or DII) represented in the tammar wallaby WFDC2 protein was analyzed using sequence similarity of Da (amino terminal end) and Db (carboxyl terminal end) (Fig. 1B). This analysis revealed that the first 4-DSC domain (Da) has high similarity to the DIII in the tammar wallaby and the second 4-DSC domain (Db) has high similarity to the DII in the tammar wallaby. 3.2. Expression profile of WFDC2 The expression profile of the WFDC2 gene in the tammar wallaby mammary gland during the lactation cycle and involution showed that there is low WFDC2 gene expression in the non

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-pregnant wallaby mammary gland, but is elevated in pregnancy (phase 1) and in early lactation (phase 2A). Subsequently WFDC2 gene expression was down regulated in mid lactation (phase 2B), which is consistent with findings from Sharp et al. (2007). WFDC2 gene expression in the mammary gland was shown to increase slowly towards the end of phase 3 and then increased markedly during involution (Fig. 2). 3.3. Identification of WFDC2 proteins WFDC2 intact protein, WFDC2 domain II protein and WFDC2 domain III protein that had been eluted from the anti-FLAG column were examined by silver staining of SDS–PAGE gels. The intact protein electrophoresed with an apparent size of 13 kD which was consistent with glycosylated protein (Bingle and Vyakarnam, 2008). The two WFDC2 domain proteins electrophoresed with an apparent size of 6 kD (data not shown). 3.4. Bacteriostatic effects of WFDC2 proteins The WFDC2 protein and domain III and II proteins were analyzed for antibacterial activity against a range of bacterial species that represented gram positive bacteria and gram negative bacteria. S. enterica, S. aureus and Pseudomonas aeruginsoa bacterial strains were chosen as they are pathogenic and can cause infection in either the mammary gland or the gut (Jean et al., 2006; Delgado et al., 2011; Harji et al., 2010). In contrast E. faecalis was chosen as a commensal bacteria in the gut of the tammar wallaby (Yadav et al., 1972). Antibacterial assays were employed using the conditioned media of WFDC2 intact protein, WFDC2 domain II protein and WFDC2 domain III protein. Antibacterial activity of the WFDC2 proteins was compared to the no treatment control using a twotailed t-test where P < 0.05 was considered significant (Table 3). WFDC2 intact protein and WFDC2 domain II protein was found to have statistically significant (Table 3) antibacterial activity against S. enterica (Fig. 3A), P. aeruginosa (Fig. 3B) and S. aureus (Fig. 3C). However, WFDC2 domain III protein showed no significant antibacterial activity against any of these strains of bacteria. In contrast WFDC2 intact protein, WFDC2 domain II protein and WFDC2 domain III protein all showed no statistically significant difference (P > 0.05) against E. faecalis (Fig. 3D). Therefore, antibacterial activity lies within the domain II of WFDC2 and not domain III and WFDC2 has strain specific activity with no apparent broad range antibacterial activity. 4. Discussion Milk has a major role in providing appropriate nutrition for the developing young but is now apparent that milk includes a range of bioactives that also have a role in regulating development of the young and promoting function and protection of the mammary gland (Piper et al., 2007; Baldi et al., 2005). The outcomes of the current study are consistent with WFDC2 having multiple roles in both the young and the mammary gland and the unique reproductive strategy of the marsupial better allows an assignment of function to this protein. The WFDC2 proteins are part of a large family of proteins identified in many species (Bingle and Vyakarnam, 2008) and are comprised of two 4-DSC domains (Sharp et al., 2007; Bingle et al., 2002). The phylogenetic analysis of the domains showed that each species has the same configuration of a domain III at the amino terminal end and a domain II at the carboxyl terminal end and suggests that these proteins may have similar conserved domain functions. The current study showed tammar wallaby WFDC2 protein had antibacterial activity and that this activity was exclusive to domain II at the carboxyl terminal

Fig. 1. Phylogenetic tree showing the evolutionary relatedness between WFDC2 proteins and different taxa. (A) Phylogram showing WFDC2 proteins grouped according to phylogenetic relatedness. Beetle WFDC2 was used as an outgroup to root the tree. (B) Radial tree (beetle outgroup) showing WFDC2 proteins grouped according to relatedness performed using maximum likelihood. Domains II and III are circled and labelled.

end. There have been extensive studies showing antibacterial activities of a number of WFDC family members including, SLPI

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Fig. 2. RT-PCR expression of WFDC2 and GAPDH. (A) RNA was extracted from tamamr wallaby mammary tissue at different stages of pregnancy and lactation (one animal per time point; total of two animals for non-pregnant virgin, eight animals for phase 1, seven animals for phase 2A, three animals for phase 2B, two animals for phase 3 and five animals for involution) and RT-PCR was used to show the expression profile. The WFDC2 expression profile shows that the gene is downregulated in the non-pregnant animal and becomes elevated in pregnancy (phase 1) and early lactation (phase 2A), expression is down regulated in mid-late lactation (phase 2B–3) and becomes elevated in involution. GAPDH was used as a housekeeping gene. (B) The relative expression of WFDC2 against GAPDH was used to normalize the expression of WFDC2 and semiquantitated by reading densitometry values of band intensity, WFDC2 densitometry values were normalized against the GAPDH densitometry values to show relative WFDC2 gene expression.

Table 3 T-test results of WFDC2 protein, WFDC2 domain II and WFDC2 domain III on antibacterial activity endpoint time.

WFDC2 protein WFDC2 DII protein WFDC2 DIII protein

Salmonella enterica

Pseudomonas aeruginosa

Staphylococcus aureus

Enterococcus faecalis

4.82  105

0.0076

6.53  106

0.789

2.19  105

0.0038

4.84  106

0.139

0.71

0.381

0.642

0.143

(secretory leukocyte protease inhibitor), Elafin and Eppin (Bingle and Vyakarnam, 2008; Williams et al., 2006; Rugarli et al., 2002). The domain structure of SLPI is similar to WFDC2 and earlier studies have shown SPLI has antibacterial activity localized to the amino terminal end 4-DSC domain while the carboxyl terminal end 4-DSC domain was found to contain no antibacterial activity (Hiemstra et al., 1996). The timing of elevated expression of the WFDC2 gene during pregnancy, early lactation (phase 2A) and during involution correlates with stages of the lactation cycle that have increased risk of infection (Basden et al., 1997; Daly et al., 2007; Old and Deane, 2000). Mastitis is the most common mammary infection and S. aureus, Streptococcus spp. and E. coli are the most frequently isolated pathogens in bovine and human mastitis (Barkema et al., 2009; Borm et al., 2006; Bradley and Green, 2001), however in the

tammar wallaby mastitis is yet to be observed. The tammar WFDC2 intact protein and WFDC2 domain II protein were shown to have significant antibacterial activity against the pathogenic bacteria S. aureus, S. enterica and P. aeruginosa. However, there was no antibacterial activity against the commensal E. faecalis indicating that WFDC2 targets specific strains of bacteria. Infections from the gram-positive S. aureus are usually caused from the same strain that the animal carries as a commensal and infections can affect the bloodstream, skin, soft tissues and lower respiratory tracts, (Plata et al., 2009; Williams et al., 1959) and antibacterial proteins like WFDC2 in milk may be a factor why mastitis hasn’t been observed in tammar wallabies. The Gram-negative bacteria S. enterica is highly host adapted and is known for its extreme invasiveness and pathogenic nature in humans frequently causing septicemic disorders and is increasingly becoming resistant to antibiotic treatments (Jean et al., 2006). In contrast P. aeruginosa is a Gram-negative opportunistic pathogen associated with a broad spectrum of infections in humans. The majority of infections caused by P. aeruginosa are seen in immune-suppressed individuals and the bacteria has the ability to rapidly develop resistance to multiple classes of antibiotics (Kerr and Snelling, 2009). Although E. faecalis is a Gram-positive bacteria, WFDC2 showed no antibacterial activity against this strain. E. faecalis is regarded as a harmless commensal found in the gastrointestinal tract of the human, tammar wallaby and other animals (Fanaro et al., 2003; Yadav et al., 1972). Studies by Old and Deane (1998) identified 30 strains of bacteria in the pouch of the tammar wallaby, including some potentially pathogenic bacteria such as Staphylococcus epidermidis,

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B

Salmonella enterica 180000 160000 140000 120000 100000 80000 60000 40000 20000 0

WFDC2 pTarget

* *

WFDC2 DIII WFDC2 DII

1

2

3

4

Pseudomonas aeruginosa

Fluorescence (RFU)

Fluorescence (RFU)

A

190000 170000 150000 130000 110000 90000 70000 50000 30000 10000

WFDC2

* *

2

Time (hours)

3

4

5

Time (hours)

D

Staphylococcus aureus

Enterococcus faecalis 25000

205000 185000 165000 145000 125000 105000 85000 65000 45000 25000

WFDC2 pTarget WFDC2 DIII

** 1

2

WFDC2 DII

Fluorescence (RFU)

Fluorescence (RFU)

C

WFDC2 DIII WFDC2 DII

1

5

pTarget

20000

WFDC2

15000

pTarget

10000

WFDC2 DIII

5000

WFDC2 DII

0

3

0

Time (hours)

1

2

3

4

Time (hours)

Fig. 3. Bacteriostatic activity of WFDC2 protein. (A) Bacteriostatic assay using Salmonella enterica; WFDC2 protein, WFDC2 domain II showed significant inhibition of growth of the bacteria and WFDC2 DIII had no significant inhibition of growth when compared the empty vector (pTarget) P < 0.05. (B) Bacteriostatic assay of Staphylococcus aureus; WFDC2 protein, WFDC2 domain II showed significant inhibition of growth of the bacteria and WFDC2 DIII had no significant inhibition of growth when compared the empty vector (pTarget) P < 0.05. (C) Bacteriostatic assay of Pseudomonas aeruginosa; WFDC2 protein, WFDC2 domain II showed significant inhibition of growth of the bacteria and WFDC2 DIII had no significant inhibition of growth when compared the empty vector (pTarget) P < 0.05. (D) Bacteriostatic assay of Enterococcus faecalis; WFDC2 protein, WFDC2 domain II and WFDC2 domain III all had no significant inhibition of growth on the bacteria when compared the empty vector (pTarget) P > 0.05 (⁄statistically significant result P < 0.05). Each assay was performed in triplicate and data is shown as means with standard error bars shown.

P. aeruginosa and Streptococcus spp. Until approximately 90 days of age the pouch young is very immature and does not have a fully functioning immune system (Basden et al., 1997). At this time the pouch young is reliant on immune protection provided by milk to prevent infection and the antibacterial effect of WFDC2 directed to potentially pathogenic bacteria like P. aeruginosa is consistent with this role (Daly et al., 2007; Old and Deane, 2000, 1998). The pouch young subsequently develops immune competency at a time that correlated with the observed down regulation of WFDC2 (phase 2B) as it is no longer required by the young and by this stage the young detaches from the teat and no longer requires antimicrobial protection from the milk. Studies by Yadav et al. (1972) found that microbial flora in the gut of developing young of the marsupials Quokka (Setonix brachyurus) and tammar wallaby was present in the early stages of development. Microbial flora in the gut of the young was observed as early as 50 days of age (phase 2A) in the tammar wallaby and as early as 21 days in the quokka suggesting that the microbes present in the gut of the pouch young are not pathogenic in the tammar wallaby and the Quokka. The predominant microbes isolated from the gut of the quokka and tammar wallaby were E. coli and E. faecalis. Staphylococcus spp., Salmonella spp. and P. aeruginosa were not present in the gut of either the quokka and tammar wallaby. Interestingly, WFDC2 is expressed when microbial flora is present and while WFDC2 is antibacterial against S. aureus, S. enterica and P. aeruginosa it seems that WFDC2 may contribute to the absence of these bacterial strains in the gut of the developing young. WFDC2 had no antibacterial effect on E. faecalis and this bacterial strain is found in the gut of the tammar wallaby. The

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