Identification and functional characterization of a novel monotreme-specific antibacterial protein expressed during lactation

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.Swathi Bisana, Satish Kumar, Peggy Rismiller, Stewart C. Nicol, Christophe Lefèvre, Kevin R. Nicholas, Julie A. Shar

Identification of EchAMP protein in echidna milk.

<p>A1, A2 and B1, B2 represent milk samples collected from two lactating echidnas at two different time points during their late-lactation phase. 70 µg protein of each sample was electrophoresed for 3 hours at 100V using a 12% SDS-Polyacrylamide gel. Bands E3 and E4 were excised from the gel, subjected to in-gel trypsin digestion and anlaysed by LTQ Orbitrap Velos. The spectra of peptides from these bands showed a significant match with the predicted EchAMP protein with high confidence levels.</p

Detection of EchAMP protein in conditioned media.

<p>(<b>A</b>) The EchAMP protein present in conditioned media (CM) was detected by silver staining. No corresponding band was seen in vector conditioned or control (Ctrl) HEK293T conditioned media. The EchAMP protein was found to be higher in conditioned media collected 48 hours post transfection as compared to the one collected at 24 hours. (<b>B</b>) Purification of EchAMP protein using Anti-Flag M2 Affinity Gel. EchAMP protein was purified from the conditioned media collected 48 hours post transfection using an Anti- Flag M2 Affinity Gel column. Silver staining of samples from each stage of the purification procedure run on a 15% SDS- polyacrylamide gel show the presence of the purified EchAMP protein in the eluates.</p

<b>Table1.</b> GRAVY of EchAMP protein.

<p>GRAVY (grand average of hydropathicity) is the computed mean of hydrophobicity and hydrophilicity values for individual amino acid residues. A negative GRAVY indicates hydrophilicity while a positive value indicates hydrophobicity. aa: amino acids.</p

Antibacterial assays.

<p>(<b>A</b>) Bacteriostatic activity using <i>E.coli 2348/69</i>: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>B</b>) Bacteriostatic activity using <i>Salmonella enterica 43971</i>: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>C</b>) Bacteriostatic activity using <i>Staphylococcus aureus 29213</i>: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>D</b>) Bacteriostatic activity using <i>Staphylococcus aureus 25923</i> : EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>E</b>) Bacteriostatic activity using <i>Staphylococcus epidermidis</i> : EchAMP showed highly significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>F</b>) Bacteriostatic activity using <i>Pseudomonas aeruginosa 27853</i>: EchAMP showed highly significant inhibition of growth as compared to the empty vector (pcDNA3) <i>P</i><0.05 (<b>G</b>) Bacteriostatic activity using <i>Enterococcus faecalis 10100</i>: EchAMP showed no inhibition of growth as compared to the empty vector and bacitracin <i>P>0.05</i> (* Statistically significant result <i>P</i><0.05). Each assay was performed in triplicate and the experiments were repeated at least thrice. Standard error bars are indicated.</p