A proteomic study of innate immune protection in the Tammar Wallaby (Macropus eugenii)

This study has taken a proteomics approach to investigating two aspects of innate immune protection in a model marsupial the tammar wallaby, Macropus eugenii. The proteins of neutrophils and their granules have been documented using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). The first step in this project required development o f protocols for the effective isolation of neutrophils and their granules. Fifty three abundant proteins were initially identified from neutrophils and subsequently a range of protocols including stimulation with PMA, Ionomycin and calcium as well as differential centrifugation and cell lysis were used to isolate granule proteins. Five antimicrobial proteins of granule origin were identified along with a number of proteins associated with the process of exocytosis. The identification of these proteins from the neutrophils in the tammar wallaby clearly shows the degree of conservation of such proteins across different mammal species. -- The second portion of this project was aimed at examining the unique nature of the marsupial pouch in protecting the young immunologically incompetent animal. Pouch secretions collected at major stages of the reproductive cycle showed varying levels of antimicrobial activity primarily against Gram negative E. coli but not against the Gram positive S. aureus. Greatest antimicrobial activity was observed in samples collected at oestrus, the anticipated time of birth of the young animal. Subsequent proteomic analysis, using 2DE and LC-MS/MS, led to confident identification of a range of peptides matched to (3-lactoglobulin. As the likely origin of (3-lactoglobulin could be mammary gland or digested products from the gut of the pouch young, samples from these sources were also analyzed. In parallel with this portion of the study the changes in the skin proteome (the secretome) of the pouch were investigated through proteomic analysis of secretions from pouch skin of immature, mature reproductively active and post-reproductive females and form a non-pouch skin site. A limited number of proteins could be reliably identified although clear differences in the patterns of secretion were observed at these different life stages. Of the proteins that could be identified, globins were present at all stages with dermcidin, a known potent antimicrobial identified in an opportunistic sample collected from a common wombat, Vombatus ursinus. Limited though the successful identification of proteins secreted into the pouch has been, this project has clearly shown that (i) secretions from this site are unique (ii) the types of proteins secreted vary dependent on the reproductive maturity of the female and (iii) there is demonstrable antimicrobial activity against Gram negative organisms although the active component could not be specifically identified.