The complement system is found in both invertebrates and vertebrates, and functions in both innate and adaptive immune responses. This thesis involves determining the presence of a complement system in non-eutherian mammals using publicly available non-eutherian genomes, followed by analysing the expression and effectiveness of the complement system in a model dasyurid, the red-tailed phascogale (Phascogale calura). Data mining was used to look for complement sequences in non-eutherian mammals, Polymerase Chain Reaction (PCR) to elucidate major complement component cDNA sequences, real-time PCR to determine the expression of those major complement components in developing phascogales, haemolytic assays to detect the presence of a functional complement system, and antimicrobial susceptibility testing to determine the antimicrobial capabilities of phascogale serum. Use of online search tools and algorithms resulted in the successful identification of 27-47 complement and complement-related sequences in gray short-tailed opossum (Monodelphis domestica), tammar wallaby (Macropus eugenii), Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus) and platypus (Ornithorhynchus anatinus) genomes. Key complement proteins, specifically involved in the Classical, Alternative and Lectin Complement pathways, were also compared with homologous human protein sequences, which then allowed for the identification of functionally important motifs and conserved sequences. The identification of complement sequences in non-eutherian genomes possibly indicate a comparable complement system to eutherian mammals, therefore the expression and functionality of the complement system was investigated in a marsupial. Real-time PCR (qPCR) has the ability to quantitate gene expression, indicating the presence of a functional gene product in a tissue. However experimental gene expression needs to be normalised against the expression of two stable reference genes. The stability of reference genes should have unregulated transcription in the various experimental conditions including differences in age, sex and tissues. Hence determining the stability of reference genes in different tissues and ages would be useful for marsupial gene expression studies. The expression of reference genes; Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), Actin-I² (ACTB), 18S rRNA (18S), 28S rRNA (28S) and Ribosomal Protein L13a (RPL13A), were investigated in liver, lung, small intestine and spleen tissues of phascogales, and at least two stable reference genes were determined per tissue. The expression stability differed according to tissue sample and age, suggesting that the analysis of reference genes according to the type of tissue and ages needs to be considered in every gene expression study. Combining the expression in juveniles and adults, GAPDH was most stable in liver and lung tissues, and 18S in small intestine and spleen tissues. The relative expression of complement component 1, subcomponent r (C1r), complement component properdin (CFP), mannan-binding lectin serine peptidase 2 (MASP2) and complement component 3 (C3) were examined in pouch young, juvenile and adult phascogales using qPCR. The complement components were chosen because of their importance in the Classical, Alternative and Lectin Complement pathways. Average complement expression of all four complement components in whole body tissues showed a significant increase in developing pouch young. However complement expression decreased in adults compared to juveniles. All juvenile and adult phascogales were males, and may indicate a link between complement expression and male semelparity. Partial cDNA sequences for C3 and MASP2 were also extracted from phascogale liver tissue using RACE-PCR, and comparisons with respective eutherian homologues revealed the conservation of functionally important sequences and motifs. This is the first time MASP2 has been suggested to be present and expressed in a marsupial, which indicates the presence of a functional Lectin Complement pathway in the phascogale. Complement proteins have been found in mammalian serum. The effectiveness of phascogale complement serum was tested using standard haemolytic assays. The presence of a Classical and Alternative Complement pathway was confirmed, with female phascogale serum being more effective at lysing foreign erythrocytes, reinforcing the possible link between the complement system and semelparity. The results obtained indicate that the function of the Classical and Alternative Complement pathways in phascogales is comparable to other vertebrates. Along with complement proteins, eutherian serum contains Acute Phase Proteins (APPs) capable of fighting bacterial infections. The presence of APPs, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were identified in marsupial genomes, and expression of Hp and CRP were confirmed in phascogale tissues from birth to adulthood using PCR. Commercially available kits were used to determine the levels of Hp and SAA. Results indicate that female phascogales had higher Hp protein levels compared to males, and SAA protein levels in both males and females indicated that all phascogales sampled were clinically healthy at the time serum samples were collected. Both complement proteins and APPs contribute to the antimicrobial properties observed in serum. The introduction of adult male and female phascogale serum to bacteria resulted in the growth inhibition of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus. The effectiveness of antimicrobial properties in serum was dependent on the duration serum was introduced to bacteria at 37°C. Growth inhibition was highest for K. pneumoniae and lowest for P. aeruginosa. All four bacteria exhibited growth inhibition, confirming the antimicrobial function of phascogale serum. The results of this thesis have contributed to marsupial immunological research. Not many studies have focussed on the marsupial complement system; however this thesis has demonstrated the presence, function and effectiveness of the complement system in marsupials, and some initial research on complement in marsupial young. Marsupial young have no adaptive immune system, yet complement components were still expressed in 1-day old phascogales. In addition, the expression and levels of APPs in adult red-tailed phascogale serum have now been reported. This thesis shows that a greater emphasis should be placed on marsupial complement research, and other complement-associated components. Further research will enhance our understanding of the importance the marsupial complement system, and diseases associated with it.
| Date of Award | 2016 |
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| Original language | English |
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