Purification and Functional Analysis of BPIFA2

Short PLUNC 2, recently renamed BPIFA2, is predominantly expressed in the serous acinar cells and interlobular ducts of the major salivary glands and secreted abundantly into saliva. The original hypothesis that the structure of BPIFA2 is similar to that of the N-terminal of BPI and LBP led to the suggestion that it would also play a role in the innate immune defence of the oral cavity and upper airway. The function of BPIFA2 has not, however, been fully elucidated and thus the aim of this thesis was to develop a protocol for the purification of BPIFA2 from whole saliva, in its native form, to fully determine if it does have similar functions to BPI and LBP. Based on the current literature, a number of purification methods were assessed including precipitation, column chromatography and electrophoresis. Native polyacrylamide gel electrophoresis and electro-elution gave the highest yields of pure protein, which was then used in a variety of functional assays including binding, growth inhibition, bacterial killing, agglutination and biofilm disruption. A novelty of this study was that a range of bacteria were used including gram-positive and gram-negative bacteria and commensal and non-commensal oral bacteria. In addition, Der p 7, a dust mite allergen also shown to have structural similarities to the N-terminal domain of BPI and LBP, was used to develop an assay to examine the effect of BPIFA2 on the TLR-4 pathway in the presence of LPS. Although the allergen was initially used as a positive control for the assay system we were able to show for the first time that Der p 7 can mimic the action of LBP in the CD14-MD2-TLR4 pathway in response to gram-negative bacterial LPS.
The most significant and novel finding of this thesis was the effect of BPIFA2 on gram-positive bacteria, particularly S. mutans, a known causative agent of dental caries. Reduced bacterial viability, increased agglutination and altered biofilm quality were all observed in the presence of BPIFA2. These results suggest a role for BPIFA2 in innate immunity, not against gram-negative bacteria as originally hypothesised, but against gram-positive bacteria.