Analysis of the Bactericidal Activity of Naive Rabbit Serum against Staphylococcus aureus

Staphylococcus aureus is a commensal bacterium that can also act as an opportunistic pathogen, causing a wide range of diseases in humans and in economically important livestock such as cows and rabbits. S. aureus quickly develops resistance against antibiotics and also evades the immune system of the host. As current treatments are difficult and expensive, new antibiotics, vaccines and the use of elements of the immune system are currently being studied as novel control approaches. A novel bactericidal mechanism able to kill S. aureus present in naive rabbit serum (NRS) was previously discovered in our laboratory. The bactericidal activity of the NRS had been characterized and the importance of the wall teichoic acids (WTA) on the bacteria for susceptibility was identified previously, but the mechanism involved in the bacterial killing remained unknown. In this study the role of the WTA as the bacterial receptor that interacts with the ‘killing factor’ present in the NRS was established. The importance of WTA modifications were also determined in pathogen resistance to the NRS killing mechanism. Use of bacterial cell wall material as an affinity matrix led to the identification of secreted phospholipase A2 (sPLA2) as a potential part of the bactericidal mechanism. The use of a range of inhibitors and specific antibodies confirmed sPLA2 as part of the killing mechanism. Purified sPLA2 from a number of species was tested and cobra sPLA2 (cvPLA2) was able to kill S. aureus in buffer but led to bacterial growth when added to NRS. This suggests that cvPLA2 interacts with rabbit serum components and this results in inactivation of both cvPLA2 and the bactericidal activity of the NRS. Annexin A1, a known regulator of PLA2, was identified through mass spectrometry as a serum protein also bound to the cell wall affinity matrix. This protein enhanced the bactericidal activity of cvPLA2 against S. aureus by direct protein-protein interaction. A model of NRS staphylococcal activity is presented.