Date of Award

2010

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Steven W. Kerrigan

Funder/Sponsor

Health Research Board of Ireland

Keywords

Cardiovascular Infections, Streptococcus Gordonii

Abstract

Numerous studies have implicated bacteria in cardiovascular disease; however the mechanisms involved have yet to be elucidated. Infective Endocarditis (IE) is characterised by the formation of platelet-bacteria thrombi on a heart valve which, if untreated, can lead to valve failure or the formation of infected emboli. The oral bacterium, Streptococcus gordonii, is amongst the most common pathogens isolated from IE patients. S. gordonii is a member of the group of oral bacteria that occur primarily in dental plaque. The ability of S. gordonii to contribute to a growing platelet fibrin thrombus is a critical step in the development of IE, however the molecular mechanisms of rapid platelet recruitment and activation is poorly understood. Here we describe novel interactions between S. gordonii and human blood platelets. Previous studies have demonstrated that platelets roll on immobilised S. gordonii through a unique interaction between S. gordonii surface protein Hsa and platelet GPlba (Takahashi et al., 2004). Following rolling platelets eventually come to a stop and firmly adhere to the S. gordonii. The identity of the protein responsible for supporting firm adhesion is unknown. Here we describe the identification of a novel cell wall protein, platelet adherence protein A (PadA) which contains a domain homologous to the A1 domain of von Willebrand Factor. A PadA deficient mutant (ApadA) induced platelet aggregation to the same extent as the parent S. gordoniistrain DLI. However, the ability of ApadA to adhere to platelets was significantly reduced. Adherence of ApadA to immobilised glycocalicin (purified GPlba) was unaffected as compared to DL1 whereas adhesion to immobilised allbP3 was greatly reduced. Parent strain DL1 adhered to CHO cells stably transfected with allbP3, however ApadA failed to do so. Strain DL1 adhesion to CHO allbP3 cells was inhibited by an RGD peptide and abciximab. These results describe the identification of a novel S. gordonii cell wall protein PadA, which binds to platelet allbP3. lmmunoreceptor tyrosine-based activation motif containing proteins such as FcyRlla, have been shown to play an essential role in transmitting activating signals into platelets following firm adhesion in order to amplify and strengthen the interaction (Boylan et al., 2008). When platelets adhered to S. gordonii strong tyrosine phosphorylation of the ITAM region of FcyRlla, as well as phosphorylation of the downstream effectors, Syk and phospholipase Cy2 was observed. Inhibition of either allbP3 or FcyRlla ablated dense granule release and platelet spreading. These results suggest that when platelets adhere to immobilised S. gordonii it results in the generation of an outside-in signal that induces cytoplasmic-dependent phosphorylation of FcyRlla which leads to platelet spreading and dense granule release. S. gordonii maintains the ability to induce platelet aggregation following the deletion of Hsa and PadA from the bacterial surface, suggesting that other bacterial proteins present are capable of inducing platelet aggregation. Using a proteomic approach we identified several potential candidates of interest. Peptide searches identified streptococcal surface protein AIB (SspAIB) proteins present in the aggregating strain DL1 and missing in the nonaggregating strain Blackburn. Lactococcus lactis does not induce platelet aggregation, therefore acts as a good surrogate host for expression of proteins of interest. L. lactis expressing SspNB induced an allbP3d ependent platelet aggregation similar to that seen with DLI. L. lactis expressing SspNB failed to support platelet adhesion. These results highlight the specific nature of SspNB in inducing platelet aggregation. Another protein identified of particular interest was fructose bisphophate aldolase (FBA), as it has recently been shown to bind lung epithelial cells through flamingo cadherin. We purified recombinant FBA (rFBA) and expressed FBA in L. lactis. L. lactis FBA and rFBA did not induce platelet aggregation or support platelet adhesion, suggesting S. gordonii-induced platelet aggregation is a multifactorial event and FBA alone is not sufficient to induce platelet aggregation. In this thesis we describe several interactions between S. gordonii and platelets which we believe play critical roles in the development of a growing thrombus in IE patients. Unravelling the mechanisms involved in platelet bacterial interactions may aid in the development of novel or improved therapeutics for the treatment of blood borne disease.

Creative Commons License


This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

13 MB

Comments

Submitted the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland