Infective endocarditis (IE) is a life-threatening infectious disease that is associated with morbidity and mortality, and affects individuals who are pre-disposed to cardiac defects. It is thought that transient bacteria gain access through dental procedures as well as through other modes to the blood stream and reach the heart valves and colonize, resulting in life threatening IE. Even with aggressive antibiotic treatment 36% of the patients succumb to the disease, resulting in death. In IE, the camouflage provided by the thrombus formation provides a safe haven from the host immune system for the bacteria to grow within the accumulated platelets. In addition, the poor penetration of antibiotics compounds the difficulties in treating IE patients. Therefore, to treat IE patients the need exists to develop other methodologies to intervene and impede bacterial attachment to platelets. As the primary adherence of bacteria to platelets begins with microbial surface proteins, they are considered to play a vital role in IE disease progression. It is here that the elucidation of the structures of bacterial surface proteins involved in platelet adhesion, as well as the structure of its co-complex with the human receptor would begin to unravel the mysteries behind this interaction. This characterization could lead to the development of inhibitors that prevent bacterial attachment and subsequent platelet activation, and open an avenue for the development of therapeutics to treat IE patients. In this resubmission application we present progress and propose to study one of the surface proteins, PadA on Streptococcus gordonii that interacts with platelets. PadA plays an important role in adhesion, and its interaction through RGD-like motif with the platelet integrin GpIIbIIIa instigates outside-in signaling, that results i platelet spreading and granule release. Apart from PadA, two other homologous proteins Hsa and GspB (from DL1 and Challis strains) also interact with platelets through the GPIba integrin, and are believed to mediate platelet rolling. Our long range hypothesis is that "Streptococcus gordonii's surface proteins (GspB, HsA and PadA) synergistically interact with platelets in a novel manner that results in both the inside-out and outside-in signaling resulting in activation and spreading of platelets". To begin to address this long range hypothesis, in this proposal we focus on PadA, and set out to structurally characterize the N-terminal platelet adherence region as well as narrow down the region of interaction within the platelet integrin GpIIbIIIa through two specific aims.
Aim 1 : Determine the structure of PadA's N-terminal platelet adherence region;
Aim 2 : Narrow down the region of PadA's adherence on GpIIbIIIa. The structure of PadA and its interaction with platelet integrin GpIIbIIIa, would represent a major step in the direction towards understanding bacterial-platelet interaction, and could serve as a model for other bacterial surface protein interactions with platelets.
In this application, we propose to structurally and functionally characterize on the platelet adherence protein A of Streptococcus gordonii, a pathogen that is commonly observed in infective endocarditis patients. The elucidation of the structure of PadA and determination of the interaction between PadA with the platelet integrin GpIIbIIIa would allow us to define the mechanism of this interaction, and aid in design of therapeutics to impede bacterial adhesion. The structure of PadA and its interaction with platelet integrin GpIIbIIIa, represents a major step in the direction towards understanding bacterial-platelet interaction at atomic resolution, and could serve as a model for other bacterial surface proteins.