Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Verification of the clinical implementation of the respiratory gated beam delivery technique with synchrotron-based proton irradiation

The clinical advantage for protons over conventional high-energy x-rays stems from their unique depth-dose distribution, which delivers essentially no dose beyond the end of range. In order to achieve it, accurate localization of the tumor volume relative to the proton beam is necessary. For cases where the tumor moves with respiration, the resultant dose distribution is sensitive to such motion. One way to reduce uncertainty caused by respiratory motion is to use gated beam delivery. The main goal of this dissertation is to evaluate the respiratory gating technique in both passive scattering and scanning delivery mode. Our hypothesis for the study was that optimization of the parameters of synchrotron operation and respiratory gating can lead to greater efficiency and accuracy of respiratory gating for all modes of synchrotron-based proton treatment delivery. The hypothesis is tested in two specific aims. The specific aim #1 is to assess the efficiency of respiratory-gated proton beam delivery and optimize the synchrotron operations for the gated proton therapy. A simulation study was performed and introduced an efficient synchrotron operation pattern, called variable Tcyc. In addition, the simulation study estimated the efficiency in the respiratory gated scanning beam delivery mode as well. The specific aim #2 is to assess the accuracy of beam delivery in respiratory-gated proton therapy. The simulation study was extended to the passive scattering mode to estimate the quality of pulsed beam delivery to the residual motion for several synchrotron operation patterns with the gating technique. The results showed that variable Tcyc operation can offer good reproducible beam delivery to the residual motion at a certain phase of the motion. For respiratory gated scanning beam delivery, the impact of motion on the dose distributions by scanned beams was investigated by measurement. The results showed the threshold for motion for a variety of scan patterns and the proper number of paintings for normal and respiratory gated beam deliveries. The results of specific aims 1 and 2 provided supporting data for implementation of the respiratory gating beam delivery technique into both passive and scanning modes and the validation of the hypothesis.