Characterization of Amyloid Fibrils in Seminal Fluid and Their Interaction With Pathogens

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2013.Several proteolytic cleavage products of prostatic acid phosphatase and semenogelin have been identified in seminal fluid that are cationic in nature and have amyloidogenic propensity. These cationic amyloid fibrils, the best described of which is the semen derived enhancer of viral infection (SEVI), have been shown to electrostatically interact with human immunodeficiency virus type 1 (HIV-1) and enhance infection in vitro by allowing virus to associate with the target cells more efficiently. Here, we used biophysical characterization of short self-assembling amyloid fibrils to determine the fibril phenotypes capable of enhancing HIV-1 infection. We also developed new tools to study fibril function. To do this, we screened a targeted library of small molecules in order to identify compounds that efficiently bound to SEVI. Our lead compound bound SEVI and reduced SEVI-mediated enhancement of HIV-1 infection. Additionally, there was enhanced fluorescence from the small molecule when bound to fibrils, suggesting that it may have utility as a novel detection reagent for the identification of SEVI and related fibrils in biological samples. The biological function of semen cationic amyloid fibrils is currently unknown. Since they share characteristics of other antimicrobial peptides, we hypothesized that they may also possess antimicrobial properties. We found that SEVI did not directly inhibit the growth of bacteria, but exerted indirect antimicrobial effects, consistent with a role in promoting bacterial clearance. Specifically, it bound both Gram-positive and Gram-negative bacteria in a charge-dependent manner, promoted their aggregation, facilitated phagocytic uptake by macrophages and neutrophils, enhanced bacterially induced pro-inflammatory cytokine product from macrophage, and protected against vaginal infection in a N. gonorrhoeae murine model. Collectively the data reported in this thesis shed light on the fundamental mechanisms by which cationic amyloid fibrils enhance HIV-1 infection, and suggest that these fibrils act principally by neutralizing charge repulsion between the virus and the host cell, and not by accelerating the sedimentation rate of bound virus particles. Additionally, we identified a novel small molecule that binds efficiently to SEVI and other fibrils, and is also capable of inhibiting SEVImediated enhancement of HIV infection. Finally, we demonstrated that SEVI has indirect antimicrobial activity, suggesting that the natural biologic function(s) of semen derived cationic amyloid fibrils may include a role in the prevention of bacterial infections in the human reproductive tract