Dye, Keane2022-06-032022-06-032022-06-02vt_gsexam:34921http://hdl.handle.net/10919/110412Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors.ETDenIn CopyrightType IV piliMyxococcus xanthusPilBbiofilmanti-virulenceThe Type IV Pilus Assembly ATPase PilB as a Regulator of Biofilm Formation and an Antivirulence TargetDissertation