Disease caused by antibiotic resistant (ABR) bacteria has become a widespread global public health issue as humanity's existing collection of effective antibiotics dwindles. ABR bacteria are responsible for approximately 5 million deaths worldwide annually, which is predicted to reach 10 million yearly by 2050. Antivirulence therapeutics have been explored in recent times as another approach to tackling the global ABR pandemic by disrupting the function of virulence factors that promote disease development. The bacterial type IV pilus (T4P) is a prevalent virulence factor in many ABR pathogens, contributing to bacterial pathogenesis by facilitating cell motility, surface adhesion, and biofilm formation. Critically, the T4P facilitates early stages of disease, providing a means to invade and colonize a host. T4P assembly is driven by the PilB/PilF motor ATPase that localizes to the cytoplasmic face of the inner membrane to drive pilus biogenesis by ATP hydrolysis. The thesis work here explores computational and experimental methods for the discovery of antivirulence therapeutics targeting the T4P assembly ATPase PilB. A computational model of Chloracidobacterium thermophilum PilB was generated by homology modeling and molecular docking was performed to analyze the binding characteristics of six anti-PilB inhibitory compounds identified in previous studies. Computational docking aligns with the existing body of work and reveals important protein-ligand interactions and characteristics, particularly involving the ATP binding domain of PilB. This work supports the use of PilB in structure-based virtual screening to identify novel compounds targeting PilB. Additionally, through heterologous expression and chromatography methods, the ATPase core of Neisseria gonorrhoeae PilF was successfully expressed and purified as an active ATPase. This work optimized conditions for its ATPase activity in vitro. Additionally, this thesis documents the experimental attempt to express and purify Clostridioides difficile PilB as an active ATPase. Two of the seven C. difficile PilB variant proteins expressed led to soluble protein while one construct remains to be explored. The results of these studies provide insight for future methodology design for antivirulence therapeutic research targeting the T4P assembly ATPase using both in silico and in vitro methods. / Master of Science / Antibiotic resistant bacterial infections are responsible for nearly 5 million deaths worldwide every year. These infections are becoming increasingly more difficult to treat as bacterial pathogens acquire greater means to overcome our dwindling antibiotic repertoire. This has prompted researchers to explore alternative therapeutic strategies, including the antivirulence approach that aims to disable the function or production of bacterial virulence factors. Virulence factors serve as arms and armor that help bacteria cause disease, but they may be disrupted in such a way that renders potentially pathogenic bacteria harmless to humans. One major virulence factor in many antibiotic resistant bacteria is the type IV pilus (T4P), which is important in the early stages of host invasion by mediating adhesion and biofilm formation. This work explores both computational and experimental strategies to antivirulence drug discovery targeting the T4P, specifically the primary motor protein PilB/PilF. Newly identified PilB inhibitors were evaluated by molecular docking and molecular dynamics simulation to assess the use of PilB for drug discovery via virtual screening in silico. This revealed key characteristics and protein-ligand interactions that contribute to successful PilB inhibition and supports the use of CtPilB for structure-based virtual screening. Additionally, the PilF motor protein from Neisseria gonorrhoeae was successfully purified and demonstrated to be active for inhibitor discovery in the future. This work also covers efforts to establish Clostridioides difficile PilB as potential model enzyme for inhibitor discovery in the future.

The type IV pilus (T4P) is a dynamic long thin fiber found on the surface of many bacterial groups. T4P is a versatile nanomachine; it plays many important roles such as for surface attachment, virulence factor, and surface motility apparatus. This research focuses on understanding the kinetics of PilB, the T4P assembly ATPase. PilB crystal structure exhibits an elongated hexamer with 2-fold symmetry indicating a symmetric rotary mechanism model. Except for its structure, the symmetric rotary mechanism of PilB has not been demonstrated experimentally. Its conformation and relatively low activity constrained previous in vitro studies of PilB. This study identified PilB from thermophilic organism Chloracidobacterium thermophilum (Ct) to be a model for in vitro studies. An active CtPilB was successfully expressed and purified as a hexamer. Malachite green phosphate assay was used to examine CtPilB ATPase activity. The examination indicated that CtPilB is a robust ATPase with a complex kinetics profile. The profile has a stepwise incline in ATPase activity as a function of [ATP] that led to a decline in higher [ATP]. The decline was confirmed to be a substrate inhibition by the enzyme-coupled assay. As for the incline, the detailed mechanism is still less clear to explain the multiphasic profile. The overall incline did not conform with classical Michaelis-Menten kinetic but the first part of the incline was shown to conform with Michaelis-Menten kinetics. The complex kinetics profile of PilB is consistent with the symmetric rotary mechanism of catalysis. / Master of Science