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.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.