The Type 4 pilus (T4P) in Neisseria gonorrhoeae, and other bacterial species, is a protein system responsible for host-cell adhesion of the pathogen. Insight into the structure of this system necessary for N. gonorrhoeae pathogenesis can aid the development of novel therapeutic avenues. PilC, the adhesin located at the tip of the T4P, is essential for the initiation of pilus assembly, DNA transformation, and host-cell adhesion. It is believed to interact with a complex of minor pilin proteins to initiate pilus assembly, but the mechanisms of this process are unclear. My project aims to develop an amber-codon suppression system to investigate the function of PilC and its interactions with minor pilins and host cells. Based on computational modeling, the last 12 amino acids of PilC form a beta-strand that binds to the minor pilin PilK to initiate piliation. I designed a mutated version of the PilC gene by inserting an amber stop codon (sequence “TAG”) before the genetic code for this beta-strand. When expressed in gonorrhoeae, the mutated gene leads to a loss of T4P. Next, I aim to genetically modify an existing tRNA to read an amber stop codon. I hypothesize that such a tRNA, known as an “amber suppressor,” when expressed in the non-piliated cell, should rescue the defect in PilC by reading the amber stop codon, thus enabling translation of the complete, functional protein. The resulting cell should change from non-piliated to piliated, confirming that the final beta-strand of PilC is essential for T4P formation. Once I develop a functional amber-suppressor system in N. gonorrhoeae, I intend to study other domains of PilC and the minor pilins essential to T4P biogenesis, by extending the system to enable site-specific incorporation of non-canonical amino acids with useful properties.

Eukaryotic cells contain many membrane-bound organelles and rely on precise vesicle trafficking to transport cargo between them and maintain organelle function and identity. Functional defects in Adaptor Protein complex 3 (AP-3) disrupt vesicle trafficking, leading to disorders such as albinism, seizures, and neutropenia. In Saccharomyces cerevisiae, AP-3 carries cargo from the late Golgi to the lysosomal vacuole, but how it dissociates from the carrier vesicle is not clear. Adenosine diphosphate (ADP)-ribosylation factor 1 (ARF1) regulates AP-3 recruitment and shedding, relying on GTPase-activating proteins (GAPs) for proper function. AGE2, an ARF1 GAP, functions redundantly with GCS1 to regulate ARF1 (Schoppe, 2020), thus AP-3 trafficking. This study aims to identify the interaction site between AP-3 and AGE2 to better understand AP-3 shedding molecularly. Using AlphaFold3, the Merz lab predicted a conserved alpha-helix region in the AP-3 subunit Apl5 C-terminal domain (CTD) as a potential interaction site. To test this hypothesis, I introduced substitution mutations in Apl5 CTD and conducted spinning disc confocal microscope experiments to assess AP-3 pathway defects with a GNSI reporter, which enables to quantify AP-3 function via fluorescence distribution. My results show no statistically significant difference in trafficking defects between wild-type and mutant strains, suggesting that the predicted site is either not a binding site, or not necessary for AP-3 and AGE2 function. Although this study yielded a negative result, it refines our understanding of AP-3 shedding. Future studies will explore alternative regions on Apl5 subunit of AP-3 to identify the true interaction site and uncover the molecular mechanism of AP-3 shedding.