Bachelor of Science (BS), Ohio University, 2024, Biological Sciences

Our strain AP312 was isolated from a rhesus macaque bite wound. Phylogenetic analysis of isolate AP312 and additional monkey-derived Neisseria isolates indicate that AP312 likely represents a new commensal species in the genus. The isolate was found to form wrinkled colonies upon certain medias at around 48 hours yet AP312 wrinkles sooner at around 24 hours on TSA 5% sheep's blood. We made a transposon mutagenesis library to search for mutant strains with altered colony morphology and transposon insertion sites were identified using inverse PCR and Sanger sequencing. For example, some colonies appear smooth, some colonies appear smaller, and some colonies portray altered wrinkling compared to the WT. My research aimed to assess AP312 by identifying knocked out genes that lead to phenotypical differences in the mutant strains, conducting biofilm formation assays, growth curves, and crude pilin preparation. Three genetic knockouts were explored out of many mutants screened. These three genes are L-lactate dehydrogenase (ldh), UDP-galactose-4-epimerase (galE), and a type IV minor pilin J (pilJ). Future efforts will assess mutant strains for in vitro phenotypes including biofilm formation in different environments, exopolysaccharide secretion, and transmission electron microscopy. These mutated genes we have identified are conserved in pathogenic Neisseria species. Genetic analysis of colony morphology mutants in AP312 provides a tool for identifying the function of orthologs that have not been studied in great detail in the pathogenic species.

Committee: Soichi Tanda (Advisor) Subjects: Biology; Genetics

Doctor of Philosophy (PhD), Ohio University, 2021, Molecular and Cellular Biology (Arts and Sciences)

Antimicrobial resistant Neisseria gonorrhoeae (Ng) is an urgent threat to public health worldwide. Ng causes gonorrhea, the second most prevalent sexually transmitted bacterial infection. Left untreated, gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, sterility, and in men can increase susceptibility to HIV. The current recommended treatment is a dual therapy with two antibiotics azithromycin and ceftriaxone. However, many countries are reporting cases with reduced effectiveness of this treatment method. This has placed a greater urgency on the development of preventative therapeutics. There is currently no commercial vaccine available for Ng infection. The development of preventative treatment strategies has long been impeded by Ng's ability to evade the immune system, particularly the complement system. Ng is an obligate human pathogen and has evolved numerous mechanisms to interact with host-derived molecules that negatively regulate the activation of the immune system. Previous characterization of these mechanisms has focused on one class of complement inhibitors, fluid-phase inhibitors. To fully understand Ng immune evasion and the host molecules that contribute to it, investigating the role of the other class of complement inhibitors, membrane-associated complement inhibitors (mCIs) is needed. mCIs like CD46, CD55, and CD59 are expressed by human epithelial cells to prevent formation of the membrane attack complex on the surface of human cells. I hypothesized that these proteins may be sequestered by Ng during infection and create localized regions of immune activation allowing Ng to persist. To test this, we used a co-culture serum bactericidal assay (ccSBA) where human epithelial cells were infected with Ng, treated with normal human serum, a vehicle for the components of the complement system, and monitored bacterial viability over time. Using the ccSBA we demonstrated that increasing host expression of mCIs resulted in higher level (open full item for complete abstract)

Committee: Nathan Weyand (Advisor) Subjects: Biology; Immunology; Microbiology; Molecular Biology

Bachelor of Science (BS), Ohio University, 2021, Biological Sciences

Neisseria gonorrhoeae is the causative agent of gonorrhea, the second most common bacterial sexually transmitted infection worldwide following chlamydia. Antibiotic resistance to N. gonorrhoeae is growing rapidly, even affecting countries with advanced healthcare systems. N. gonorrhoeae has been labeled as an imminent threat due to its high levels of antibiotic resistance. If gonorrhea is left untreated, it can cause sterility, ectopic pregnancy, and pelvic inflammatory disease. Many times, N. gonorrhoeae is not easily noticed due to its ability to cause asymptomatic infections. Some individuals infected asymptomatically are pharyngeal carriers. Currently, there is no vaccine. Research efforts are focused on vaccine development and other preventative therapeutics. N. gonorrhoeae may cause infection by attaching to human epithelial cells in the urogenital tract. N. gonorrhoeae persistent mechanisms in humans frequently rely on its ability to evade the human immune defenses such as the complement cascade. The aim of this project is to focus on complement regulatory proteins, which are proteins that human cells express to protect cells from autologous complement damage. Our previous data suggests that N. gonorrhoeae is stealing complement regulatory proteins, like CD46, CD55, and CD59 to prevent immune activation, allowing the bacteria to persist and cause infection. This project studies the effect of microRNAs, such as miR-200b and miR-200c, on the membrane complement proteins. This was accomplished by transfecting our model cell line, MCF7, with microRNA expression plasmids. The aim of this research is to understand the effect of complement regulatory proteins on N. gonorrhoeae survival.

Committee: Nathan Weyand (Advisor); Soichi Tanda (Other) Subjects: Biology; Biomedical Research; Cellular Biology; Genetics; Health; Immunology; Medicine; Microbiology; Molecular Biology

Master of Science (MS), Ohio University, 2020, Biological Sciences (Arts and Sciences)

Neisseria gonorrhoeae is the causative agent of gonorrhea infection. The Center for Disease Control estimates that over 800,000 gonorrhea infections occur in the United States annually. Treatment of this pathogen is confounded by increasing levels of antibiotic resistance worldwide. To inform the development of novel therapeutics, future research must discern the mechanisms Neisseria gonorrhoeae uses to evade host immunity during infection. This work seeks to develop an assay to determine if Neisseria gonorrhoeae can utilize membrane-bound complement inhibitors expressed by epithelial cells to evade complement. Membrane-bound complement inhibitors typically protect human cells from aberrant complement activation. Additionally, flow cytometry is used to better characterize cell lines with differing surface expression levels of the membrane- bound complement inhibitors CD46, CD55, and CD59. By manipulating human cells and bacterial strains in this novel assay, we are able to elucidate the mechanisms that Neisseria gonorrhoeae uses to evade complement activation. This work is beginning to describe a previously unreported link between Neisseria gonorrhoeae and membrane- bound complement inhibitors expressed on infected epithelial cells.

Committee: Nathan Weyand Dr. (Advisor); Ronan Carroll Dr. (Committee Member); Mark Berryman Dr. (Committee Member) Subjects: Biology; Immunology; Microbiology

Master of Science (M.S.), University of Dayton, 2013, Chemistry

Complete and faithful replication of a cell's genetic information is an essential process. Many enzymes are involved in the process of successfully duplicating a cell's genetic information. Helicases, DNA polymerases, ligases, nucleases, and DNA binding proteins all play a role in DNA replication. However, the integrity of these enzymes can be compromised when they encounter DNA damage, which in general could be caused by chemical mutagens, ionizing radiations, or reactive oxidative species. Bacterial cells use a pathway called "DNA replication restart" to resume DNA replication following a disruptive encounter of the DNA replication enzymes with DNA damage. This pathway is catalyzed by primosome proteins, including PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The importance of DNA replication restart for bacterial cell survival is demonstrated by the inability of strains that carry mutations in key primosome genes to grow and resist DNA damaging agents. Furthermore, this pathway is specific for bacterial cells: human cells don't use the same replication restart pathway and they don't encode genes for the primosome proteins that function in bacteria. Since DNA replication restart pathways are essential for bacterial cell growth and survival and are notably absent in human cells, we seek to answer the following question: can bacterial DNA replication restart pathways be targeted with novel antibacterial compounds? In order to answer this question, we have developed an enzyme based assay for high-throughput inhibitor screening to identify compounds that block the function of the primosome proteins PriA and PriB. Several interesting lead compounds have already been identified from the preliminary screening. In this study, the lead compounds have been validated as legitimate inhibitors and characterized with respect to their potency and mechanism of action.

Committee: Matthew Lopper (Advisor); Gary Crosson (Committee Member); Mark Masthay (Committee Member); Shawn Swavey (Committee Member) Subjects: Biochemistry; Chemistry

Master of Science (M.S.), University of Dayton, 2012, Chemistry

Neisseria gonorrhoeae is the causative organism of gonorrheal infection. This pathogen shows remarkable resistance to the oxidative damaging agents released by the neutrophils. The Bacterial genome is one of the important targets of these agents and studies indicate that DNA replication restart pathways help in bypassing the hazardous effects of these oxidative agents. The DNA replication restart pathway helps to reload and restart the stalled or derailed replication machinery onto the DNA which is blocked by obstructions such as single stranded nicks, double stranded breaks, or oxidized bases. In N. gonorrhoeae, the primosome proteins PriA and PriB carry out the DNA replication restart process by forming a nucleoprotein complex with the DNA. PriA, a helicase protein, binds and partially unwinds the DNA at the fork where the replication machinery will be reloaded. PriB serves to stimulate PriAs DNA binding and unwinding activity. PriA, PriB and the DNA form a ternary nucleo-protein complex in this process. In, E. coli the PriB has a high affinity interaction with ssDNA and a low affinity interaction with PriA, while N. gonorrhoeae PriB has a high affinity interaction with PriA and a low affinity interaction with ssDNA. The first part of my research contributed to understand the features of this affinity reversal. I performed pull down experiments, DNA unwinding assays and ATP hydrolysis experiments using single point alanine mutants of PriB. From my work, I have provided evidence that in N. gonorrhoeae, PriBs ssDNA binding activity is not required for PriB stimulation of PriA helicase, unlike what is seen in E. coli. Evidence that PriA provides resistance against oxidative damaging agents bolsters the importance of DNA replication restart pathway for the survival of this disease causing bacteria. Developing antibiotics that target this pathway could be a vital event in the field of drug discovery research. In this process, we have developed an enzyme-based assay to (open full item for complete abstract)

Committee: Matthew E. Lopper PhD (Advisor); Madhuri Kango-Singh PhD (Committee Member); Shawn M. Swavey PhD (Committee Member) Subjects: Biochemistry

Master of Science (M.S.), University of Dayton, 2011, Chemistry

DNA replication restart pathways enable bacterial cells to reinitiate DNA replication when replication has been disrupted due to encounters with DNA damage, thereby allowing complete and faithful duplication of the cell's genetic information. Neisseria gonorrhoeae is a bacterium that is highly adapted to survive oxidative damage to its DNA incurred by attack from immune cells in infected individuals, suggesting that DNA replication restart pathways might play a critical role in N. gonorrhoeae pathogenicity. The bacterial helicase, PriA, is a key primosome protein that plays essential roles in DNA replication restart pathways. However, little is known of the mechanism by which PriA performs these roles in N. gonorrhoeae. I performed equilibrium DNA binding assays and DNA unwinding assays to provide insight into the mechanisms by which PriA functions in DNA replication restart pathways. I report that DNA binding by PriA is strongly dependent on the structure of the DNA. DNA substrates that resemble a DNA replication fork with a three-way branch are bound with higher affinity than partial duplex structures or single-stranded DNA. PriA-catalyzed DNA unwinding is also DNA structure-specific, and PriA-catalyzed unwinding decreases upon increasing the length of the duplex DNA, indicating that PriA is a low-processivity helicase. Another primosome protein, PriB, strongly stimulates the helicase activity of PriA, and this activity might facilitate reloading of the replication machinery by PriA at repaired replication forks. Stimulation of PriA by PriB appears to occur through a mechanism that is distinct from that used by the well-studied E. coli primosome proteins.

Committee: Matthew Lopper PhD (Advisor); Madhuri Kango-Singh PhD (Committee Member); Shawn Swavey PhD (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2012, Medicine: Epidemiology (Environmental Health)

Preterm birth is the leading cause of perinatal morbidity and mortality. The risks of preterm birth are heterogeneous and there remains much to elucidate regarding etiology and epidemiology of this perinatal condition. Maternal genitourinary tract infection may reduce the length of gestation through premature rupture of membranes and/or preterm labor. Specifically, antenatal infection with Chlamydia trachomatis and/or Neisseria gonorrhoeae, both high prevalence sexually-transmitted infections, may convey a higher risk of preterm birth. Early detection and treatment of these infections without recurrent/persistent infection during pregnancy may serve as an effective intervention that protects against preterm birth. The objective of this study was to characterize the association between early antenatal detection and eradication of Chlamydia trachomatis and/or Neisseria gonorrhoeae infection and the likelihood of preterm birth among pregnant women in an urban county. A retrospective cohort study was conducted in Hamilton County, Ohio (2006-2011) to evaluate the risk of preterm birth among women whose infections were detected and eradicated at or before 20 weeks gestation (intervention cohort). The risk of preterm birth in this intervention cohort was compared to that in a cohort of women whose infections were detected late (> 20 weeks gestation) or were recurrent/persistent (= 20 weeks and >20 weeks gestation) during pregnancy. A novel approach was used to measure the timing and frequency of Chlamydia trachomatis and/or Neisseria gonorrhoeae detection and subsequent adverse pregnancy outcomes through the use of linked population-based public health data. The study population contained 3,912 linked birth-communicable disease records (i.e., maternal Chlamydia trachomatis and/or Neisseria gonorrhoeae infection). The relative risk of moderate to late preterm birth (32-36 weeks gestation) was 0.54 (95% CI: 0.37-0.80) for women in the intervention cohort who were 19 years (open full item for complete abstract)

Committee: Kim Dietrich Ph.D. (Committee Chair); Aimin Chen Ph.D. (Committee Member); Shantini Gamage Ph.D. (Committee Member); Paul Succop Ph.D. (Committee Member) Subjects: Epidemiology