Deoxyribonucleic acid isolated from bacteriophage lambda can infect Escherichia coli K12 in the presence of adsorbed helper phage (Kaiser and Hogness, 1960; Kaiser, 1962). The manner in which lambda DNA enters the cell and the possible role of helper phage in the penetration process is still not clear. Kinetic studies conducted in this laboratory during the initial stages of infection by lambda DNA demonstrate a requirement for helper function during the penetration of an infectious molecule. Further investigation into this problem is needed and was the major objective of this study.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18675号 / 医博第3947号 / 新制||医||1007(附属図書館) / 31608 / 京都大学大学院医学研究科医学専攻 / (主査)教授 清水 章, 教授 渡邉 大, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Staphylococcus aureus is a common bacterium found on the skin and mucosal membranes of about 20% of the population. S. aureus growth on the skin is harmless, but if it bypasses the skin it can causes life-threatening diseases such as pneumonia, meningitis, bacteremia, and sepsis. Antibiotic-resistant strains of S. aureus, called Methicillin Resistant S. aureus (MRSA), are resistant to most antibiotics except vancomycin. However, vancomycin resistant strains of MRSA are becoming more common. In this study, 12 phages were isolated capable of infecting human S. aureus and/or MRSA strains. Five phages were discovered through mitomycin C induction of prophages and seven phages were found through enrichment of environmental samples. Primary S. aureus strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates. S. aureus isolates were tested for susceptibility to oxacillin in order to determine methicillin-resistance. Experiments were performed to assess the host range and killing potential of newly discovered phage. The M1M4 phage had the broadest host range and lysed 12% of the S. aureus strains that were tested. The host ranges were reinforced by spectrophotometric assay data which showed a reduction in bacterial optical density of 1.3 OD600. The phages were used to decontaminate MRSA from fomites (glass and cloth) and successfully reduced colony forming units by 1-2 logs, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

No / The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1(CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.

Thesis advisor: Jianmin Gao / Protein-protein interactions (PPIs) are essential for all biological functions. Developing peptides that disrupt these PPIs is an avid research effort, as peptides possess several advantages over small molecules and monoclonal antibodies. Peptide phage display is a useful tool in identifying peptides for targeting PPIs. This technology displays up to 10^10 unique polypeptides on the surface of bacteriophage, which after several rounds of panning enriches high affinity peptide sequences towards a target protein. Phage display is classically done on immobilized discrete protein; however, we propose to use this technology to identify peptides ligands for overexpressed oncogenic proteins on live cells in-vitro. This is a more accurate representation of the therapeutic target landscape and resembles how the peptide will interact with the receptor in-vivo. Several groups have explored live cell panning, such as Ruoslahti et al. and Cieslewicz et al., and while they demonstrate the capabilities of in-vitro style phage display, there are areas for improvement. We intend to improve on this previous work by 1. Identifying a peptide ligand against specific receptor/protein, and 2. By incorporating the use of covalent phage libraries to elucidate a high affinity binder. This work will be accomplished using the mammalian epidermal oncogenic cell line, A431, that is known to overexpress epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) is responsible for cellular proliferation, survival, differentiation and metastasis, which makes it an attractive target to inhibit oncogenic proliferation. Despite successfully marketed monoclonal antibodies and tyrosine kinase inhibitors, EGFR can mutate and develop resistance as diseases progress; this phenomenon, in addition to the benefits of peptides as therapeutics, are driving factors for pursuing this project. Despite our best efforts using non-covalent phage libraries to identify a viable ligand, screening against EGFR extracellular domain (ECD) has proven to be more difficult than anticipated. We hypothesize that non-covalent phage libraries do not possess any sequences with a high enough binding affinity for this protein, and that the use of covalent libraries will be needed to pull out a positive hit. Due to these findings, we have successfully constructed two phage libraries, a ACX7C and a ACX7C-TEV, where the latter introduced a TEV protease cleavage site on the C’-terminal side of the randomized amino acids suitable for covalent warhead modification and screening. Further, we have begun work on constructing an EGF-displaying phage construct to aid in optimizing a live cell panning protocol. In the future, we plan to evaluate ligand affinity and protein density, as well as determine the optimal covalent warhead/peptide combination for live cell screenings. With this information, we intend to apply this to other oncogenic cell lines, such as MCF-10CA1a, to identify potent peptide ligands for overexpressed oncogenic proteins. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Jianman Gao / The interaction between the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the human angiotensin-converting enzyme 2 (hACE2) receptor is an advantageous target for the development of therapies for COVID-19. We used an anti spike receptor binding domain (S RBD) antibody (AM122) to competitively elute phage binding to the S RBD in phage display screening to identify a novel peptide that binds the S protein and hACE2 interaction. We identified a peptide sequence (P1: CPLEYHTC) as a possible hit, and the KD was determined to be 2.667 μM, indicating the potential of this peptide sequence as a therapeutic agent. However, we found no inhibition of the spike protein and hACE2 receptor interaction, suggesting that the peptide may not directly bind to the hACE2 binding site on S RBD. Although further studies are needed, the competitive elution method in phage display screening appears to be an effective method for elucidating onsite peptide sequences that target protein-protein interactions (PPIs). / Thesis (BA) — Boston College, 2023. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Chemistry.

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