Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Vincent A. Fischetti
Keywords
pathogenic bacteria, Paenibacillus bacteria, Clostridium bacteria, bacteriophage endolysins, diagnostic tools, antibiotic resistance
Abstract
Pathogenic bacteria represent a significant and increasing global health and safety concern. In addition, growing antibiotic resistance poses a serious threat to our ability to prevent and treat bacterial infection and disease. Addressing these threats will require the development and application of novel therapeutics and clinical approaches. The focus of our research is on one such novel approach, the use of bacteriophage endolysins as diagnostic (lysidiagnostics) and therapeutic (enzybiotics) tools for pathogens of human disease. Endolysins are modular proteins that facilitate release of progeny phage by digesting the bacterial host cell wall. Structurally, endolysins are composed of an enzymatically active domain (EAD) and a cell wall binding domain (CBD). It is the variable architecture of these protein domains that confers functionality and specificity toward the bacterial host. This work describes studies of endolysins against two pathogens of human disease, Paenibacillus, primarily Paenibacillus thiaminolyticus (P. thiaminolyticus) and Clostridium, specifically Clostridium perfringens (C. perfringens). Paenibacilli are gram-positive, aerobic or facultatively anaerobic, spore-forming bacilli of the soil microbiome. While not traditionally considered human pathogens, they can cause paenibacilliosis in neonates, an emerging disease presenting with sepsis, meningitis complicated by post-infectious hydrocephalus (PIH), and high mortality. Infections by anaerobic bacterial pathogens are primarily caused by gram-positive, toxinogenic, spore-forming members of the Clostridium genus, all of which can cause life-threatening disease. Their pathogenicity is largely mediated through the production of potent exotoxins, which can result in a wide spectrum of clinical manifestations ranging from gastrointestinal illness and enterotoxemia to neurotoxicity and tissue necrosis. Identification of multi-drug resistant (MDR) strains of this primarily food-borne pathogen serves as a bellwether for potentially widespread disease with high lethality. In this thesis, we identified and characterized endolysins for the selective killing of pathogenic Paenibacillus (Chapter 2) and Clostridium (Chapter 4), employing several existing methods. In addition, it was necessary to develop and optimize additional methods and protocols to complement and advance this research. These included: (1) bioinformatics and protein structural analyses to identify endolysin candidates, (2) mid- and high-throughput techniques for protein expression and purification, (3) screening assays for initial characterization of hydrolytic activity, and (4) modifications of existing methods to optimize in vitro bactericidal activity. For P. thiaminolyticus, we identified the first endolysin to be described for this organism (LysPt9) that could achieve >3-logs of bactericidal activity at endolysin concentrations < 100 μg/mL. This endolysin was active against three environmental P. thiaminolyticus strains, the MbaleIII strain derived from neonatal cerebral spinal fluid (CSF), and several other Paenibacilli. To our knowledge, this is the first described endolysin against P. thiaminolyticus that could be optimized for use in therapeutic development. We also identified and characterized endolysins for C. perfringens that are strong candidates for future use in a murine gnotobiotic gastrointestinal (GI) model of C. perfringens infection. These potent candidates are characterized by high protein yields and specific bactericidal activity in studies comparing C. perfringens strains with commensal bacteria derived from the murine gut. Additional studies assessed the use of endolysin cell binding domains (CBDs) for specific identification and potential diagnostics of P. thiaminolyticus (Chapter 3) and C. perfringens (Chapter 4). For these studies, we generated and purified fluorophore-labeled CBDs and optimized methods for fluorescence staining of their target organisms to image protein binding. Results with our candidates showed highly specific binding of fluorescently labeled CBDs to P. thiaminolyticus under conditions of mixed bacterial cultures. Similarly using labeled CBDs, we were able to specifically identify C. perfringens from other commensals in a murine GI microbiome model (Oligo-MM12). Finally, based on these studies, we performed preliminary experiments to develop a gnotobiotic murine model of C. perfringens infection for oral endolysin delivery (Appendix 7.1). Taken together, our work identifies novel therapeutic candidates and diagnostic tools to control and identify two major pathogenic bacteria, P. thiaminolyticus and C. perfringens.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Soellner-Szwed, Sarah, "Bacteriophage Endolysins: Anti-Infectives and Lysidiagnostics for the Post-Antibiotic ERA" (2026). Student Theses and Dissertations. 845.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/845
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy