Dissertations, Theses, and Capstone Projects

Date of Degree

6-2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Akira Kawamura

Committee Members

Moriya Tsuji

Wayne Harding

David R. Mootoo

Haiping Cheng

Subject Categories

Biochemistry | Immunology and Infectious Disease | Microbiology

Keywords

Natural Products, Vaccine Adjuvants, Anti-inflammatory Phytochemicals, Biofilms

Abstract

Medicinal plants and their associated microbiomes constitute a rich source of bioactive natural products with applications in immunotherapy and microbial ecology. Herbal formulations have long been valued for safe immune enhancement, while bacterial signaling molecules govern community behaviors critical to pathogenesis, symbiosis, and environmental processes. Bacterial lipopolysaccharides (LPS) from plant-associated Gram-negative bacteria have emerged as potent TLR4 agonists, yet their endotoxic potential limits direct use, and diketopiperazines (DKPs) serve as versatile quorum-sensing mediators capable of modulating biofilms in mixed microbial systems.

The work in this thesis comprises two independent but complementary research strands. The first strand pursued the engineering of safer TLR4 agonist-based vaccine adjuvants derived from plant-associated bacterial LPS, drawing inspiration from the clinically established safety and immunostimulatory efficacy of the Japanese Kampo formulation Juzen-taiho-to (JTT). The second strand independently explored DKP mixtures as modulators of bacterial biofilm formation, elucidating stereochemical influences on microbial communication in ecologically relevant contexts.

Chapter 1 introduces the dual challenges addressed: the need for next-generation vaccine adjuvants that retain TLR4-mediated immunogenicity while minimizing proinflammatory toxicity, and the need to decode small-molecule signals like DKPs that regulate biofilm dynamics in complex microbial communities. It reviews established natural-product adjuvants (e.g., MPLA, QS-21) and their limitations in toxicity, stability, and scalability, while highlighting the understudied roles of plant microbiome-derived LPS and cyclic dipeptides in immune and microbial modulation.

Chapter 2 examines the immunostimulatory basis of JTT through in vitro macrophage assays (RAW 264.7 and THP-1 cells). Confirming that bacterial LPS drives JTT’s immune activity, the chapter demonstrates that co-occurring anti-inflammatory phytochemicals (AIPs) such as quercetin, scopoletin, liquiritigenin, and others selectively attenuate MyD88-dependent proinflammatory cytokine expression (IL-6) while largely preserving TRIF-dependent protective responses (CCL5). Species-specific differences and mechanistic insights (lipid raft remodeling, Nrf2/HO-1 activation, nuclear receptor crosstalk) position LPS-AIP combinations as tunable, naturally balanced TLR4 agonists.

Chapter 3 extends this line of inquiry to withaferin A (WA) from ashwagandha, another immunomodulatory herb. Dose-response studies in THP-1 cells show WA concentration-dependently suppresses LPS-induced IL-6 and CCL5 expression, with structural comparisons to diphosphoryl lipid A reinforcing LPS’s relevance as a full physiological ligand. These parallel findings underscore the recurring theme that plant metabolites can mitigate bacterial LPS endotoxicity, supporting the development of herbal-derived adjuvant platforms.

Chapter 4 advances the adjuvant strand to in vivo models. Intramuscular co-administration of low-dose LPS with quercetin or scopoletin significantly boosted antigen-specific IFN-γ responses in a PyCSP malaria model, revealing synergistic enhancement of cellular immunity. In contrast, intranasal low-dose LPS reduced SARS-CoV-2 lung viral titers without systemic inflammation, but AIP co-administration unexpectedly abrogated this mucosal protection, highlighting route-dependent outcomes and the complexities of translating in vitro modulation to physiological contexts.

Chapter 5 independently investigates DKP stereochemistry in microbial communication. Using an optimized crystal violet biofilm assay on E. coli, individual DKPs showed limited activity at submicromolar concentrations (0.05–0.1 µg/mL), but the trans racemic mixture of cyclo(L-Phe-D-Pro) and cyclo(D-Phe-L-Pro) consistently inhibited biofilm formation. These results suggest stereospecific synergies, potentially via quorum-sensing receptor interactions, membrane disruption, or differential stability, offering a pathway to targeted biofilm control distinct from the adjuvant work.

In conclusion, this thesis advances two separate frontiers. One demonstrates that plant-associated bacterial LPS, when combined with anti-inflammatory phytochemicals from medicinal herbs like JTT and ashwagandha, can yield safe, tunable TLR4 agonists for vaccine adjuvants, with in vitro selectivity and in vivo immunogenicity validated across parenteral and mucosal routes. The other establishes DKP mixtures as stereochemically nuanced modulators of bacterial biofilms, providing new leads for disrupting pathogenic communities or enhancing beneficial ones. Although arising from shared interest in plant-microbe interactions, these strands remain conceptually independent, together broadening our understanding of natural microbial products for immunological and ecological applications.

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