Oligosaccharide-mediated modulation of Staphylococcus aureus biofilm formation and epithelial inflammatory responses

Abstract

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen whose treatment is increasingly complicated by antimicrobial resistance and persistent biofilm formation. Carbohydrate-based molecules, including non-digestible oligosaccharides (NDOs) and human milk oligosaccharides (HMOs), have gained attention for their potential to interfere with bacterial adhesion and biofilm formation and modulate host immune responses. This thesis investigated how the amino sugars glucosamine (GAM) and N-acetylglucosamine (NGAM) and the HMOs 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), and lacto-N-tetraose (LNT) influence S. aureus biofilm formation and inflammatory responses in human intestinal epithelial cells. In Chapter 1, the scientific and clinical background is established, focusing on S. aureus, antimicrobial resistance, biofilm formation, and lipoteichoic acid (LTA)-induced inflammatory signaling through Toll-like receptor 2 (TLR2). Carbohydrate-based molecules are introduced as potential alternative or complementary approaches for affecting bacterial adhesion, biofilm development, and host immune signaling. In Chapter 2, available in vivo evidence for the antibacterial and antiviral properties of NDOs, including HMOs, is reviewed. Proposed mechanisms include soluble decoy receptor activity, interference with microbial growth and biofilm formation, toxin binding, strengthening epithelial barrier function, and modulation of immune and microbiota responses. Despite evidence from animal and some human studies, clinical evidence and understanding of structure-activity relationships remain limited. In Chapter 3, the effects of GAM and NGAM on S. aureus Wood 46 biofilm formation are investigated using RNA sequencing. GAM strongly inhibited biofilm formation, whereas NGAM showed weaker activity. GAM caused broader changes in bacterial adhesion and central metabolism, suppressing genes involved in the tricarboxylic acid cycle, arginine biosynthesis, and urea metabolism. Suppression of urease-associated pathways was accompanied by reduced environmental pH, suggesting impaired bacterial pH homeostasis. In Chapter 4, the effects of 2′-FL, 3′-SL, and LNT on S. aureus biofilm formation are investigated. 3′-SL produced the strongest inhibition, followed by 2′-FL, while LNT showed the weakest activity. All three HMOs downregulated the icaADBC operon, while compound-specific effects occurred in adhesion, metabolism, and stress-response pathways. Notably, 3′-SL strongly suppressed sdrC and sdrD and affected arginine biosynthesis. In Chapter 5, selected HMOs were investigated for their effects on inflammatory responses in HT-29 intestinal epithelial cells stimulated with LTA, Poly(I:C), or LPS. 2′-FL and LNT reduced LTA-induced IL-8 secretion, whereas 3′-SL did not. The HMOs did not substantially reduce Poly(I:C)- or LPS-induced IL-8 responses. Transcriptomic analysis showed that 2′-FL attenuated inflammatory and interferon-associated responses, including GBP4 and CXCL11, whereas LNT affected oxidative phosphorylation, thermogenesis, and proteasome-related pathways. Finally, Chapter 6 integrates the findings, demonstrating structure-dependent effects of carbohydrates on bacterial biofilms and host inflammatory responses. Importantly, strong anti-biofilm activity did not necessarily correspond to strong anti-inflammatory activity. Overall, even small structural differences substantially altered biological activity, supporting the development of targeted carbohydrate-based strategies to weaken bacterial biofilms and modulate infection-associated inflammation.