Functional and molecular responses of human neutrophils to Fusobacterium nucleatum subspecies

Muchová, Mária ORCID: 0009-0000-5983-0090 (2024). Functional and molecular responses of human neutrophils to Fusobacterium nucleatum subspecies. University of Birmingham. Ph.D.

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Abstract

Fusobacterium nucleatum is a Gram-negative, commensal oral anaerobe, which is a key structural member of dental plaque biofilms. It is also considered an opportunistic pathogen due to its association with periodontitis and systemic extraoral diseases, such as cardiovascular disease and colorectal cancer. Five subspecies are currently recognised: animalis, fusiforme, nucleatum, polymorphum and vincentii. These subspecies, found in the oral cavity and in the extraoral tissues, interact with host innate immune cells, such as neutrophils, which form the first line of defence against microbes. Neutrophils possess a powerful antimicrobial arsenal to remove the invaders, however, excessive responses can cause collateral tissue damage and lead to chronic inflammation. Subspecies-specific neutrophil responses toward F. nucleatum have been reported in the neutrophil-like cell line HL-60, however responses of primary human neutrophils are largely unexplored. Additionally, F. nucleatum typically resides in biofilms, yet, the immunogenicity of biofilm-grown F. nucleatum subspecies has not been addressed to date. Therefore, this doctoral thesis systematically characterised biofilm formation by all subspecies. Subsequently, functional and molecular responses of primary human neutrophils were evaluated when stimulated with planktonic and biofilm-grown F. nucleatum subspecies. Characterisation of single-subspecies biofilm formation included biomass quantification using crystal violet (CV) staining, evaluation of biofilm architecture by scanning electron microscopy (SEM) and assessment of biofilm thickness and stability using confocal laser scanning microscopy (CLSM). Adhesion proteins (adhesins) involved in F. nucleatum biofilm formation were analysed in silico bioinformatically. Formalin-inactivated (planktonic) and live F. nucleatum subspecies (planktonic and biofilm-grown) were used as stimuli to evaluate functional and molecular responses of primary neutrophils isolated from human peripheral blood. Responses of HL-60 cells were evaluated alongside primary neutrophils for comparison.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):