The role of mature mycolic acids in mycobacterial pellicle biofilm formation and interactions with the human complement system

Halkerston, Rachel Keira ORCID: 0000-0001-6420-3331 (2022). The role of mature mycolic acids in mycobacterial pellicle biofilm formation and interactions with the human complement system. University of Birmingham. Ph.D.

Halkerston2022PhD.pdf
Text - Accepted Version
Restricted to Repository staff only until 1 January 2025.
Available under License All rights reserved.
Download (13MB)

Abstract

M. tuberculosis cell wall components are essential for host-pathogen interactions and modulating the immune response. M. tuberculosis may persist as pellicle-like biofilms within the peripheries of human cavities, and expectorated bacteria from these biofilms may establish infection in a new host. The complement system is an early cascade of the innate immune response, which plays an important role in M. tuberculosis access to alveolar macrophages. The impact of mycobacterial biofilms on complement activation has not been studied in-depth. This thesis aims to evaluate the role of mycolic acid maturation in pellicle biofilm formation and interactions of the mycobacterial cell envelope with the human complement system.
Mycolic acids are a major cell wall component, and their accumulation is a hallmark of pellicle biofilm formation. M. smegmatis, a surrogate organism for M. tuberculosis, was mutated in gene MSMEG4722 (ortholog of M. tuberculosis Rv2509) (ΔMSMEG4722), causing the loss of mature mycolic acids for mycolic acid intermediates (Bhatt et al., 2008). As a result, ΔMSMEG4722 pellicle biofilms could form on the air-liquid interface but could not mature and produced significantly less extracellular material.
Biochemical changes in the cell envelope of ΔMSMEG4722 were examined using thin layer chromatography. Changes included alteration to the distribution of glycolipids (TDM, TMM and GMM) within the apolar lipid fractions, occurring in bacteria cultured in Middlebrook 7H9 and as planktonic cells and pellicle biofilms. Within pellicle biofilms, mature free mycolic acids and free mycolic acid intermediates/ pre-mycolates decarboxylated to unsaturated ketones accumulated in the apolar lipid fractions for M. smegmatis mc2 155 and ΔMSMEG4722, respectively.
Flow cytometry and ELISA demonstrated increased complement system activation, both on whole bacterial cells and the apolar lipid fractions of ΔMSMEG4722 compared to M. smegmatis mc2 155. Increased complement activator binding was also observed. When examining M. smegmatis mc2 155 pellicle biofilms, comparable complement activation was observed on planktonic cells and pellicle biofilm-derived cells. However, overall activator binding was lower, and there was decreased complement activation by the apolar lipid fractions. ΔMSMEG4722, in contrast, showed higher complement activation by both phenotypes compared to M. smegmatis mc2 155, but activator binding was only increased on biofilm-derived cells and both phenotypes demonstrated decreased complement activation by the outer apolar lipid fraction.
The implications of these findings in understanding mycobacterial pellicle biofilm formation and deciphering mycobacterial cell wall component activation of the complement system are discussed.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):

Supervisor(s)	Email	ORCID
Bacon, Joanna	UNSPECIFIED	orcid.org/0000-0002-2217-228X
Taylor, Stephen	UNSPECIFIED	orcid.org/0000-0002-9936-8539
Alderwick, Luke	UNSPECIFIED	orcid.org/0000-0002-1257-6053
Besra, Gurdyal S.	UNSPECIFIED	UNSPECIFIED