Molecular understanding of fungal cell wall metabolism by gut Bacteroides

Ph. D. Thesis.The human gut is populated with a vast community of microbes, the microbiota. The Bacteroidetes play a prominent role in the breakdown of complex carbohydrates. Fungi are also normal members of the gut microbiota. Fungal cell wall proteins are extensively mannosylated, forming a highly branched matrix of mannan. Previous data from our lab show that the common gut bacterium, Bacteroides thetaiotaomicron (Bt) degrades cell wall mannan from S. cerevisiae via a selfish mechanism. Bt does this by releasing large oligosaccharides at the cell surface, which are transported into the periplasmic space to be further depolymerised. Firstly, the degradation of mannan from a common human pathogen, Candida albicans was investigated. Here, novel enzymes from the Glycoside Hydrolase 130 family, which target β-1,2-mannosidic linkages found in C. albicans mannan, have been biochemically characterised. Bt was genetically manipulated to examine its ability to utilise mannan from C. albicans. This work demonstrated that degradation of C. albicans mannan in Bt requires an additional regulatory mechanism. Secondly, I discovered that another gut Bacteroides, B. salyersiae (Bs), degrades yeast α-mannan in a mechanism contrasting to the ‘selfish’ strategy. Unlike Bt, Bs releases a range of smaller manno-oligosaccharides into the extracellular milieu, which are utilised as ‘public goods’ by other members of the gut microbiota. Biochemical characterisation of this alternative mechanism revealed that proteins orchestrating mannan breakdown contain signals, targeting them for the Bacteroidetes specific type 9 secretion system (T9SS). Our analysis revealed that Bs directs at least 109 proteins of diverse functions to the T9SS, implicating its role in a plethora of metabolic processes. This is the first description of T9SS in a human gut Bacteroides. This work demonstrates that members of the microbiota have developed multiple strategies for utilisation of the same carbon to survive in a highly populated human gut