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An endo-acting galactanase with a type II signal peptide confers keystone status to arabinogalactan degrading bacteria of the human gut microbiota

ABSTRACT: Glycans, the major source of energy for the human gut microbiota (HGM), are metabolised by numerous members of the Bacteroides genus. Arabinogalactan proteins (AGPs) are a ubiquitous and highly heterogenous group of plant glycans in which a B1,3-galactan backbone and B1,6-galactan side chains are conserved features. Diversity is provided by the extensive and highly variable nature of the sugars that decorate both the backbone and side chain galactans. The mechanisms by which nutritionally relevant AGPs are degraded at a cellular and biochemical level are poorly understood, as is the impact of this process on the ecology of the HGM. Here we have explored how the HGM organism Bacteroides thetaiotaomicron metabolises simple and highly complex AGPs. We propose a sequential degradative model in which a repertoire of exo-acting family GH43 B1,3-galactanases releases backbone galactose units that are attached to the side chains. The oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or glycoside hydrolase. We identified an a-L-arabinofuranosidase and B-D-glucuronidase that are the founders of two previously undiscovered glycoside hydrolase families. The crystal structures of the backbone exo-B1,3-galactanases identified a key specificity determinant and reveals significant departure from the canonical catalytic apparatus of other family GH43 enzymes. Growth studies of 20 HGM Bacteroidetes species on a complex AGP revealed three keystone organisms that facilitated utilisation of fragments of the glycan by the 17 other bacteria, which thus acted as recipients. The ability to function as a keystone organism was conferred by a surface endo-B1,3-galactanase, which, when engineered into a recipient enabled this bacterium to utilise complex AGPs and facilitate the growth of the other Bacteroidetes species. This study underpins future pre- and probiotic strategies to exploit AGPs to manipulate the function of the HGM.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Bacteroides Cellulosilyticus Bacteroides Thetaiotaomicron (strain Atcc 29148 / Dsm 2079 / Nctc 10582 / E50 / Vpi-5482)

TISSUE(S): Cell Culture

SUBMITTER: Matthias Trost

LAB HEAD: Matthias Trost

PROVIDER: PXD010274 | Pride | 2018-07-03

REPOSITORIES: Pride

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			Action	DRS
	20180525_TH_B_cellu_WT_0_01.raw	Raw
	20180525_TH_B_cellu_WT_0_02.raw	Raw
	20180525_TH_B_cellu_WT_2_01.raw	Raw
	20180525_TH_B_cellu_WT_2_02.raw	Raw
	20180525_TH_B_tetha_00844_0_01.raw	Raw

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Project description:Mycoplasma pneumoniae is a significant cause of pneumonia, particularly in children, the elderly and the immunocompromised, and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model pathogen where extensive ‘omics’ studies have been conducted to gain insight into how minimal genome self-replicating organisms function. Here we conducted an N-terminome study to determine which proteins are targets of processing events in M. pneumoniae. We identified 6,973 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Nearly half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events which predominately target negatively charged residues in P1′ (D and E) with lysine or serine/alanine in P2′ and P3′ positions. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Mycoplasma pneumoniae culture conditions The M. pneumoniae (strain M129) cells were cultured in modified Hayflick’s medium at 37 °C in tissue culture flasks using established culture conditions63. Modified Hayflick’s medium contained 21 g PPLO broth base without crystal violet, 5 g of D-glucose, 4 mL of 0.5% phenol red, 100 mL of liquid yeast extract (150 g/L), 200 mL heat-inactivated horse serum (56 °C, 30 min) supplemented with 1 g ampicillin (Sigma, A5354, St. Louis, MO, USA) per litre. M. pneumoniae cells were harvested at mid-log phase by washing adherant cells 3 times with PBS and collected using cell scaping.

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An endo-acting galactanase with a type II signal peptide confers keystone status to arabinogalactan degrading bacteria of the human gut microbiota

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