Title: Glycoproteins in probiotic bacteria - Exploring the glycosylation potential of Lactobacillus rhamnosus GG by genomics and glycoproteomics
Other Titles: Glycoproteïnen in bacteriën - Onderzoek naar het glycosylatiepotentieelvan Lactobacillus rhamnosus GG via genoom- en glycoproteoomanalyse
Authors: Tytgat, Hanne
Issue Date: 16-Apr-2015
Abstract: A wide variety of glycoconjugates covers the cell wall of prokaryotes, forming a unique species-specific barcode. These glycoconjugates are important to establish specific interactions with the environment. Most studies of bacterial glycobiology focus on pathogens. Here, the importance of glycosylation in the microbiota isolate and model probiotic strain Lactobacillus rhamnosus GG is investigated, with a focus on glycoproteins. To shed light on how and why L. rhamnosus GG glycosylates part of its proteome, a multidisciplinary study was performed.
A systematic in silico workflow was designed to mine the genome of L. rhamnosus GG for genes encoding glycosyltransferases, the key enzymes of glycobiology. Moreover, our method enabled the prediction of the substrate classes of these putative glycosyltransferases. Ultimately, we could identify 48 putative glycosyltransferases, of which 20 could be linked to their potential substrate and 8 putatively to a protein substrate. This genetic study was complemented by a second, now wetlab, proteomic screening, in which the fractionated proteome of L. rhamnosus GG was screened for glycosylated proteins, showing that the glycoproteome might encompass at least 40 glycoproteins, apart from the well-studied Msp1 glycoprotein.
To also perform a dedicated study on the functional importance of glycosylation in L. rhamnosus GG, the glycosylation of the SpaCBA pili was explored. A multidisciplinary approach enabled the confirmation of the glycosylation of these pili with mannose and fucose-residues. Moreover, we could show that their glycosylation is crucial for the interaction with the DC-SIGN immune lectin receptor present on dendritic cells.
Taken together, we were able to achieve several breakthroughs by optimizing and designing new approaches. Our study generated important new insights in the glycosylation potential and the potential role of protein glycosylation in bacteria, like the potential role of glycosylation in the modulation of multiprotein complexes and interaction with immune lectin receptors. This work contributed to a better knowledge on the importance of glycoproteins in the interaction of probiotics and members of the microbiota with the host and the knowledge on bacterial protein glycosylation in general.
Table of Contents: Index

ii dankwoord
vi abstract
viii samenvatting
x list of abbreviations

1 one - outline
11 two - the sweet tooth of bacteria: common themes in bacterial glycoconjugates
65 three - a network-based approach to identify substrate classes of bacteria glycosyltransferases
97 four - endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection
107 five - systematic exploration of the glycoproteome of a beneficial gut microbe
133 six - glycosylated heterotrimeric pili of L. rhamnosus GG mediate DC-SIGN interaction
157 seven - general discussion

178 references
202 addendum
204 list of publications
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics
Molecular Physiology of Plants and Micro-organisms Section - miscellaneous

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