Lactobacillus buchneri Cell Wall Glycobiology

Lactobacillus buchneri CD034 is a Gram-positive lactic acid bacterium (LAB) with GRAS (generally regarded as safe) status that was isolated from silage samples. Due to its possible utilization in food sciences L. buchneri is an ideal candidate organism for cell surface display of biofunctional compounds. Prior to establishment of such a system it is required to completely characterize both the cell surface and cell wall architeture of this organism at the biochemical and molecular level. Preliminary investigations have shown that L. buchneri CD034 is completely covered by a 2D crystalline cell surface (S-)layer. The S-layer protein monomers are obviously modified by covalently attached carbohydrate chains. Previous analyses of a related organism, where both the S-layer glycan structure and a decapeptide O-glycosylation sequence have been identified, support such an assumption. This decapeptide O-glycosylation sequence was also found in a recent protein sequence analysis of the L. buchneri CD034 S-layer protein. The current project proposal is based on the knowledge of a recently performed whole genome sequence analysis of this organism (CD Laboratory for Genetically Modified Lactic Acid Bacteria; Prof. Reingard Grabherr) and the availability of molecular tool for genetic modification of L. buchneri CD034. There are three main topics of the project which have not been analyzed in any LAB so far. (i) The complete structural characterization of the S-layer glycans of L. buchneri CD034 and the functional analysis of key enzymes of the S-layer glycosylation pathway, based on a preliminary annotation of carbohydrate-active enzymes,. (ii) Biochemical and molecular analysis of the anchor molecule between S-layer and cell wall of L. buchneri CD034 . In this context novel, poorly investigated neutral oligosaccharides will be considered in addition to classical secondary cell wall polymers such as teichoic and lipoteichoic acids. (iii) If exopolysaccharides (EPS) are present, the biochemical and molecular characterization of these should be performed on this S-layer carrying organism. When L. buchneri CD034 is used as a cell surface display system, depending on the possible presence of the EPS on the cell wall, it might be necessary to shut-down EPS biosynthesis. In conclusion, with this GRAS organism the application potential of biofunctional S-layer glycoproteins should be considerably extended.

The goals of the recently finished research project P24305-B20 comprised the structural and functional characterization of selected cell surface compounds of Lactobacillus buchneri CD034 which play a crucial role in the bacterias interaction with the surrounding environment. This organism, isolated from silage material, is a Gram-positive, lactic acid bacterium (LAB) with GRAS (generally regarded as safe) status. Because of its possible usage in food industry, L. buchnerii CD 034 is an ideal candidate for in vivo cell surface display of biofunctional materials. In concrete terms, four thematic foci were dealt with in the said project: (1) Structural characterization of the surface (S-)layer glycan of L. buchnerii CD 034. Preliminary investigations have shown that this strain is completely covered with a 2D-crystalline S-layer. On a defined dekapeptide sequence of the S-layer protein, short glucose-containing glycan chains were found to be O-glycosidically linked to the protein. This also confirmed previous results from a related L. buchneri strain. (2) Investigations to explain the anchoring mechanism of the S-layer glycoprotein (SGP) to the cell wall of L. buchnerii CD 034. Until now there existed no generally valid models for lactic acid bacteria. Besides classical secondary cell wall polymers (SCWPs) such as teichoic and lipoteichoic acids, neutral glycans/glycoproteins were also discussed as anchoring structure. To clarify this issue, the SGP/SCWP complex of L. buchnerii CD 034 was isolated and the in vivo interaction between SGP, SCWP and peptidoglycan was defined for the first time for LAB by single molecule force spectroscopy. (3) Analysis of possible exopolysaccharide (EPS) production by L. buchnerii CD 034. The electronmicroscopical analysis of cells did not reveal the presence of EPS on the cell surface, although in the genome a cluster for synthesis of a previously unknown glycan was identified additional analyses are required. (4) The usage of the GRAS organism L. buchneri considerably extended the application potential of biofunctionalized S-layers. Having the S-layer of L. buchnerii CD 034 as carrier for a peanut allergen Ara h 2-derived peptide made possible the construction of a model vaccine for a peanut allergen-specific immunotherapy; first immunological tests of that material were positive.To extend our general knowledge on prokaryotic protein glycosylation for glycan engineering purposes in side projects specific glycosylation related aspects of S-layer glycoprotein-carrying bacterial and archaeal organisms were investigated.