S-layer nanoglycobiology of bacteria

Abstract

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.

Figure 1

Illustration of prerequisites and strategies for the production of S-layer neoglycoproteins.

Figure 2

Bacterial S-layer glycan structures and attachment sites of S-layer proteins.

Figure 3

SDS-PAGE analysis of S-layer protein glycosylation patterns on intact bacterial cells by (A) Coomassie Blue staining and by (B) periodic acid-Schiff (PAS) staining reaction. Lanes 1, 8, molecular mass standard; lanes 2, 9, A. thermoaerophilus L420-91^T; lanes 3, 10, A. thermoaerophilus DSM 10155/G⁺; lanes 4, 11, P. alvei CCM 2051^T; lanes 5, 12, G. stearothermophilus NRS 2004/3a; lanes 6, 13, G. tepidamans GS5-97^T, lanes 7, 14, T. thermosaccharolyticum E207-71. Amounts of 5 μg (A) and 10 μg (B) were loaded onto the gel.

Figure 4

Genetic organization of slg gene clusters of (A) G. stearothermophilus NRS 2004/3a; (B) G. tepidamans GS5-97^T; (C) A. thermoaerophilus L420-91^T; (D) A. thermoaerophilus DSM 10155/G⁺; and (E) T. thermosaccharolyticum E207-71. The identical color code for the components involved in S-layer glycoprotein glycan biosynthesis is used in Figures 4, 6 and in Table 1: light blue, monosaccharide biosynthesis; red, glycan assembly; orange, glycan transfer; green, export; gray, transposase; black, unknown function; dark blue, elongated glycan chain; pink, linkage glycose; yellow, S-layer protein.

Figure 5

Nucleotide sugar biosynthetic pathways of activated sugars required for S-layer glycoprotein glycan biosyntheses utilizing dTDP-4-dehydro-6-deoxyglucose as key intermediate.

Figure 6

Schematic representation of the proposed biosynthesis route of S-layer glycoproteins, exemplified with G. stearothermophilus NRS 2004/3a. The identical color code for the components involved in S-layer glycoprotein glycan biosynthesis is used in Figures 4, 6 and in Table 1: light blue, monosaccharide biosynthesis; red, glycan assembly; orange, glycan transfer; green, export; gray, transposase; black, unknown function; dark blue, elongated glycan chain; pink, linkage glycose; yellow, S-layer protein.