The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions ([6][1]).

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S-Layer Proteins

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archeaobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.

Crystalline bacterial cell-surface layers.

The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kerneis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors involved in adherence of lactobacilli to human Caco-2 cells

Summary: In a previous study, electron microscopic examinations of thin sections of Lactobacillus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identified as a layer of a non-glycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (S-layer) lattice (a = 4·5 nm, b = 9·6 nm, γ = 77 °) which completely covers the cell surface. Treatment with 5 m-LiCl extracted the S-layer protein from intact cells efficiently and selectively. Viability did not decrease significantly. Moreover, the S-layer reappeared when treated cells were allowed to grow again. In vitro self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in vivo. The purified S-layer protein had a high content (44%) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid.

S-layer of Lactobacillus helveticus ATCC 12046: isolation, chemical characterization and re-formation after extraction with lithium chloride

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside. Images

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Bacillus stearothermophilus PV72 carries a crystalline surface layer (S-layer) with hexagonal symmetry (Sleytr and Messner 1983). Sites of insertion of newly synthesized subunits into the regular lattice during cell growth have been determined using the indirect fluorescent antibody technique (Hughes and Stokes 1971) and the protein A/colloidal gold marker system (Muhlpfort 1982, Smit and Agabian 1982). In both systems, cells from a logarithmic growing culture have been incubated with anti-S-layer serum in order to completely cover the cells and then reincubated in fresh medium. Samples were extracted at intervals and reacted with fluoresceinisothiocyanate conjugated goat-anti-rabbit-IgG or protein A/colloidal gold conjugate. S-layer material incorporated into the lattice after reincubation had no IgG attached to it and therefore did not bind the marker.

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains.

Various aspects of surface properties of the S-layer-carrying Bacillus stearothermophilus PV72 and of an S-layer-deficient mutant (strain PV72/T5) have been tested by adsorption assays on solid surfaces, electrostatic interaction chromatography and hydrophobic interaction chromatography. The adsorption assays have shown that cell adhesion of the S-layer-carrying strain was less influenced by environmental changes than it was with the S-layer-deficient mutant. Electrostatic interaction chromatography indicated that both strains have positively and negatively charged groups exposed on the cell surface but the S-layer-carrying strain reveals more positively charged groups than does the S-layer-deficient mutant. Hydrophobic interaction chromatography showed that both strains have a hydrophilic surface but that the hydrophilic properties are more pronounced with the strain lacking an S-layer.

Influence of an S-layer on surface properties of Bacillus stearothermophilus.

Dietary spermidine is emerging as a substance with promising health-promoting and healthy ageing-promoting properties. Its autophagy-inducing potential has been especially well-documented. This process is suspected to be at the root of the life-prolonging effects of fasting and in-vivo spermidine supplementation. In humans, high dietary spermidine intake is associated with a lifespan increase of 5.7 years. Numerous studies provide indications of its effects on the cardiovascular, nervous, and immune systems as well as on the formation and development of tumors. Nevertheless, no health claims are currently authorized in the EU for spermidine or spermidine-rich functional foods. When ingested through food or dietary supplements, dosage appears to play an important role in replicating the favorable effects. Currently, there are ongoing proceedings regarding the novelty of a certain manufacturing process of spermidine supplements, but many other plant extracts with high spermidine content from different sources are marketed without the need for novel food authorization.  This article discusses the varying test results obtained for the spermidine concentration of popular spermidine supplements. Soybean extract seems to be slightly superior in terms of spermidine concentration, but one wheat germ-based product stood out in this analysis due to a 10 times higher spermidine concentration than comparable products.Keywords: spermidine, food supplements, novel food, botanicals, functional foods, autophagy, spermidine-rich wheat germ extract 

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Karin Gruber

Papers

S-layer of Lactobacillus helveticus ATCC 12046: isolation, chemical characterization and re-formation after extraction with lithium chloride

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains.

Influence of an S-layer on surface properties of Bacillus stearothermophilus.

Spermidine content of selected dietary supplements: potential for improvement?