The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.     

Organization of mature external glands on the trap and other organs of the bladderwortUtricularia monanthos

Summary The mature dome-shaped glands which cover the outer surfaces of the trap, leaves, anchor and runner stolons inU. monanthos are described using conventional and some high voltage transmission electron microscopy. The glands occur as scattered ordinary external glands and as a compact clump of vestibule glands at the entrance to the doorway. Each gland rests on a basal epidermal cell and consists of a single pedestal and terminal cell. Vestibule and leaf glands differ slightly from the other glands mainly in the structure of the outer wall of the terminal cell. Nuclear crystals are prominent and the cytoplasm of the pedestal and terminal cells contains tubular structures usually aggregated near the nucleus. The pedestal cell is a transfer cell with short wall protuberances on the outer wall, conspicuous mitochondria and a heavily impregnated lateral wall.The terminal cell often has an outer wall that is greatly thickened and a protoplast that may degenerate early. In the most developed cells the protoplast remains active for a long period and the outer wall is differentiated into several layers. The outermost layer is cuticularized consisting of an open meshwork of deposits. In leaf glands a local polysaccharide mass is usually developed within the cuticularized region. The inner non-impregnated region of the outer wall may show four layers. In vestibule glands fewer layers are present and the wall shows prominent lamellations. Some ordinary external glands differentiate a sponge-like substructure within the inner wall.The ultrastructure and function of the glands are discussed. We support the concept that mature external glands are responsible for secreting water, with those on traps being particularly active during the resetting of the organ. Our work provides a structural basis for recent suggestions by other workers that the mechanism of secretion probably involves establishing a standing osmotic gradient within the gland.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

Production of Slime Polysaccharides by Shigella dysenteriae Type 1

Electron microscopy of ruthenium red-stained ultrathin section of strains of Shigella dysenteriae type 1 grown in the Casamino Acids-yeast extract broth medium showed the presence of an extracellular slime layer. The slime appeared as a dense sheath covering bacteria. The presence of slime promoted hemagglutinating activity of the bacteria. The slime polysaccharide (SPS) isolated from the cell-free culture supernatant or the bacterial surface was less than 162,000 daltons in size and immunochemically similar. The SPS showed cross-reaction with lipopolysaccharide (LPS) antigen in immunological tests; however, it also appeared to be different from LPS since it did not contain 2-keto-3-deoxyoctonate, a core sugar of LPS. A different pattern of separation from LPS was also observed by silver staining of SDS-polyacrylamide gels. From these data it appeared that either LPS and SPS are contaminated with each other or that SPS is the polysaccharide portion of LPS.     

Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure

The role of the outer membrane and lipopolysaccharide (LPS) in the interaction between the small cationic antimicrobial peptide magainin 2 and the Gram-negative cell envelope was studied by FT-IR spectroscopy. Magainin 2 alters the thermotropic properties of the outer membrane-peptidoglycan complexes from wild-type Salmonella typhimurium and a series of LPS mutants which display differential susceptibility to the bactericidal activity of cationic antibiotics. These results are correlated with the LPS phosphorylation pattern and charge (characterized by high-resolution 31P NMR) and outer membrane lipid composition, and are compared to the bactericidal susceptibility. LPS mutants show a progressive loss of resistance to killing by magainin 2 as the length of the LPS polysaccharide moiety decreases. Disordering of the outer membrane lipid fatty acyl chains by magainin 2, however, depends primarily upon the magnitude of LPS charge rather than the length of the LPS polysaccharide, contradicting the proposal by Weiss et al. [Weiss, J., Beckerdite-Quagiata, S., & Elsbach, P. (1980) J. Clin. Invest. 65, 619-628] that the sugar side chain of LPS shields the negative charges of the outer membrane surface. While disruption of outer membrane structure most likely is not the primary factor leading to cell death, the susceptibility of Gram-negative cells to magainin 2 is associated with factors that facilitate the transport of the peptide across the outer membrane, such as the magnitude and location of LPS charge, the concentration of LPS in the outer membrane, outer membrane molecular architecture, and the presence or absence of the O-antigen side chain.     

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag⁺/Cl^? colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔH_ITC upon interacting with free Ag⁺ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag⁺ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag⁺/Cl^? colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag⁺/Cl^? colloids. Biomineralization of Ag⁺/Cl^? colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

A combined FT-IR microscopy and principal component analysis on softwood cell walls

A combined FT-IR microscopy and principle component analysis was used to investigate chemical variations between softwood species as well as types of wood cell walls; latewood tracheids, earlywood tracheids and earlywood ray parenchyma cells. The method allowed us to detect small spectral differences between cell types rather than species and to predict characteristic chemical components of each cell type. The method enabled information to be obtained which allowed a evaluation of the polysaccharide composition even in lignified woody plant cell walls.     

Adhesion of Desulfovibrio desulfuricans and Pseudomonas fluorescens to mild steel surfaces

The adhesion of micro-organisms to metal surfaces has been shown to be important in the corrosion process, but the cell surface structures participating in this adhesion have not previously been identified. Evidence is presented that a bacterial substance taking part in the initial adhesion of Pseudomonas fluorescens and Desulfovibrio desulfuricans (New Jersey) to mild steel is polysaccharide in nature. It is likely that this is present in the outer membrane of the bacterial cells as lipopolysaccharide.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.     

Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.

Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.     

A genetic approach for analysing surface-exposed regions of the OmpC protein of Escherichia coli K-12

Phage attachment sites on bacterial cell surfaces are provided by the exposed regions of outer membrane proteins and lipopolysaccharide (LPS). We have identified surface exposed residues of OmpC that are important for phage binding. This was accomplished by employing a genetic scheme in which two simultaneous selections enriched for ompC mutants defective in phage attachment, but retained functional channels. Mutational alterations were clustered in three regions of the OmpC protein. These regions also showed the greatest divergence from the analogous regions of the highly related OmpF and PhoE proteins. The majority of alterations (8 out of 11) occurred in a region of OmpC that is predicted to form a large exterior loop (loop 4). Interestingly, while the removal of this loop prevented phage binding, the deletion conferred enhanced channel activities.   Another type of phage-resistant mutants synthesized defective LPS molecules. Biochemical analysis of mutant LPS revealed it to be of the Re-type LPS, lacking the heptose moieties from the LPS inner core. As a result of this LPS defect, many outer membrane proteins were present in somewhat reduced levels. The phage resistance seen in these mutants could be a result of both the presence of defective LPS and reduced OmpC levels.     

Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa

The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results demonstrated that the rhamnolipid at concentrations below and above CMC provoked a multi-component response of the bacterial cells without affecting their growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F, Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa. In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine.     

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell^{1, 2}. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli^3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence^6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods⁹. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann¹⁰, is ideal for LPS extraction.     

Cell wall ultrastructure of flocculent and non-flocculent Schizosaccharomyces pombe strains. Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure

Scanning and transmission electron microscopic studies revealed the presence of slime-like, amorphous material on the surface of Schizosaccahromyces pombe RIVE 4-2-1 cells, independently, whether they were in flocculated or in non-flocculated state. Close contact of the adjacent cells via the merging outermost cell wall layers was found, however, only in the case of floc formation, which was induced by cultivating the cells in the presence of 6% (v/v) ethanol. Irreversible loss of the flocculation ability of the cells by treatment with proteinases suggests that proteinaceous cell surface molecules as lectins contribute to the cell-to-cell interaction during flocculation. Both proteinase K and pronase treatments removed a distinct outer layer of the cell wall, which indicated that the protein moieties of the phosphogalactomannan outer surface layer has a crucial role in the maintenance of cell wall integrity. In the case of lysing enzyme treatment the removal of the outermost layer was also observed as the first step of the cell wall digestion, while driselase treatment resulted in almost complete digestion of the cell wall.     

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.     

An electron microscopic study of surface polysaccharides in Bacteroides

The electron microscopic appearance of the cell surface of Bacteroides strains and Klebsiella pneumoniae stained with ruthenium red or colloidal iron is described. The effect of polymyxin B (PMB) was also registered. It was found that all Bacteroides strains have a polysaccharide lined 'micro-capsule' external to the outer membrane which could aggregate and form blebs. The blebs so formed were distinct from other types of bleb formed in Klebsiella involving the outer membrane and induced by PMB. Such types of PMB alterations were not induced in Bacteroides.     

Epithelial cell contact-induced alterations in Salmonella enterica serovar Typhi lipopolysaccharide are critical for bacterial internalization

The invasion of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells depends on the cystic fibrosis transmembrane conductance regulator (CFTR) protein as an epithelial receptor. In the case of P. aeruginosa , the bacterial ligand for CFTR is the outer core oligosaccharide portion of the lipopolysaccharide (LPS). To determine whether serovar Typhi LPS is also a bacterial ligand mediating internalization, we used both P. aeruginosa and serovar Typhi LPS as a competitive inhibitor of serovar Typhi invasion into the epithelial cell line T84. P. aeruginosa LPS containing a complete core efficiently inhibited serovar Typhi invasion. However, neither killed wild-type Typhi cells nor purified LPS were effective inhibitors. LPS from mutant Typhi strains defective in O side-chain synthesis, but with an apparently normal core, was capable of inhibiting invasion, but LPS obtained from a deeper rough mutant strain with alterations in fast-migrating core oligosaccharide failed to inhibit invasion. Lastly, exposure of wild-type serovar Typhi to T84 cultures before heat killing resulted in a structural alteration in its LPS that allowed the heat-killed cells to inhibit invasion of wild-type serovar Typhi. These data indicate that the serovar Typhi LPS core, like the P. aeruginosa LPS core, is a ligand mediating internalization of bacteria by epithelial cells, and that exposure of this ligand on wild-type Typhi is induced by the bacteria's interaction with host cells.