Bacterial cell walls and membranes. Discovery of the teichoic acids.

Teichoic acids are major wall components of most Gram-positive bacteria. Their discovery followed that of their nucleotide precursors. Lipoteichoic acids associated with the cell membrane were discovered at the same time. Events leading to these discoveries and the probable function of teichoic acids in cation control are described.

Adherence of Streptococcus agalactiae to synchronously growing human cell monolayers without lipoteichoic acid involvement.

Freshly isolated virulent and nonvirulent strains of Streptococcus agalactiae type III were used to study differences in coccal adherence to synchronously dividing, subconfluent human embryonic amnion and fetal lung monolayers in vitro. The adherence frequency by virulent isolates of mid-logarithmically growing cocci to amnion cells varied markedly with host cell age, being highest shortly after eucaryotic cell division. This variation was not observed with lung cell monolayers, suggesting that cyclic production or exposure of coccal receptor sites on the eucaryotic cell surface with age is not a common property of all primary human cells in vitro. However, and regardless of age, not all cells within these synchronously dividing populations bound virulent cocci, indicating that a very small segment of a population may always be unresponsive to host cell interactions with a coccal pathogen. By comparison, adherence of nonvirulent coccal isolates to amnion and lung cells remained constant and of a very low order, regardless of host cell age. Maximal adherence of virulent S. agalactiae to young host cells occurred at early and mid-logarithmic phases of growth. However, at the late stationary growth phase, adherence was reduced to almost that of nonvirulent isolates. Pretreatment of virulent S. agalactiae with anti-lipoteichoic acid (LTA) serum failed to inhibit coccal adherence to these different host cells. Heat negated adherence. Group B coccal LTA was cytotoxic for these host cells. However, pretreatment of amnion and lung cells with nontoxic levels of this amphiphile did not prevent attachment of virulent cocci. Finally, coccal pretreatment with pronase abrogated adherence to either host cell even though surface-exposed LTA was uneffected, as observed by the indirect fluorescent-antibody procedure. Likewise, no observable difference in surface LTA was detected when fresh isolates of virulent and nonvirulent coccal strains were compared by this procedure. These studies suggest that protein involvement, rather than LTA, is primarily responsible for mediating virulent S. agalactiae type III attachment to these synchronously growing, subconfluent eucaryotic monolayers in vitro.

Adherence of group B streptococci to adult and neonatal epithelial cells mediated by lipoteichoic acid.

We have investigated the role of lipoteichoic acid in mediating the adherence of different serotypes of group B streptococci to human adult and neonatal epithelial cells. Pretreatment of neonatal buccal and vaginal epithelial cells with lipoteichoic acid, but not with deacylated lipoteichoic acid, induced a marked inhibition in the adherence of all strains tested. Pretreatment of bacteria with substances known to bind lipoteichoic acid, such as monoclonal and polyclonal antipolyglycerophosphate antibodies and albumin, also resulted in adherence inhibition. Group B streptococci adhered in 6- to 10-fold-higher numbers to buccal epithelial cells from neonates older than 3 days than to those from neonates less than 1 day old. This increase in receptiveness for group B streptococci was paralleled by an increased ability of epithelial cells from older neonates to bind group B streptococcal lipoteichoic acid. These data suggest a role for the lipid portion of lipoteichoic acid in the adherence of different serotypes of group B streptococci to vaginal and neonatal epithelial cells.

Lipoteichoic acid from Bacillus subtilis subsp. niger WM: isolation and effects on cell wall autolysis and turnover.

Lipoteichoic acid (LTA) was extracted by means of hot aqueous phenol from Bacillus subtilis subsp. niger WM cells grown under various conditions in chemostat culture. The extracts were partially purified by nuclease treatment and gel permeation chromatography. Chemical analyses revealed a composition consistent with a polyglycerol phosphate polymer. The influence on autolysis of the LTAs thus obtained was studied with both whole cells and autolysin-containing native walls of B. subtilis subsp. niger WM. Lysis rates of phosphate-limited cells could be reduced to about 40% of the control rate by the addition of LTA, whereas lysis of cells grown under phosphate-sufficient conditions was affected to a much lesser extent. The lysis of native walls prepared from variously grown cells proved to be fairly insensitive to the addition of LTA. The effect of LTA on wall turnover was studied by following the release of radioactively labeled wall material during exponential growth. The most obvious effect of LTA was a lowered first-order rate of release of labeled wall material; calculations according to the model for cell wall turnover in Bacillus spp. formulated by De Boer et al. (W. R. De Boer, F. J. Kruyssen, and J. T. M. Wouters, J. Bacteriol. 145:50-60, 1981) revealed changes in wall geometry and not in turnover rate in the presence of LTA.

Mediation of Staphylococcus saprophyticus adherence to uroepithelial cells by lipoteichoic acid.

Treatment of uroepithelial cells with lipoteichoic acid from Staphylococcus saprophyticus resulted in a decrease in the adherence of this organism. Similar effects were observed when bacteria were pretreated with the lipoteichoic acid ligands albumin and anti-polyglycerophosphate monoclonal antibodies. Lipoteichoic acid might behave as an adhesin of S. saprophyticus.

Relationship of cell surface morphology and composition of Streptococcus salivarius K+ to adherence and hydrophobicity.

The cell surfaces of a range of variants of Streptococcus salivarius HB, altered in cell wall antigen composition, were compared with those of the parent with respect to adherence, ability to adsorb to hexadecane, morphology, and exposure of lipoteichoic acid (LTA). Adherence to host surfaces was measured by using both saliva-coated hydroxyapatite beads and tissue-cultured HeLa cells, and interbacterial adherence was measured by using Veillonella alcalescens V1 cells. Progressive loss of the protease-sensitive fibril classes was generally associated with decreasing ability to adsorb to hexadecane. However, increased exposure of protein antigen C (AgC) increased the apparent hydrophobicity of the cell. This correlated with the finding that AgC was the most hydrophobic of the solubilized fibrillar cell wall antigens. Collectively, this demonstrates that adsorption to hydrophobic ligands is directly related to the density of the fibrillar layer on the cells and the properties and surface exposure of specific fibril classes. The involvement of hydrophobic interactions in AgC-associated attachment was suggested by its sensitivity to low levels of the hydrophobic bond-breaking agent tetramethyl urea, although the reduction was not to the level of adherence observed with strains lacking AgC. However, hydrophobicity was less essential to other adherence reactions. Circumstantial evidence, including immunoelectron microscopy, showing that LTA was virtually absent from the fibrillar layer, whole-cell enzyme-linked immunosorbent assay, suggesting that surface exposure of LTA related inversely to the density of the fibrillar layer, and agarose gel electrophoresis, showing that LTA was not specifically associated with protein fibrillar antigens, strongly suggested that LTA does not confer hydrophobic properties to these cells and is not involved in adherence reactions associated with the cell wall protein antigens.

Cell surface hydrophobicity of group D and viridans streptococci isolated from patients with septicaemia.

Sixty-three strains of Group D streptococci and viridans streptococci isolated from blood cultures during a two year period were typed to the species level with conventional biochemical tests and API Strep. Streptococcus faecalis was the most common species isolated followed by S. sanguis, S. mitis and S. constellatus (S. milleri). One of the two isolates of S. faecium was a contamination. The reported increasing frequency of this organism and other Group D and viridans streptococci as well as the association of S. bovis with malignant bowel disease indicate the need for full identification of streptococcal isolates from blood cultures. Pronounced surface hydrophobicity as measured with the salt aggregation test (SAT) was expressed by 59/63 (94%) of the blood culture isolates whereas strains isolated from commercial fermentation products and strains passaged several times were hydrophilic. In the presence of human serum albumin which binds to lipoteichoic acid only one strain decreased in surface hydrophobicity. The surface hydrophobicity of two strains even slightly increased indicating that lipoteichoic acid but marginally contributes to surface hydrophobicity of streptococcal cells from these species.

Critical micelle concentrations of lipoteichoic acids.

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methods involving spectral changes of an added merocyanine dye probe, to have critical micelle concentrations in the range of 1 to 10 micrograms/ml, suggesting that acylated LTAs in their monomer forms may represent the major configuration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained did not differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findings in relation to the biological properties of LTA is discussed.

Staphylococcus aureus adherence to influenza A virus-infected and control cell cultures: evidence for multiple adhesins.

During major epidemics with influenza, there is an increased number of pneumonias due to Staphylococcus aureus with a subsequent high mortality rate. We have postulated that influenza A virus infection of host cells promotes the adherence of S. aureus ultimately resulting in bacterial superinfection. In the present study we compared the adherence of seven strains of 3H-labeled S. aureus to Madin-Darby canine kidney (MDCK) cell monolayers, uninfected and infected with influenza A/FM/1/47 virus. Test strains included: Cowan I; a Cowan I protein A-deficient mutant (PA-); EMS, a protein A and clumping factor-deficient mutant; HSmR; 52A5, a teichoic acid-deficient mutant of HSmR; M, an encapsulated strain; and, No. 1071, a clinical isolate. By radioassay, six of the seven strains demonstrated significantly enhanced adherence to virus-infected cell monolayers compared to uninfected controls; only the M strain was adherence negative. Surface hydrophobicity of the staphylococci did not correlate with their ability to adhere. Four strains of labeled staphylococci (Cowan I, PA-, EMS, and No. 1071), untreated or treated with 2.5% trypsin, 1.25% protease, or by autoclaving, were tested in the radioassay. Protease treatment, which was more effective than trypsin treatment, reduced adherence of all four test strains by 74-96%. Results of heat treatment suggested the presence of both thermolabile and thermostable adhesins. Staphylococcal thermal extracts, profiled by anion-exchange HPLC, were used to pretreat monolayers in a blocking radioassay. Adherence was decreased to control cells (9-78%) and to virus-infected cells (56-90%). The data suggest that multiple distinct surface proteins mediate the binding of S. aureus to uninfected and influenza A virus-infected cells.

Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci.

The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype III strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA through competitive interactions; however, 10-fold greater levels of the same material were required to displace virulent [3H]LTA from HEC and HFC surfaces. These observed differences in binding avidity were shown to be due to longer LTA chains (30 to 35 glycerolphosphate units) in virulent strains when compared with LTA chains (10 to 12 glycerolphosphate units) of asymptomatic and avirulent strains. Thus, LTA appears to enhance the ability of virulent group B streptococci to bind to HEC and HFC with stronger avidity by virtue of the increased length of the cell-associated polymers synthesized by these strains. Mild enzymatic treatment of HEC and HFC with trypsin or periodate abolished LTA binding, which suggests the presence of a certain glycoprotein receptor(s) for LTA which does not appear to be present on human adult buccal epithelial cells. These data may therefore partially explain the increased susceptibility of newborn infants to group B streptococcal infections.

Role of sucrose in plaque formation.

Results are presented which support the concept that the bacterial enzyme glucosyltransferase (GTF) plays a crucial role in sucrose induced plaque formation. GTF was shown to adhere strongly to anionic, hydrophobic and polysaccharide solid materials, and to be able to produce glucans in the adsorbed state. It appears conceivable that GTF adsorb to teeth and produce glucans. Glucan chains on the surface of the bacteria and glucans on the tooth surfaces interact (pack) and form a strong binding mechanism. The rigid alpha 1,3 linked glucans produced by Streptococcus mutans are particularly suited for interaction of this kind. This mechanism could account for sucrose-induced binding of bacteria to enamel, pellicle covered enamel and preformed plaque. S. mutans would adhere particularly strongly to tooth surfaces in the presence of sucrose, according to this model.

The role of lipoteichoic acids on the adherence of Streptococcus equi to epithelial cells.

The effect of the presence of lipoteichoic acids (LTA) on a strain of Streptococcus equi was investigated. The LTA were extracted in a crude form from the S. equi strain and were found to sensitize sheep red blood cells so that they agglutinated with antibodies specific to purified LTA of group A streptococci. The crude LTA preparation was also able to inhibit the specific haemagglutination reaction involving group A streptococcal LTA and LTA antibodies. Neither the purified LTA from group A streptococci nor the anti-LTA serum interfered with the adherence of S. equi to equine epithelial cells.

Mediation of staphylococcal adherence to mucosal cells by lipoteichoic acid.

Staphylococcal lipoteichoic acid markedly reduced adherence by Staphylococcus aureus to buccal cells in vitro, suggesting that lipoteichoic acid mediates adherence by that bacterium. Adherence inhibition by lipoteichoic acid was lost after deacylation of the preparation, suggesting that fatty acids on the molecule are essential to binding.

Hydrophobic interactions of group A streptococci with hexadecane droplets.

The adherence of Streptococcus pyogenes cells to hexadecane droplets was measured by vortexing water suspensions of streptococci with hexadecane. It was found that adherence of the organisms to hexadecane droplets was abolished by pretreating the organisms with trypsin, pepsin at pH 4.5, or HCl solutions at 95 degrees C. Streptococcal adherence was best expressed in organisms harvested during the stationary phase of growth and was inhibited by fatty acid-free albumin because of the interaction of the protein with the streptococcal surfaces. The data suggest that adherence to hexadecane droplets measures the availability on the surface of S. pyogenes cells of lipophilic residues that are either hydrophobic regions of surface protein structures or, more likely, glycolipids complexed with and oriented by surface proteins.

Lipoteichoic acid is the major cell wall component responsible for surface hydrophobicity of group A streptococci.

The contribution of lipoteichoic acid (LTA) to the hydrophobic surface properties of group A streptococci was investigated in aqueous dextran-polyethylene glycol two-phase systems. Enzymatic digestions were performed to characterize the hydrophobic surface structure. The results obtained indicated that LTA is a major factor responsible for the hydrophobic character of the cell surface of group A streptococci. This was further supported by the similarity of partition in polymer two-phase systems between whole group A streptococci and tritiated LTA extracted from a group A streptococcal strain. Surface LTA was also determined on intact organisms by a new method measuring the adsorption of antibodies to LTA to the bacterial surface. A correlation was found between the content of surface LTA and the hydrophobicity of the group A streptococci. We conclude that surface-associated LTA is the major factor determining surface hydrophobicity of group A streptococci.

[The adherence of bacteria to mucosal surfaces (author's transl)]. / Die Adhärenz von Bakterien an Schleimhautoberflächen.

A review on the significance of the bacterial adherence of the host parasite-relationship. Special attention is drawn to the following topics: bacterial adhesion as a principle of pathogenicity, the specificity of the reaction, the nature of adhesion-receptors. Furthermore, the non specific mucosal defense and the possibilities of preventative measures are discussed.

The adherence of group A streptococci to oropharyngeal cells: the lipoteichoic acid adhesin and fibronectin receptor.

The attachment of group A streptococci to oropharyngeal epithelial cells is mediated by adhesive molecules (adhesins) on the surfaces of the micro-organisms that interact with receptor molecules on the epithelial cells. The evidence that the adhesin is composed of lipoteichoic acid (LTA) complexed with bacterial cell surface proteins is as follows: (a) Among the purified cell wall substances tested, only LTA was able to inhibit attachment; (b) treatment of streptococci with anti LTA but not with antibody against other surface substances blocks attachment; (c) LTa forms complexes with purified M protein, the most abundant protein on the surface of virulent streptococci; (d) the lipid moieties of LTA, which mediate attachment, remain free in the M protein-LTA complexes to interact with receptor analogues, such as serum albumin. The evidence that the receptor for the LTA mediated binding of streptococci resides in fibronectin molecules on oropharyngeal cells is as follows: (a) the addition ot adhesion test mixtures of fibronection inhibits binding; (b) the number of streptococci capable of attaching is directly proportional to the amount of fibronectin present on epithelial cells; (c) purified fibronectin immobilized on latex beads agglutinates suspensions of streptococci; (d) radiolabeled fibronectin binds to group A streptococci; (e) both the agglutination of fibronectin-beads and the binding of fibronectin to streptococci is blocked by LTA, the streptococcal adhesin.