Adherence ofStaphylococcus epidermidis to pharyngeal epithelial cells mediated by lipoteichoic acid

Prior treatment of pharyngeal epithelial cells (PEC) with lipoteichoic acid (LTA) derived fromStaphylococcus epidermidis produced a marked inhibition of adherence of the homologous strain and two heterologous strains. The inhibition was dose dependent and saturable with 100 µg/ml of LTA. However, pretreatment of PEC with deacylated LTA did not block the adherence of the three strains tested. A similar but less marked blocking effect on the adherence ofS. epidermidis to PEC was also observed with LTAs derived fromS. aureus andStreptococcus pyogenes. On treatment of bacteria with substances capable of binding to LTA, such as polyclonal mouse anti-LTA antibodies or with human albumin, a marked inhibition of bacterial adherence was observed. Immunofluorescence studies showed that anti-LTA antiserum bound readily to the surface of bacterial cells. These findings provide clear evidence that the lipid component of LTA located on the bacterial surface is centrally involved in the adherence ofS. epidermidis to human mucosal cells.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Glycoconjugates as possible receptors forStreptococcus pyogenes

The adherence capacities of M-protein-positive (M+) and M-protein-negative (M-) strains ofStreptococcus pyogenes were compared in human epithelial cells obtained from the pharynx (PEC) or from the buccal mucosa (BEC). Adherence to PEC was related to the presence of M protein (40.5±1.1 M+ and 17.8±0.6 M–S. pyogenes per PEC), whereas BEC showed adherence equally for M+ and M– strains. Different receptor sites may thus be involved on the two cell types. Preincubation of the bacteria with disialogangliosides (1 mg/ml), orN-acetylgalactosamine, ord-galactose (10 g/ml) resulted in diminished adherence of M+ strains to PEC but not to BEC. Chromatography ond-galactose-Sepharose 6B showed specific binding only of M+ group A streptococcal strains to gel beads. M– group A, and groups C and G streptococci did not bind. These observations suggest that the receptors on PEC for group A streptococci are distinct from those on BEC, and that most probably the attachment ofS. pyogenes to human pharyngeal cells occurs by specific, lectin-like binding to galactose residues on epithelial cells.     

Antibodies to phospholipids and liposomes: binding of antibodies to cells

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.     

Toxinogenicity and markers of pathogenicity of Pseudomonas aeruginosa strains isolated from patients with tumor diseases

Potential virulence factors (elastase, proteinase, lipase, phospholipase C, alginate) as well as surface properties (hydrophobicity, motility) were determined in 103Pseudomonas aeruginosa strains isolated from patients with cancer. Nontypable strains were the dominant group (60%), followed by serotypes O11 (17%), O12 (7%) and O4 (5%). Seventy-one strains (69%) produced high level of elastase (10–60 mg/L), 87% of the strains possessed high activity of proteinase (bacterial) (10–250 mg/L) and 69% of the strains demonstrated higher level of lipase (20–150 U/mL); these elevated levels of enzymes were associated mainly with nontypable strains. On the other hand, 79% of the strains did not produce or produced only a low level of phospholipase C and 60% of isolates did not manifest any or very low production of alginate. Hydrophobicity demonstrated by adherence of the bacteria to xylene was shown by 69% of strains; 94% of strains aggregated with ammonium sulfate. Motility in the range of 31–80 mm was found in 76 strains (74%). The considerable virulence of testedP. aeruginosa strains was confirmed. The nontypable strains manifested the most frequent group with high level of elastase, proteinase, lipase, hydrophobicity and motility.     

Comparison of the adherence of three Lactobacillus strains to Caco-2 and Int-407 human intestinal cell lines

F. SAREM, L.O. SAREM-DAMERDJI AND J.P. NICOLAS. 1996. Adhesion of three Lactobacillus strains onto human epithelial intestinal Caco-2 and Int-407 cell lines was compared. More adhesion occurred onto Int-407. The trypsin and sodium periodate pretreatment of bacteria revealed different mechanisms of adhesion depending on the Caco-2 and Int-407, involving carbohydrates and proteins. The absence of adherence for one Lactobacillus strain onto both cell lines indicated the specificity of the adhesion. Electron microscopic observations showed that bacteria adhered by underlying the brush border microvilli of the Caco-2 surface contrasting onto the Int-407 which entrapped and surrounded them by fimbrial extracellular cell matrix material.     

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.     

Propranolol-sensitive and phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets from rat adipocytes

Norepinephrine, epinephrine, and isoproterenol at concentrations of 5.5 x 10(-8) M were found to elicit lipolysis in a cell-free system containing lipid droplets from fat cells and lipase solution. In the cell-free system, the beta-blockers propranolol and dichloroisoproterenol at concentrations of 1 microM inhibited lipolysis induced by norepinephrine, whereas similar concentrations of the alpha-blockers phenoxybenzamine and yohimbine did not inhibit lipolysis. The binding of norepinephrine to endogenous lipid droplets was inhibited by propranolol, but not by phenoxybenzamine. We concluded that the propranolol-sensitive, phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets is involved in lipolysis in fat cells. Treatment of endogenous lipid droplets with phospholipase C, but not phospholipase D, trypsin, chymotrypsin, or neuraminidase, inhibited the propranolol-sensitive binding of norepinephrine to the droplets. These results suggest that the phosphate group of phospholipid in endogenous lipid droplets may be the site of propranolol-sensitive binding of norepinephrine. The physiological significance of the propranolol-sensitive binding is discussed.     

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.     

The effect of the cag pathogenicity island on binding of Helicobacter pylori to gastric epithelial cells and the subsequent induction of apoptosis

BACKGROUND: Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. METHODS: AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. RESULTS: cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. CONCLUSIONS: cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties.     

Binding ability of Clostridium botulinum neurotoxin to the synaptosome upon treatment of various kinds of the enzymes

The binding ability of Cl. botulinum neurotoxin to synaptosomes upon treatment with various enzymes (neuraminidase, trypsin, and β-bungarotoxin containing phospholipase A₂ activity) was studied. When synaptosomes were treated with neuraminidase, their ability to bind toxin decreased; trypsin and β-bungarotoxin had slightly week or no effect. The decrease in toxin-binding ability of synaptosomes was paralleled by a release of sialic acid from the synaptosomes by the neuraminidase treatment. The toxin-binding ability of synaptosomes treated with neuraminidase was lower than untreated ones at a high concentration of sodium chloride. The binding of the toxin to synaptosomes occurred at least at the two types of structural sites, one site which contained sialic acid, and other site which was sensitive to high ionic strength. It may be possible that another binding state except these is present at the synapse.     

Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Ability of various oral bacteria to bind human plasma fibronectin

The present study describes the ability of various oral bacteria to bind human plasma fibronectin (PFN). Avid binding of 125I-PFN was found for Streptococcus mutans (serotypes a to h), Streptococcus sanguis, group A Streptococcus pyogenes and Staphylococcus aureus, while other gram-positive bacterial species tested demonstrated only weak or negligible PFN binding ability. Two gram-negative bacterial species, Bacteroides gingivalis and Escherichia coli, did not significantly bind PFN. 125I-PFN binding to S. mutans 6715 cells was decreased by pretreatment with unlabeled PFN, and the radiolabeled PFN bound to the cell surface was released on addition of unlabeled PFN. Strong inhibition of 125I-PFN binding to S. mutans 6715 cells was obtained by protease pretreatment, while partial inhibition was also observed following treatment with acid, alkali, lipase, and monoclonal anti-polyglycerophosphate. These results suggest that PFN binding to S. mutans cells is reversible and that PFN receptors on the cell surface appear to be heat-stable multiple proteins.     

Recognitory bacterial surface lectins which mediate its mannose-specific adherence to eukaryotic cells

Cell surface protein were found to play a role in the sugar-specific molecular mechanism by which bacteria adhere to mammalian cells. We have demonstrated that at least three different types of lectin-like proteins mediate the mannose-sensitive adherence of gram negative bacteria to epithelial cells. One group of such lectins was shown in our study to be associated with the bacterial flagellum. Flagella isolated from Escherichia coli 7343 and Serratia marcescens 8347 exhibited mannose-sensitive agglutination of yeast cells; however, the flagella of the two bacteria differ in the molecular structure of their protein subunits. Another class of lectins comprises the bacterial fimbriae (also known as type 1 pili), which were previously shown to facilitate the mannose-sensitive adherence of various bacteria to mammalian cells. Fimbriae isolated from E. coli 346 were reversibly dissociated by saturated guanidine hydrochloride to their protein subunits. The dissociated subunits retained in part their mannose-binding ability, and were reassembled into fimbriae-like structures by removal of the denaturant under specific conditions. Mannose-sensitive yeast agglutinating activity of E. coli 2699, as well as of its isolated outer membranes devoid of fimbriae or flagella, was abolished by pretreatment with trypsin. It is therefore believed that the mannose-sensitive adherence of these bacteria is mediated also by lectin-like proteins associated directly with the outer membrane.     

Lectin binding by eosinophils

Lectins which identify carbohydrates and glycoproteins have been used to characterize specific components of the surface of guinea pig peritoneal exudate eosinophils. Agglutination of eosinophils purified by discontinuous metrizamide gradients was scored microscopically. Wheat germ agglutinin (WGA) was most effective (0.05 micrograms/ml). However, higher concentrations of soybean lectin and concanavalin A (Con A) were also effective. No differences in lectin binding were noted between eosinophils harvested from uninfected animals, Trichinella spiralis-infected animals, or animals receiving weekly intraperitoneal injections of polymyxin B. Neuraminidase pretreatment to remove surface sialic acid reduced agglutination by WGA. Eosinophils did not adhere to WGA-coated Sepharose beads; however, they did adhere to Con A-coated beads. Pretreatment with neuraminidase did not affect the adherence of eosinophils to plastic surfaces, suggesting that sialic acid does not play an important role in adherence. Formation of lectin-inhibitor complexes within the incubation mixture complicated interpretation of studies of binding to plastic surfaces. These studies demonstrate that lectin binding sites are present on the surface of eosinophils. Lectin-type binding may be important in interactions between eosinophils and noningestible parasites.     

Effect of trace elements on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells

Trace elements have significant effect on the physiology of bacteria. Variation in the concentration of trace elements may affect the expression of virulence by microorganisms. The effect of trace elements on hydrophobicity and adherence of E.coli to uroepithelial cells was studied. Increasing concentrations of Ca2+, Mg2+, Fe3+ and Zn2+ significantly decreased the surface hydrophobicity. Toxic trace elements like Co2+, Cu2+, Mn2+ and Ni2+ did not alter surface hydrophobicity. With regards to adherence of E.coli to uroepithelial cells, only Mg2+ had significant effect. Toxic trace elements decreased the rate of cell adherence. The pathogenic strains of E.coli showed higher surface hydrophobicity and better cell adherence compared to the nonpathogenic strains. There was good correlation between surface hydrophobicity and cell adherence at higher concentrations (0.1 to 0.2mM) of Fe2+ and Zn2+. The results indicated that trace elements can significantly affect surface hydrophobicity and adherence of E.coli to uroepithelial cells. Such effect may have a significant impact on the initial stages of bacterial infection.