A preliminary investigation on the chemical composition of the cell surface of five enteropathogenic Escherichia coli serotypes.

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100 degree C for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.     

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.     

Characterization and mapping of a major Na+/H+ antiporter gene of Escherichia coli.

Using in vivo assays, we show that the Na+/H+ antiporter activity of the Escherichia coli mutant HIT-1 is reduced dramatically compared with activity in wild-type cells. An isogenic nhaA (formerly antA) deletion strain, however, is not significantly different from wild type in this respect. We call the locus affecting Na+/H+ antiporter activity of the HIT-1 mutant nhaB. The nhaB activity exhibits no pH dependence in the range between 7.0 and 8.5, whereas that of the nhaA gene increases considerably at pH levels above 8.0. Mutants with defects in nhaB grow normally on agar media containing 0.5 M NaCl, but nhaA mutants are sensitive to 0.5 M NaCl. We have mapped the nhaB mutation of HIT-1 to 25.6 min on the E. coli map. It is unlinked to the nhaA region, which is located at about 0.5 min. Since a cell with a mutation in nhaB alone is essentially Na+/H+ antiporter negative up to pH 8.0, we conclude that nhaB is required for the major Na+/H+ antiporter activity in the usual physiological pH range.     

Heterogeneity of human monocytes: differences in response to IFN-gamma

Human peripheral blood monocytes can be separated into two subpopulations which differ in the efficiency of their adherence to glass after 16 hours of incubation. The adherent subpopulation was found to be about twice as effective in binding mannose-resistant E. coli 0-124, mannose-sensitive E. coli 0-128 and opsonised E. coli than the nonadherent one. In addition, reduction of cytochrome C in response to E. coli binding or 12-myristate 13-acetate (PMA) stimulation was two fold higher in adherent cells. The binding of E. coli O-124 and the superoxide generation stimulated by E. coli were inhibited by the addition of mannose only in the adherent monocytes, indicating the presence of mannose receptors on the cell surface in the adherent subpopulation. The treatment of the nonadherent cells with 0.1-1000 U/ml of Interferon (IFN-gamma) for 24 hours resulted in a dose dependent increase in superoxide generation. After 72 hours of incubation with IFN-gamma (1000 U/ml) the amount of superoxide generated by the nonadherent cells was elevated to 20.5 +/- 1.4 nmoles/10(6) cells/15 min, similar to that of the adherent cells (24.5 +/- 1.2 nmoles/10(6) cells/15 min untreated adherent monocytes). The generation of superoxide in the IFN-gamma treated nonadherent monocytes stimulated by E. coli 0-128 was significantly reduced by addition of mannose.     

Proton stoichiometry in the reduction of the FAD and disulfide of Escherichia coli thioredoxin reductase. Evidence for a base at the active site

The oxidation-reduction midpoint potentials, Em, of the FAD and active site disulfide couples of Escherichia coli thioredoxin reductase have been determined from pH 5.5 to 8.5. The FAD and disulfide couples have similar Em values and thus a linked equilibrium of four microscopic enzyme oxidation-reduction states exists. The binding of phenylmercuric acetate to one enzyme form could be monitored which allowed solving the four microscopic Em values. The Em values at pH 7.0 and 12 degrees C of the four couples of thioredoxin reductase are: (S)2-enzyme-FAD/FADH2 = -0.243 V, (SH)2-enzyme-FAD/FADH2 = -0.260 V, (FAD)-enzyme-(S)2/(SH)2 = -0.254 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.271 V. Thus, at pH 7.0, the FAD and disulfide moieties have a 0.017-V negative interaction and Em values which are different by 0.011 V. The delta Em/delta pH of the FAD couples E2m and E3m are about 0.060 V/pH throughout the pH range studied, showing an approximately 2-proton stoichiometry of reduction of the enzyme FAD. The delta Em/delta pH of the disulfide couples E1m and E4m are about 0.052 V/pH from pH 5.5 to 8.5, showing an apparently nonintegral proton stoichiometry of reduction of 1.8 in this pH range. This proton stoichiometry suggests the presence of a base with an ionization behavior that is linked to the oxidation-reduction state of the disulfide. A novel method is presented for determining the pK values on oxidized and reduced enzyme which agrees with the less accurate classical method. The proton stoichiometry results are consistent with the presence of a thiol-base ion pair in which the pK of the base is elevated from 7.6 in disulfide containing enzyme to greater than 8.5 upon forming an ion pair with a thiol anion of pK 7.0 generated upon reduction of the disulfide. The fluorescence of the FAD in thioredoxin reductase decreases as the pH is lowered with a pK of 7.0, direct evidence for a base near the FAD probably distinct from the base interacting with the dithiol.     

Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.     

Acid intracellular vesicles and the cytolysis of mammalian target cells by Entamoeba histolytica trophozoites

Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 +/- 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to greater than or equal to 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 microM). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4 degrees C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 micrograms/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 micrograms for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

Xylem Sap pH Increase: A Drought Signal Received at the Apoplastic Face of the Guard Cell That Involves the Suppression of Saturable Abscisic Acid Uptake by the Epidermal Symplast

Drought increased the pH of Commelina communis xylem sap from 6.1 to 6.7. Conductances of transpiring leaves were 50% lower in pH 7.0 than in pH 6.0 buffers, but bulk leaf abscisic acid (ABA) concentration and shoot water status were unaffected by pH. Stomatal apertures of isolated abaxial epidermis incubated on simple buffers increased with external pH, so in vivo this must be overridden by alternative pH effects. Reductions in leaf transpiration rate at pH 7.0 were dependent on the presence of 10-8 mol dm-3 ABA in the xylem stream. We inferred that at pH 7.0 leaf apoplastic ABA concentrations increased: pH did not affect distributions of ABA among leaf tissues, but isolated epidermis and mesophyll tissue took up more 3H-ABA from pH 6.0 than from pH 7.0 buffers. The apoplastic ABA increase at pH 7.0 may result from reduced symplastic sequestration. A portion of 3H-ABA uptake by the epidermis was saturable at pH 6.0 but not at pH 7.0. An ABA uptake carrier may contribute to ABA sequestration by the leaf symplast of well-watered plants, and its inactivity at pH 7.0 may favor apoplastic ABA accumulation in draughted plants. Effects of external pH on stomatal apertures in the isolated epidermis indicate that published data supporting a role for internal guard cell ABA receptors should be reassessed.     

The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins.     

Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of CysE enhances biofilm formation in Escherichia coli

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.     

pH值对沼液培养的普通小球藻生长及油含量积累的影响

以50％的沼液为普通小球藻的全营养培养基,考察培养基的起始pH值对小球藻生长及油脂含量的影响,普通小球藻对不同初始pH的沼液中氮、磷的去除情况。设定了2组实验,一组只调节初始接种培养液的pH,分别为6.0、6.5、7.0、7.5、8.0、8.5;另一组将培养液pH分别固定在6.0、6.5、7.0、7.5、8.0、8.5,pH用稀HCl和NaOH进行调节。研究发现在pH 6.5和pH 7.0的偏酸环境有利于小球藻生长,而pH在7.0~8.5的偏碱性条件下有利于小球藻油脂的积累,因此综合小球藻生长和油脂积累2个因素,得到最适合小球藻生长和油脂积累的pH为7.0。培养结束后沼液中氮磷的去除率分别达到了95％和97％,沼液中的总氮由原来的134.91 mg/L降至4.86 mg/L,总磷由10.19 mg/L降到0.32 mg/L。     

Steady-state kinetic properties of FoF1-ATPase: the pH effect.

1. The kinetic properties of FoF1-ATPase from submitochondrial particles isolated from rat heart were studied, with emphasis to the pH effect. The velocity data were treated according to the Hill equation, and the results were discussed on the basis of the knowledge on the soluble F1-ATPase properties. 2. Three kinetic phases were observed in the range of pH 6.0-8.5, with apparent dissociation constant values (K0.5) of 0.001, 0.04 and 1.5 mM (respectively sites I, II and III) at pH 7.0. Their contribution to the total activity of the enzyme were pH-dependent on the range of 6.0-7.0, but not from 7.0 to 8.5, where the maximal velocity (V) for site III was some 4-fold larger than for site II, and the total V of sites II and III was some 40-fold larger than V assumed for site I. Therefore, two catalytic sites seem to participate significantly in the catalysis at steady-state condition. 3. Azide increased the sites II and III K0.5 values as well as decreased the site III V. In the presence of bicarbonate these two sites were not distinguishable, and the kinetic parameters at pH 7.0 were similar to those for sites II and III combined. Both azide and bicarbonate did not have a significant effect on site I, and this behavior was not pH-dependent. 4. The studies on the effect of pH on the kinetic parameters showed the following results: (1) the optimum pH for V was around 8.5; (2) decrease in the K0.5 values at pH below 7.0 for site II, and increase at pH over 7.0 for sites II and III; (3) in the pH range of 6.0-8.5 the Hill coefficient increased for site II, decreased for site III, and an intermediary effect was observed for the sites II and III combined, with a Michaelis-Menten behavior in the highest affinity pH, which was found in the physiological range.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation

Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here we show that the FcRgamma adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRgamma(-/-) mice show increased survival during peritonitis, owing to markedly increased E. coli phagocytosis and killing and to lower production of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The FcRgamma-associated receptor that inhibits E. coli phagocytosis is FcgammaRIII (also called CD16), and its absence protects mice from sepsis. FcgammaRIII binds E. coli, and this interaction induces FcRgamma phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-alpha production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRgamma on macrophages as the class A scavenger receptor MARCO. E. coli-FcgammaRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcgammaRIII, E. coli triggers an inhibitory FcRgamma pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-alpha secretion.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Sensitivity of some marine bacteria, a moderate halophile, and Escherichia coli to uncouplers at alkaline pH.

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus 118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicola NRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, its concentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transport at pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatest reduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of amino acid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences between the protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values. 3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The results support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+ antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase which is stimulated by Na+ at pH 8.5.     

Phagocytosis of concanavalin A-treated erythrocytes is mediated by the Fc gamma receptor

Impaired Fc gamma receptor-mediated phagocytosis has been reported in monocytes from HLA-DR2- and -DR3-positive disease-free individuals compared to normals without these B cell alloantigens. We have noted, however, a decrease in the ingestion of concanavalin A (Con A)-treated rabbit erythrocytes (E-Con A) in the same immunogenetically defined groups (DR2 vs Other: 2.94 +/- 0.84 erythrocytes/monocyte vs 4.16 +/- 1.37, p less than 0.003; DR3 vs Other: 3.35 +/- 1.51 vs 4.16 +/- 1.37, p less than 0.04). These data raised the possibility that carbohydrate-lectin interactions might trigger ingestion mediated by the Fc gamma receptor. To test this hypothesis, we performed receptor modulation and monosaccharide blocking experiments. Modulation of the Fc gamma receptor off the apical cell surface of monocytes by adherence to solid-phase IgG aggregates specifically reduced internalization of E-Con A and IgG-sensitized erythrocytes (EA) to 9.1% and 10.6% of control, respectively (p less than 0.001). Internalization of wheat germ agglutinin-treated erythrocytes, tannic acid-treated erythrocytes, and zymosan was not inhibited. In reciprocal modulation experiments using solid-phase Con A, no effects on phagocytosis of any particle was observed. alpha-Methyl mannoside, 0.1 M in PBS, did not inhibit the internalization of EA but blocked ingestion of E-Con A by 97% (p less than 0.001). Other monosaccharides had little or no effect on the ingestion of any of the phagocytic probes. These data demonstrate that a mechanism integrally involving the Fc gamma receptor mediates the ingestion of E-Con A by human monocytes. This Fc receptor has an oligosaccharide(s) with an exposed mannose which may be functionally significant. Whereas the mannose moiety does not play a crucial role in the interaction of the Fc gamma receptor with the Fc portion of IgG, engagement of the receptor via mannose can initiate internalization. Our findings raise the possibility that nonimmune functions may utilize classical immune system receptors through carbohydrate interactions. Furthermore, the ability of the Fc gamma receptor to trigger internalization is defective in HLA-DR2 and -DR3 normals, whether the receptor is ligated at its classical ligand-binding site or by way of its carbohydrate moieties.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 microM) than mannose (Kd = 2.3 microM). Exploration of the binding affinities of alpha- d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.