Potent Inhibition of Scrapie-Associated PrP Accumulation by Congo Red

Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using scrapie-infected neuroblastoma cells as a model system, we found that Congo red, an amyloid-binding dye, potently inhibits the accumulation of the scrapie-associated, protease-resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease-resistant PrP detected in the cultures by greater than 90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease-resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.     

Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.     

Correlation between Congo red binding as virulence marker in Shigella species and Sereny test.

Six variants of nutrient agar were tested in order to chose the suitable media for Congo red binding test. Trypto-soy Eiken, T.S.A - Cantacuzino Institute and B.T.S.D. (a medium prepared with Difco ingredients) are appropriate to distinguish between virulent Crb+ and avirulent Crb- strains. Congo red binding was compared with Sereny test using 25 Shigella strains. The strains were inoculated onto trypto-soy agar Eiken plates with 0.01% Congo red, incubated 24 hours at 37 degrees C. A number of each kind (Crb+ and Crb-) of colonies developed by every strain was subcultured on nutrient agar and Sereny test was performed with these cultures. As expected, all 84 Crb+ colonies in vivo tested, produced keratoconjunctivitis. In the case of Crb- colonies a proper correlation with Sereny negative test was observed in 57 out of 73 colonies (78.2%) to which 10.9% (8 out of 73) less virulent (evoking illness in only one of the two inoculated eyes) colonies may be added. As our results confirmed that loss of pigmentation was consistently accompanied by loss or diminishing of virulence, we consider that Congo red binding may be used as an alternative of in vivo test for establishing the virulence of Shigellae in the routine practice of microbiology laboratories which usually are not provided with cell cultures or animals. Its reduced cost is an important advantage, too.     

Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red [corrected]

Congo red and certain sulfated glycans are potent inhibitors of protease-resistant PrP accumulation in scrapie-infected cells. One hypothesis is that these inhibitors act by blocking the association between protease-resistant PrP and sulfated glycosaminoglycans or proteoglycans (e.g., heparan sulfate proteoglycan) that is observed in amyloid plaques of scrapie-infected brain tissue. Accordingly, we have investigated whether the apparent precursor of protease-resistant PrP, protease-sensitive PrP, binds to Congo red and heparin, a highly sulfated glycosaminoglycan with an inhibitory potency like that of heparan sulfate. Protease-sensitive PrP released from the surface of mouse neuroblastoma cells bound to heparin-agarose and Congo red-glass beads. Sucrose density gradient fractionation provided evidence that at least some of the PrP capable of binding heparin-agarose was monomeric. Free Congo red blocked PrP binding to heparin and vice versa, suggesting that these ligands share a common binding site. The relative efficacies of pentosan polysulfate, Congo red, heparin, and chondroitin sulfate in blocking PrP binding to heparin-agarose corresponded with their previously demonstrated potencies in inhibiting protease-resistant PrP accumulation. These results are consistent with the idea that sulfated glycans and Congo red inhibit protease-resistant PrP accumulation by interfering with the interaction of PrP with an endogenous glycosaminoglycan or proteoglycan.     

Evidence that supramolecular Congo red is the sole ligation form of this dye for L chain lambda derived amyloid proteins.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.     

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

AIMS: The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg+) Bordetella bronchiseptica cultures. METHODS AND RESULTS: Congo red binding and urease activity of Bvg+ B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as filamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (> or = 26 nmol ml-1 of CR and < or = 2.6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO4 20 mmol l-1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO4 5 mmol l-1 cultures) could be distinguished. CONCLUSION: CR binding assay and urease activity are specific and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production.     

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).     

Inhibition of cell-cell interactions in Myxococcus xanthus by congo red

The function of molecules associated with the cell surface may be determined by examining the phenotype of cells treated with inhibitors specific to these cell surface molecules. This strategy was used to examine the function of the major Congo red receptor of the myxobacterium Myxococcus xanthus, which has a developmental cycle that involves social interactions among cells. A class of social motility mutations (A+ S-), known as dsp, may inhibit the same subcellular component as Congo red because the phenotype of wild-type cells which had been treated with Congo red resembled in several ways the phenotype of the Dsp mutants. First, Congo red inhibited agglutination of wild-type cells, whereas Dsp cells were incapable of agglutinating, even in the absence of Congo red. Second, Congo red inhibited fruiting body formation by wild-type cells and reduced the yield of myxospores. Untreated Dsp cells were unable to form fruiting bodies and produced few myxospores. Third, Congo red reduced the rate of wild-type gliding motility to a level comparable to that of untreated Dsp cells, but did not inhibit the A motility of Dsp cells. Finally, binding studies showed that Dsp cells lacked the major Congo red receptor. Wild-type cells bound Congo red with an apparent association constant of 2.4 X 10(5) M-1, while Dsp cells bound it with an apparent association constant of 8.5 X 10(3) M-1. Binding of Congo red to wild-type cells was saturated in less than 10 min and was reversible when excess Congo red was removed. These results suggest that the Congo red receptors are controlled by the S motility system and that these receptors are involved in cell cohesion, social motility, and fruiting body formation.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

Correlation between Congo red binding and contact haemolysin production in Shigella species

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.     

Protein distorsion-derived mechanism of signal discrimination in monocytes revealed using Congo red to stain activated cells

The supramolecular dye Congo red was used to check whether monocyte activation may be mediated by a torsion-dependent mechanism preventing transduction of weak random signals in cell contacts in a way corresponding to the discrimination mechanism found in complement fixation by immune complexes. Tight cell-cell contacts generating torsional effects may be expected to produce alteration of receptor structure, making them accessible for binding of supramolecular dyes. In this study, Congo red was used to observe the binding accessibility of (1) monocytes (human) induced by contact with cancer cells (HCV29T, human), (2) monocytes (mouse) stimulated by interaction with heat-aggregated IgG and (3) monocytes (mouse) activated by rosetting in the presence of an SRBC-anti-SRBC system. Microscopic studies confirmed the activation of monocytes manifested by their clustering and Congo red binding, but only tightly clustered cells appeared to attach the dye on the surface. Usually not the whole cell surface is found to be engaged in dye complexation. Staining occurs predominantly on the interfaces of reacting cells, making probable the suggestion that cell adhesion receptors are involved in dye binding. The cells in the central areas of tight clusters undergo accelerated death. In the presence of Congo red they are easily recognized as intensely fluorescent. The characteristic localization of dead cells in the central area of clusters indicates that death is not random but results from cell activation. The role of Congo red in this process remains to be clarified. The staining characteristics of monocytes after application of Congo red probably discloses the initial step in signal transduction generated by torsional movements in receptor proteins.     

Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers

Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.     

Congo red binding test in enteroinvasive and nonpathogenic Escherichia coli strains.

Out of 6 variants the appropriate media to perform Congo red binding test for enteroinvasive E. coli strains were established (trypto-soy agar Eiken, T.S.A.--Cantacuzino Institute and B.T.S.D.). 12 E. coli strains belonging to enteroinvasive O-serogroups formed on Congo red agar red-coloured, non-coloured colonies or both; cultures from 59 red colonies and 61 white colonies were inoculated in guinea pig eyes. The correlation between positive Congo red binding test and positive Sereny test was 91% (out of 59 red colonies, 47 evoked keratoconjunctivitis in both infected eyes and 7 in only one eye). The negative Congo red binding test corresponds (98.4%) to the failure to induce illness in the guinea pigs' eye (only one out of 61 Crb = colonies was Sereny positive, evoking keratoconjunctivitis in only one of the two infected eyes of a guinea pig). Comparing in vivo lack of pathogenicity in 44 E. coli strains isolated from human normal intestinal flora and negative Congo red binding test, a correlation of 72.73% on B.T.S.D. and 65.91% on T.S.A. medium was found. Developing an appropriate method based on Crb test about 70% of the nonpathogenic E. coli colonies could be eliminated from the laborious agglutination with enteroinvasive O-serogroups E. coli antisera.     

Effect of the reducing environment on the accumulation of elastin and collagen in cultured smooth-muscle cells.

We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylation is thought to be that of a reductive cofactor that maintains the proper oxidation state of molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors.     

The cause of the green polarization color of amyloid stained with Congo red

Summary Experiments done with Congo red crystals and with Congo red deposits polished in a single direction by a glass wheel have shown that the appearance of green polarization color primarily depends on near-perfect parallel alignment of the dye particles. The green polarization color was seen only in the deposits which showed a clear transition from red to colorless when examined for dichroism. Another factor was found to be the thickness of the object, as the green polarization color was not present in too thick or too thin sections of amyloid-containing tissues stained with Congo red.The phenomena can be explained by the assumption that the green polarization color is due to interference between the red ray and the red component of the white ray whenever the retardation by the object approximates half the wavelength of red light.The findings indicate that amyloid differs from other materials which are stained by Congo red in that amyloid deposits bind the dye molecules in a more orderly and parallel fashion. It is suggested that minimal amounts of amyloid which are not visible in Congo red stained sections with ordinary light microscopy and which do not give the green polarization color can best be detected by examination for dichroism in ultraviolet light after having been stained with fluorescent dyes.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

In vivo accumulation of self-assembling dye Congo red in an area marked by specific immune complexes: possible relevance to chemotherapy

Supramolecular micellar structures have been proposed as carriers in aim-oriented drug transportation to a target marked by specific immune complexes. In this study, the self-assembling dye Congo red was used as a model supramolecular carrier and its accumulation in the target was studied in vivo. The target was created in vivo as the local specific inflammation provoked by subcutaneous injection of antigen to the ear of a previously immunized rabbit. The color caused by accumulation of Congo red after its intravenous injection was registered by pictures of the ear with suitably filtered visible light shining through it to distinguish Congo red against the background color of hemoglobin. The results confirmed the expected accumulation and retention of Congo red in the inflammation area marked by deposits of specific immune complexes. The role of albumin and its possible interference with transportation of drugs through the blood by supramolecular carriers was also subjected to preliminary examination. The results revealed that albumin collaborates rather than interferes with drug transportation; this is another factor making the use of supramolecular carriers for aim-oriented chemotherapy highly promising.     

The identification and estimation of elastase in serum and plasma

1. Electrophoretic separation of partially purified elastase preparations from pancreas followed by incubation of the electrophoretogram in contact with an agar gel containing 4% of either Congo Red-stained or unstained elastin demonstrated that the enzyme which dissolves elastin can be identified with that which releases dye from the stained preparation. 2. A method for the estimation of elastase based on the release of dye from Congo Red-stained elastin is described. It is 23 times as sensitive as methods employing protein determination. 3. With the method, elastase activity can be identified in plasma and in a partially fractionated plasma protein preparation. 4. Lineweaver–Burk plots of these estimates of activity at a variety of substrate concentrations indicate that the reaction between elastase and the dyed elastin is more closely similar to that between elastase and the soluble substrate elastin rather than to that between elastase and the solid substrate. 5. Values for K_m and V_max. calculated for the enzyme present in plasma present further evidence for its identity with the pancreatic enzyme. 6. By calculation of the slopes of the Lineweaver–Burk plots for various enzyme concentrations it has proved possible to demonstrate that the inhibitor which is also present in the plasma is without effect on the enzyme when it acts on the dyed substrate.     

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.     

Investigating by circular dichroism some amyloidogenic elastin-derived polypeptides

Tamburro and coworkers have demonstrated that some elastin-derived polypeptide sequences are able to give rise, in vitro, to amyloid-like fibers. The biological relevance of this finding could be explained by the recent detection of some amyloidogenic material found in arteries of old patients affected by atherosclerosis and demonstrated to be elastin derived. In this context, the comprehension of the mechanism responsible for the amyloid-like fibrillogenesis of elastin-derived sequences is of crucial importance for the design of drugs that could inhibit the amyloidogenic process. To gain further insights into the elastin amyloidogenic process, we studied the polypeptide sequences encoded by Exon 7 and Exon 32 of the human tropoelastin gene, and we demonstrated that only Exon 32 is able to aggregate in amyloid-like fibers. Vis-UV Thioflavin T circular dichroism (CD) spectroscopy rapidly and unambiguously detected the amyloidogenic propensity of the polypeptides. To gain additional insights into the aggregation mechanism of elastin-derived amyloidogenic peptides, we carried out the kinetics of EX32 amyloid-like aggregates by using ThT dye. CD spectroscopy was also used for investigating the secondary structure of the polypeptides, thus giving useful insights into the conformations involved in amyloid-like fiber formation. Furthermore, complementary techniques such as fluorescence spectroscopy, spectral shift, and binding Congo red UV assays as well as atomic force microscopy were also used to confirm the amyloidogenic behavior of the studied polypeptides.