Inhibition of biofilm formation, quorum sensing and other virulence factors in Pseudomonas aeruginosa by polyphenols of Gynura procumbens leaves.

Quorum sensing (QS) enables virulence factors in bacteria for biofilm formation and pathogenic invasion. Therefore, quorum quenching (QQ), disruption of QS circuit, becomes an alternative antimicrobial therapy. In this study, leaf extract of Gynura procumbens (GP) was used to inhibit biofilm and virulent factors in Pseudomonas aeruginosa. The extract inhibited the biofilm production (p ≤ 0.05) in P. aeruginosa strains MZ2F and MZ4A. The minimum biofilm eradication concentration (MBEC) was recorded at 250 and 500 µg/ml while total activity was found at 288 and 144 ml/g, respectively. Moreover, a significant reduction of virulence factors (p ≤ 0.05) at sub-MBEC without affecting the growth implies the QQ action of the extract. The bioactive fractions were rich in polyphenols and tentatively identified as quercetin and myricetin (Rf=0.53-0.60). Furthermore, we employed computational methods to validate our findings and their interactions with QS receptors (LasR and RhlR). Interestingly, docking studies have also shown that quercetin and myricetin are the promising anti-QS agents out of 31 GP compounds. Notably, their binding affinity ranged between -9.77 and -10.52 kcal/mol for both QS receptors, with controls ranging from -5.40 to -8.97 kcal/mol. Besides, ΔG of quercetin and myricetin with LasR was -71.56 and -74.88 kcal/mol, respectively. Moreover, compounds were suitable drug candidates with stable binding interactions. Therefore, the anti-QS activity of GP leaves and the identified polyphenols can be used in developing QQ-based therapeutics. Communicated by Ramaswamy H. Sarma.

Computational prediction of active sites and ligands in different AHL quorum quenching lactonases and acylases.

With the emergence of multidrug-resistant 'superbug', conventional treatments become obsolete. Quorum quenching (QQ), enzyme-dependent alteration of quorum sensing (QS), is now considered as a promising antimicrobial therapy because of its potentiality to impede virulence gene expression without resulting in growth inhibition and antibiotic resistance. In our study, we intended to compare between two major QQ enzyme groups (i.e., AHL lactonases and AHL acylases) in terms of their structural and functional aspects. The amino acid composition-based principal component analysis (PCA) suggested that probably there is no structural and functional overlapping between the two groups of enzymes as well as within the lactonase enzymes but the acylases may functionally be affected by one another. In subcellular localization analysis, we also found that most lactonases are cytoplasmic while acylases are periplasmic. Investigation on the secondary structural features showed random coil dominates over alpha-helix and beta-sheet in all evaluated enzymes. For structural comparison, the tertiary structures of the selected proteins were modelled and submitted to the PMDB database (Accession ID: PM0081007 to PM0081018). Interestingly, sequence alignment revealed the presence of several conserved domains important for functions in both protein groups. In addition, three amino acid residues, namely aspartic acid, histidine, and isoleucine, were common in the active sites of all protein models while most frequent ligands were found to be 3C7, FEO, and PAC. Importantly, binding interactions of predicted ligands were similar to that of native QS signal molecules. Furthermore, hydrogen bonds analysis suggested six proteins are more stable than others. We believe that the knowledge of this comparative study could be useful for further research in the development of QSbased universal antibacterial strategies.

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

Relationship between the risk for a shrimp allergy and freshness or cooking.

Tropomyosins are defined as risk factors for shrimp allergy. However, their concentration in different preparations has not been clarified. We quantified the tropomyosin concentration in shrimp meat, which was cooked using several methods or was stored under various conditions. The results demonstrated that shrimp meat from various preparations and storage conditions maintained tropomyosin concentrations that were sufficient to cause food allergies.

Unstable mutant lysozymes are degraded through the interaction with calnexin homolog Cne1p in Saccharomyces cerevisiae.

Cne1p is a yeast homolog of calnexin, which is a constituent of endoplasmic reticulum (ER)-associated protein quality control system in mammals. Cne1p may be involved in the degradation of misfolded lysozymes in Saccharomyces cerevisiae. To test this, c-Myc-tagged lysozymes were expressed in CNE1-deficient S. cerevisiae. The expression and secretion of an unstable lysozyme mutant G49N/D66H were enhanced and its intracellular localization was changed in the CNE1-deficient strain. Furthermore, when Cne1p was co-expressed with unstable lysozyme mutants (G49N/D66H, G49N/C76A, and K13D/G49N), its affinity to the misfolded mutant proteins was revealed by co-immunoprecipitation. The interaction with Cne1p was abrogated by the addition of tunicamycin, an inhibitor of N-glycosylation, indicating that N-linked carbohydrates might be necessary for protein binding to Cne1p. These results suggest that in yeasts, Cne1p interacts with misfolded lysozyme proteins possibly causing their retention in the ER and subsequent elimination via ER-associated degradation.

LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens.

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.

Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen.

Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.

The periodontopathogenic bacterium Eikenella corrodens produces an autoinducer-2-inactivating enzyme.

Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

Genomic recombination through plasmid-encoded recombinase enhances hemolytic activity and adherence to epithelial cells in the periodontopathogenic bacterium Eikenella corrodens.

The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.

The inhibitory effects of catechins on biofilm formation by the periodontopathogenic bacterium, Eikenella corrodens.

Eikenella corrodens is a periodontopathogenic bacterium that forms biofilm even by itself. In this study, we investigated the inhibitory effects of catechins on E. corrodens biofilm formation. Biofilm formation was inhibited by the addition of 1 mM of the catechins with the pyrogallol-type B-ring and/or the galloyl group. The catechins with the galloyl group were effective at smaller doses than those with only the pyrogallol-type B-ring. An inhibitory effect was observed even when these catechins and gallic acid were added at sub-minimal inhibitory concentration (MIC) or at concentrations that showed no bactericidal effect. These results suggest that some catechins at sub-MIC might inhibit biofilm formation. No inhibitory effect of catechins at sub-MIC on biofilm formation was observed in the luxS deletion mutant. Our studies suggest that some species of catechins with the galloyl group affect autoinducer 2-mediated quorum sensing and thereby inhibit biofilm formation by E. corrodens.

Effects of various saccharides on the masking of epitope sites and uptake in the gut of cedar allergen Cry j 1-saccharide conjugates by a naturally occurring Maillard reaction.

A major allergen of Japanese cedar, Cry j 1, was conjugated with galactomannan (M(w) of 15 kDa), dextran (M(w) of 12 kDa), xyloglucan (M(w) of 1.4 kDa), and various monosaccharides through the Maillard reaction by dry-heating in 65% relative humidity. The Cry j 1-galactomannan conjugate completely masked the epitopes of the allergen in Cry j 1. The Cry j 1-dextran conjugate also masked the epitopes of Cry j 1. The small size of oligosaccharide (xyloglucan) and various monosaccharides cannot mask the epitopes of allergen Cry j 1. This suggests that the higher molecular size of attached saccharides is important to mask sterically the epitope sites. The Cry j 1-galactomannan and Cry j 1-mannose conjugates were effectively trafficked in the gut and co-localized with immune cells, such as dendritic cells in the gut, suggesting that Cry j 1-saccharide conjugates are phagocytosed via the mannose receptor in immune cells. These results suggest that the Cry j 1-galactomannan conjugate is suitable for masking the epitope sites of Cry j 1 and trafficking to immune cells in gut lumen.

Reduction of antigenicity of Cry j 1, a major allergen of Japanese cedar pollen, by thermal denaturation.

The soluble aggregates of Cry j 1, a major allergen of Japanese cedar pollen, were formed without any coagulates during heat treatment at acidic pH 5, as shown in HPLC and SDS-PAGE patterns. A remarkable change in the CD spectrum was observed between native and heat-denatured Cry j 1 at a linear rate of 1 degrees C/min from 40 to 90 degrees C. The negative peak of native Cry j 1 at 222 nm was moved to 218 nm, suggesting the transition of an alpha-helix to beta-structure during heat denaturation. The increase in beta-structure was also observed during heat denaturation by monitoring the fluorescence with Thioflavin T. These results suggest that Cry j 1 forms intermolecular cross-beta-structure between denatured proteins during heating at 90 degrees C. The antigenicity of Cry j 1 detected by dot-blotting was greatly diminished during heating at a linear rate of 1 degrees C/min from 40 to 90 degrees C without any coagulates. These results suggest that IgE epitopes exposed on the molecular surface of Cry j 1 was buried inside soluble aggregates through intermolecular beta-structure formed by heating.

Amyloid fibril formation of hen lysozyme depends on the instability of the C-helix (88-99).

Stable and unstable mutant lysozymes in long helices B and C were constructed to evaluate the effect of the helices on amyloid fibril formation at pH 2. Stable mutant N27D and unstable mutant K33D in the B-helix did not change in amyloid fibril formation. In contrast, stable mutant N93D and unstable mutant K97D in the C-helix showed big differences in behavior as to amyloid fibril formation. Stable mutant N93D showed a longer lag phase of aggregation and suppressed the amyloid fibril formation, whereas unstable mutant K97D showed a shorter lag phase of aggregation and accelerated amyloid fibril formation. These results suggest that the long C-helix is involved mainly in the alpha-helix to beta-sheet transition during amyloid formation of lysozyme.

Effects of Er:YAG laser irradiation on biofilm-forming bacteria associated with endodontic pathogens in vitro.

With the development of dental laser delivery systems that can enter into the root canals, it is possible to use Er:YAG lasers to remove the residual biofilm associated with infected root canals. We examined their effects against biofilms made of Actinomyces naeslundii, Enterococcus faecalis, Lactobacillus casei, Propionibacterium acnes, Fusobacterium nucleatum, Porphyromonas gingivalis, or Prevotella nigrescens in vitro. After Er:YAG laser irradiation with energy densities ranging between 0.38-0.98 J/cm(2), the biofilm samples on hydroxyapatite disks were quantitatively and morphologically evaluated. The Er:YAG laser was effective against biofilms of 6 of the bacterial species examined, with the exception of those formed by L. casei. After irradiation, the numbers of viable cells in the biofilms were significantly decreased, whereas atrophic changes in bacterial cells and reductions in biofilm cell density were seen morphologically. Er: YAG lasers might be suitable for clinical application as a suppressive and removal device of biofilms in endodontic treatments.

Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with alpha-helix dipoles and their secretion amounts in yeast.

The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.

Characterization of autoinducer 2 signal in Eikenella corrodens and its role in biofilm formation.

Quorum sensing (QS) is a process by which bacteria communicate using secreted chemical signaling molecules called autoinducers (AIs). By this process, many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density. In this study, the periodontal pathogen Eikenella corrodens was observed to secrete type 2 signaling molecules. An ortholog of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the E. corrodens genome. A V. harveyi bioassay showed luxS functionality in E. corrodens and the ability of luxS to complement the luxS-negative phenotype of Escherichia coli DH5alpha. AI activity was detected in the supernatant, and the maximum expression of AI-2 was observed during the late exponential phase. To determine the potential role of luxS in the colonization processes, an E. corrodens luxS mutant was constructed and tested for its capacity to form an in vitro biofilm on a polystyrene surface. The biofilm forming efficiency of the luxS mutant was approximately 1.3-fold greater than that of the wild type. These data suggest that a LuxS-dependent signal plays a role in the biofilm formation by E. corrodens.

Plasmid-mediated genomic recombination at the pilin gene locus enhances the N-acetyl-D-galactosamine-specific haemagglutination activity and the growth rate of Eikenella corrodens.

Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-D-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5-6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E. corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5-6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a genomic recombination of the type IV pilin gene locus.

Involvement of N-acetyl-D-galactosamine-specific lectin in biofilm formation by the periodontopathogenic bacterium, Eikenella corrodens.

Eikenella corrodens is known not only as one of the periodontopathogenic bacteria but also as a pathogen associated with many infectious diseases of humans. Dental plaque is a complex biofilm community comprised of many bacterial species. E. corrodens has a lectin on its cell surface that is thought to be involved in its pathogenicity. In this study, we found that E. corrodens forms a biofilm on a polystyrene surface. A biofilm was formed at the bottom of the wells in microtiter plates after 24 h. Microcolonies were observed as the amount of biofilm became larger. When anaerobic respiration was repressed due to nitrate limitation, the biofilm formed only at the air-water interface. Strain 1073 and HU, which have higher lectin activity, formed a biofilm more effectively than other strains. Biofilm formation was repressed by the addition of N-acetyl-D-galactosamine. These results suggest that the lectin on the surface of E. corrodens might be involved in biofilm formation.

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast.

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.

Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.