Experimental murine model for autoimmune enterocolitis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant

An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella 03 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed-type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ-specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.     

Hexagonal assembly of the magnesium salt of an R-form lipopolysaccharide from Klebsiella pneumoniae: its lowered stability compared with original non-electrodialyzed preparation

The magnesium salt of R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm as the original non-electrodialyzed preparation of the R-form LPS. When the magnesium salt was suspended in 50 mM glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4 C for 24 hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh-like structure at pH 3.0 or higher. In the original non-electrodialyzed preparation of the R-form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4 C for 24 hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg2+ and in stabilizing the ordered hexagonal assembly of the R-form LPS.     

Disintegration of Mg2+ -induced hexagonal assembly of an R-form lipopolysaccharide from Klebsiella pneumoniae by treatment with CaCl2

R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-: K1-), which was precipitated by the addition of 2 volumes of ethanol containing 10 mM MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R-form LPS was suspended in 50 mM tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mM or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R-form LPS was disintegrated and changed to an irregular rough, mesh-like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg-bound R-form LPS result in a tighter binding of Ca2+ to the R-form LPS and the release of Mg2+ from the R-form LPS, and as a consequence, destroys the Mg2+ -induced ordered hexagonal lattice structure of the R-form LPS.     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

Human antibody responses to R3-core epitopes on the lipopolysaccharide of Escherichia coli O157

AIMS: To establish the incidence of serum antibodies binding to the R3-core lipopolysaccharide (LPS) of verocytotoxin-producing Escherichia coli (VTEC) O157, in patients with serum antibodies to E. coli O157 LPS, and to characterize the class(es) of antibodies binding to epitopes on the R3-core. METHODS AND RESULTS: SDS-PAGE profiles of LPS prepared from VTEC O157 were used in combination with immunoblotting to detect and characterize serum antibodies binding to the R3-core LPS of VTEC O157. Of 417 sera, referred to the Laboratory of Enteric Pathogens (LEP) for routine O157 serology and found to have serum antibodies to long-chain VTEC O157 LPS, 31 had antibodies binding to the R3-core of VTEC O157 LPS. The majority of the 31 sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. Patients who did not develop haemolytic uraemic syndrome (HUS) produced antibodies of the IgM class to R3-core and IgG-class antibodies to long-chain LPS more frequently than patients with HUS. CONCLUSIONS: Only 7.4% of sera received by the LEP, and shown to have antibodies to VTEC O157 LPS, contained antibodies binding to the R3-core of VTEC LPS. Most sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. SIGNIFICANCE AND IMPACT OF THE STUDY: Patients infected with VTEC O157 produced antibodies binding to the R3-core epitopes of VTEC O157 LPS only rarely, and these antibodies are unlikely to interfere with the serodiagnosis of infections caused by these organisms.     

Serological relationships of the O antigens of Klebsiella pneumoniae O5, Escherichia coli O8 and a new O serotype of Serratia marcescens

Klebsiella pneumoniae O5, Escherichia coli O8 and Serratia marcescens 3255 were shown to cross-react in both ELISA and immunoblotting. The cross-reaction appeared to be due to the O antigen of their lipopolysaccharide (LPS). In addition, there was evidence that the reactions of these strains with their homologous antisera were due, in part, to determinants other than O polysaccharide.     

Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study

Introduction

Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.

Methods

CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.

Results

Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.

Conclusions

Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.     

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [³H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP) (500 μM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [³H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.     

An N-[(R)-(-)-2-Hydroxypropionyl]-α-L-Perosamine Homopolymer Constitutes the O Polysaccharide Chain of the Lipopolysaccharide from Vibrio cholerae O144 Which Has Antigenic Factor(s) in Common with V. cholerae O76

Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144). The LPS of O144 contained D -glucose, D -galactose, L -glycero-D -manno-heptose, D -fructose, D -quinovosamine (2-amino-2,6-dideoxy-D -gluco-pyranose) and L -perosamine (4-amino-4,6-dideoxy-L -manno-pyranose). The perosamine, a major component sugar of the LPS from O144, was in an L -configuration, as is also the case in the LPS from V. cholerae O76 (O76), in contrast to the D -configuration of the perosamine in the LPS of V. cholerae O1. A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an α(1 → 2)-linked homopolymer of (R)-(-)-2-hydroxypropionyl-L -perosamine. The serological cross-reactivity between O144 and O76 was clearly revealed by cross-agglutination and cross-agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red-blood cells that had been sensitized with the LPS from O144 and O76. In contrast, in passive hemolysis tests, the LPS of O144 did not cross-react serologically with the LPSs from other strains such as V. cholerae O1 (Ogawa and Inaba), V. cholerae O140, Vibrio bio-serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9. The LPSs from these strains consist of O polysaccharide chains composed of α(1 → 2)-linked homopolymers of D -perosamine with various N-acyl groups, and they share the Inaba antigen factor C of V. cholerae O1 in common. The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V. cholerae O1.     

The O-polysaccharide of lipopolysaccharide isolated from Vibrio fluvialis O19 is identical to that of Vibrio bioserogroup 1875 variant

A structural analysis has been carried out on the O-polysaccharide of lipopolysaccharide (LPS) isolated from Vibrio fluvialis 181-86 (Kobe) serotype O19 (O19) which has the Inaba antigen factor C of O1 V. cholerae and factors D and E in common with Vibrio bioserogroup 1875. The O-polysaccharide of O19 was characterized as an alpha (1-->2)-linked homopolymer of N-3-hydroxypropionyl-D-perosamine (4-amino-4,6-dideoxy-D-mannopyranose), which was identical to that of Vibrio bioserogroup 1875 Variant. Passive hemolysis and passive hemolysis inhibition analysis performed using anti-factor D, E and anti-factor E antisera, demonstrated that the LPS from O19 harbored O-antigenic factors identical to those of the LPS from Vibrio bioserogroup 1875 Variant.     

Mutations in the genes for synthesis of the outer core region of the lipopolysaccharide of Yersinia enterocolitica O:3

AIMS: The aim of this study was to construct non-polar frame-shift mutations in some of the individual genes responsible for the biosynthesis of the branching outer core (OC) hexasaccharide of the lipopolysaccharide (LPS) in Yersinia enterocolitica O:3 (YeO:3). METHODS AND RESULTS: Chromosomal segments of YeO:3 containing wbcN, wbcO and wbcQ genes were cloned into a suicide vector. A frame-shift mutation was introduced into each gene by modifying a unique restriction enzyme recognition site. Each recombinant plasmid with a modified OC gene was mobilized into YeO:3 to allow for allelic exchange between the modified gene and the wild type chromosomal gene. The exchange was confirmed by demonstrating the absence of the particular restriction site in the chromosome of each mutant strain. Analysis of LPS by gel electrophoresis showed that the LPS of the mutants was lacking the OC. Therefore, the constructed wbcN, wbcO and wbcQ strains are true mutants with frame-shifts in the corresponding genes. CONCLUSIONS: The products of the wbcN, wbcO and wbcQ genes are putative glycosyltransferases and, based on the present analysis, essential for the biosynthesis of the OC hexasaccharide. The absence of OC in the LPS of these mutants further supports the hypothesis that the OC hexasaccharide is a single O-antigen O-unit that is not polymerized in YeO:3. SIGNIFICANCE AND IMPACT OF THE STUDY: These mutants provide information on the unique nature of the synthesis of OC of YeO:3 LPS. They are valuable for future biochemical studies to establish the roles of the products of individual OC genes.     

Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.     

Molecular basis of recognition of human osteopontin by 23C3, a potential therapeutic antibody for treatment of rheumatoid arthritis

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem β-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody-antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.     

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.     

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n = 35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C_14:0 3-OH FA to C_16:0 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C_14:0 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C_14:0 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C_14:0 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C_14:0 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.     

Molecular characterization of the β‐lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh,Saudi Arabia

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β‐lactamases such as metallo‐β‐lactamases (MBL) and extended spectrum β‐lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β‐lactamases. Here, the prevalence of different β‐lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β‐lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi‐resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates.     

Retracted: Downregulated microRNA-135a ameliorates rheumatoid arthritis by inactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway via phosphatidylinositol 3-kinase regulatory subunit 2

Synovial fibroblasts (SFs) of rheumatoid arthritis (RA) are phenotypically aggressive, typically progressing into arthritic cartilage degradation. Throughout our study, we made explorations into the effects of microRNA-135a (miR-135a) on the SFs involved in RA by mediating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via regulation of phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2). The expression of PI3K was higher, the expression of PIK3R2 was lower, and AKT was phosphorylated in the RA synovial tissues, relative to the levels found in the normal synovial tissues. We predicted miR-135a to be a candidate miR targeting PIK3R2 using an online website, microRNA.org, which was verified with a dual-luciferase reporter gene assay. Subsequently, high miR-135a expression was observed in RA synovial tissues. To study the effect of the interaction between miR-135a and PIK3R2 in RA, the SFs isolated from RA samples were cultured and transfected with mimic, inhibitor, and small interfering RNA. The proliferation, invasion, and apoptosis of the SFs were detected after the transfection. The cells transfected with miR-135a inhibitor showed inhibited cell proliferation, migration, and invasion, while also displaying promoted cell apoptosis, G0/G1 cell ratio, and decreased S cell ratio, through upregulation of PIK3R2 and inactivation of the PI3K/AKT signaling pathway. These findings provided evidence that downregulation of miR-135a inhibits proliferation, migration, and invasion and promotes apoptosis of SFs in RA by upregulating the PIK3R2 coupled with inactivating the PI3K/AKT signaling pathway. The downregulation of miR-135a might be a potential target in the treatment of RA.     

An in silico,in vitro and in vivo investigation of indole-3-carboxaldehyde identified from the seawater bacterium Marinomonas sp. as an anti-biofilm agent against Vibrio cholerae O1

Biofilm formation is a major contributing factor in the pathogenesis of Vibrio cholerae O1 (VCO1) and therefore preventing biofilm formation could be an effective alternative strategy for controlling cholera. The present study was designed to explore seawater bacteria as a source of anti-biofilm agents against VCO1. Indole-3-carboxaldehyde (I3C) was identified as an active principle component in Marinomonas sp., which efficiently inhibited biofilm formation by VCO1 without any selection pressure. Furthermore, I3C applications also resulted in considerable collapsing of preformed pellicles. Real-time PCR studies revealed the down-regulation of virulence gene expression by modulation of the quorum-sensing pathway and enhancement of protease production, which was further confirmed by phenotypic assays. Furthermore, I3C increased the survival rate of Caenorhabditis elegans when infected with VCO1 by significantly reducing in vivo biofilm formation, which was corroborated by a survivability assay. Thus, this study revealed, for the first time, the potential of I3C as an anti-biofilm agent against VCO1.     

Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO⁻ and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature.