Identification of a bile-induced exopolysaccharide required for Salmonella biofilm formation on gallstone surfaces.

Salmonella enterica serovar Typhi can establish a chronic, asymptomatic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment. Gallstones are one of the most important risk factors for developing carriage, and we have previously demonstrated that salmonellae form biofilms on human gallstones in vitro. Thus, we hypothesize that bile-induced biofilms on gallstone surfaces promote gallbladder colonization and maintenance of the carrier state. A colanic acid/cellulose S. enterica serovar Typhimurium double mutant formed a mature biofilm on gallstones in a test tube assay and in a new, gallstone-independent assay using cholesterol-coated Eppendorf tubes. These data suggest the presence of an unidentified exopolysaccharide necessary for mature biofilm development and demonstrate specific binding affinity between salmonellae and cholesterol. Our experiments indicate that the Salmonella O-antigen capsule (yihU-yshA and yihV-yihW) is a crucial determinant in gallstone and cholesterol biofilms but that expression of this exopolysaccharide is not necessary for binding to glass or plastic. Real-time PCR revealed that growth in bile resulted in upregulation of the O-antigen capsule-encoding operon in an agfD-independent manner. Thus, the O-antigen capsule genes are bile induced, and the capsule produced by the enzymes of this operon is specifically required for biofilm formation on cholesterol gallstones. These studies provide new therapeutic targets for preventing asymptomatic serovar Typhi gallbladder carriage.

Identification of a ribosomal L10-like protein from Flavobacterium psychrophilum as a recombinant vaccine candidate for rainbow trout fry syndrome.

The psychrophilic bacterium Flavobacterium psychrophilum is a rapidly emerging, virulent pathogen of a variety of commercially important finfish species, including salmonids. No vaccines against F. psychrophilum are currently available, partly due to its recalcitrant growth in vitro. Consequently, we explored the possibility of constructing recombinant vaccines in Escherichia coli as a prophylactic biotechnological strategy to counter F. psychrophilum infections. An immunoreactive clone from a F. psychrophilum expression library was found to express a approximately 16 kDa protein antigen. A proteomics approach was taken to identify the ORF encoding the approximately 16 kDa protein. Tryptic fragments of the approximately 16 kDa protein were analyzed by MALDI-TOF mass spectrometry and compared to theoretical (in silico) tryptic fragments of translated ORFs predicted within the cloned DNA. The target protein was identified as a 166 amino acid protein (named 7-166) with homology to rplJ which encodes bacterial ribosomal protein L10. Whenhighly expressed in E. coli as an N-terminal fusion protein, this chimera reacted with convalescent rainbow trout serum. When adjuvanted and administered intraperitoneally to immature rainbow trout a high level of protection (82% RPS) was afforded against virulent F. psychrophilum challenge; thus establishing F. psychrophilumrplJ homologue 7-166 as a promising vaccine candidate for RTFS.

Identification and expression of a host-recognized antigen, FspA, from Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome, an economically important disease of immature salmonid fish for which there is no vaccine. Convalescent serum from the host, rainbow trout (Oncorhynchus mykiss), reacted strongly with a approximately 20 kDa, Flavobacterium-specific protein antigen (subsequently named FspA) from F. psychrophilum. Protein-enriched, detergent-partitioned samples were separated by two-dimensional gel electrophoresis and the protein target was excised, proteolytically cleaved and the resulting peptides analysed by MS. Quadrupole-time-of-flight MS was used to generate a fragmented peptide spectrum. The resulting peptide sequences were then used to design degenerate PCR primers to amplify the gene (fspA) of interest: 612 bp encoding 203 aa, including a putative 19 aa N-terminal signal sequence which predicted a processed 19 303.6 Da protein. FspA proved to be unique and only homologous to two unspecified sequences reported from Flavobacterium johnsoniae, although weakly homologous to a Yersinia pseudotuberculosis adhesin. An amplified gene fragment (537 bp, encoding 179 aa) was further cloned into an expression vector, expressed as a approximately 30 kDa N-terminal fusion protein and found to retain its strong reactivity with host serum antibodies. These results suggest that the surface-localized FspA may be an important subunit vaccine candidate antigen against F. psychrophilum.

The structure of the antigenic O-polysaccharide of the lipopolysaccharide of Edwardsiella ictaluri strain MT104.

The structural characterization of the antigenic O-polysaccharide component of the lipopolysaccharide produced by the fish pathogenic bacterium Edwardsiella ictaluri MT104 was undertaken by the application of NMR spectroscopy and chemical analysis. The O-chain was found to be a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in a 1:2:1 ratio having the structure: [carbohydrate structure]; see text.

Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco.

Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. A plant transformation vector was constructed with transcriptional fusion between the pathogen-responsive win3.12T promoter from poplar and the gene encoding the novel cecropin A-melittin hybrid peptide (CEMA) with strong antimicrobial activity. This promoter-transgene combination was evaluated in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) for enhanced plant resistance against a highly virulent pathogenic fungus Fusarium solani. Transgene expression in leaves was strongly increased after fungal infection or mechanical wounding, and the accumulation of CEMA transcripts was found to be systemic and positively correlated with the number of transgene insertions. A simple and efficient in vitro regeneration bioassay for preliminary screening of transgenic lines against pathogenic fungi was developed. CEMA had strong antifungal activity in vitro, inhibiting conidia germination at concentrations that were non-toxic to tobacco protoplasts. Most importantly, the expression level of the CEMA peptide in vivo, regulated by the win3.12T promoter, was sufficient to confer resistance against F. solani in transgenic tobacco. The antifungal resistance of plants with high CEMA expression was strong and reproducible. In addition, leaf tissue extracts from transgenic plants significantly reduced the number of fungal colonies arising from germinated conidia. Accumulation of CEMA peptide in transgenic tobacco had no deleterious effect on plant growth and development. This is the first report showing the application of a heterologous pathogen-inducible promoter to direct the expression of an antimicrobial peptide in plants, and the feasibility of this approach to provide disease resistance in tobacco and, possibly, other crops.

Structural characterization of the O-polysaccharide antigen of Edwardsiella tarda MT 108.

Edwardsiella tarda, a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. The lipopolysaccharide produced by the fish pathogenic strain E. tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry. The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), D-galactose (D-Gal), L-rhamnose (L-Rha), D-galacturonic acid (D-GalA) and (2S,3R)-threonine (1:1:1:1:1:1) having the structure: [structure: see text].

Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot.

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.

The structure of the glycopeptides from the fish pathogen Flavobacterium columnare.

Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: [carbohydrate structure--see text] where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.

Structural characterization of the lipopolysaccharide O-polysaccharide antigen produced by Flavobacterium columnare ATCC 43622.

The structure of the antigenic O-chain polysaccharide of Flavobacterium columnare ATCC 43622, a Gram-negative bacterium that causes columnaris disease in warm water fish, was determined by high-field 1D and 2D NMR techniques, MS, and chemical analyses. The O-chain was shown to be an unbranched linear polymer of a trisaccharide repeating unit composed of 2-acetamido-2-deoxy-d-glucuronic acid (d-GlcNAcA), 2-acetamidino-2,6-dideoxy-l-galactose (l-FucNAm) and 2-acetamido-2,6-dideoxy-d-xylo-hexos-4-ulose (d-Sug) (1 : 1 : 1), having the structure: [structure: see text].

Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus.

Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2-acetamido-3-O-acetyl-4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-beta-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino-3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2-C-7 fragment of the latter monosaccharide (B) was assigned beta-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide beta-Sug-(2-3)-l-Rha, isolated from the products of the acidic methanolysis of the LPS.

Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum.

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.