How bacteria hack the matrix and dodge the bullets of immunity.

Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential.

Effects of dietary fructo-oligosaccharide supplementation on the growth performance, haemato-immunological parameters, gut microbiota and stress resistance of common carp (Cyprinus carpio) fry.

The present study was conducted to investigate the effects of dietary fructo-oligosaccharide (FOS) (0, 1, 2 and 3%) supplementation on the growth performance, haemato-immunological parameters, cultivable autochthonous (non-adherent) intestinal microbiota and stress resistance of common carp (Cyprinus carpio) fry (3·23 (SEM 0·14) g). These parameters were measured after feeding the carp fry with the experimental diets for 7 weeks. Dietary FOS supplementation had no significant effects on the growth performance and food intake of carp fry compared with the control treatment. It also had no significant effects on the following haematological parameters: erythrocyte count; leucocyte counts (WBC); haematocrit; Hb; mean corpuscular volume; mean corpuscular Hb content; mean corpuscular Hb concentration. However, WBC and respiratory burst activity were significantly affected by dietary FOS supplementation. Evaluation of the cultivable autochthonous intestinal microbiota revealed a significant increase in the levels of total viable heterotrophic aerobic bacteria and lactic acid bacteria in fish fed diets supplemented with 2 and 3% FOS. Furthermore, dietary FOS supplementation significantly increased the survival rate and stress resistance of carp fry compared with the control treatment. These results encourage conducting further research on the administration of FOS and other prebiotics in carp fry studies.

Gram-negative versus Gram-positive bacteremia: what is more alarmin(g)?

Gram-negative bacteremia has been associated with severe sepsis, although the exact mechanism and pathophysiological differences among bacterial species are not well understood. In the previous issue of Critical Care, Abe and colleagues report results of a retrospective study that show a significantly higher incidence of Gram-negative bacteremia among adult intensive care unit patients with septic shock than in those with sepsis or severe sepsis. In this study, C-reactive protein and IL-6 levels were significantly higher in Gram-negative bacteremia than in Gram-positive bacteremia. These observations suggest a distinct immunopathophysiologic behavior of sepsis in patients with Gram-negative bacteremia that may influence clinical outcomes. Future research exploring new biomarkers and danger signals and further characterizing differences in the virulence mechanisms between Gram-negative and Gram-positive bacteria appears promising and could lead to new therapeutics and to improved clinical outcomes.

Screening of molecular repertoires by microbial surface display.

A multitude of systems for the presentation of foreign peptides or proteins on the surface of microorganisms has been developed within the past two decades. However, the majority of the bacterial surface display systems are devoted to the presentation of heterologous antigens to the immune system (vaccine generation). Bacteria are the preferable hosts for the generation of vast genetic repertoires, and their genetic manipulation and cultivation is easy. As a consequence, they provide promising systems for large-scale functional screenings, e.g. for enzyme activity or protein-protein interactions. This review will focus on examples of microbial surface display used for the screening of combinatorial repertoires. Further, we discuss future opportunities and promising candidate proteins not yet employed for that task.

Display of proteins on bacteria.

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.

Removal of bowel aerobic gram-negative bacteria is more effective than immunosuppression with cyclophosphamide and steroids to decrease natural alpha-galactosyl IgG antibodies.

Natural alpha-Galactosyl (Gal) antibodies play an important role in the rejection of pig xenografts by humans and Old World monkeys. In this study we investigate the efficacy of two different strategies to reduce the serum level of natural anti-Gal antibodies. On the one hand, removal of aerobic gram-negative bacteria from the intestinal flora, because anti-Gal antibodies appear to be produced as a result of the continuous sensitization by these microorganisms. On the other hand, we studied the effect on these antibodies of an immunosuppressive regimen of cyclophosphamide and steroids. Ten baboons were treated for three months with norfloxacin (Nor Group; n=6) or cyclophosphamide and steroids (CyP Group; n=4). A further four baboons did not receive any treatment (Control Group). Aerobic gram-negative bacteria became negative in stools of the Nor Group after two weeks of treatment, and remained undetectable until week 7. Thereafter, a gradual increase on the fecal concentration of aerobic gram-negative bacteria was observed despite the norfloxacin treatment. The mean anti-Gal IgG in the Nor Group gradually declined from week 4 to 9 to a mean of 62.7 +/- 18% of the baseline level, and during this period were significantly lower than in the CyP (P<0.02) and the Control (P<0.05) groups. No differences were observed between the three groups during the 16 weeks of follow-up in serum levels of anti-Gal IgM, hemolytic anti-pig antibodies, total IgG, IgM and IgA. In conclusion, removal of normal aerobic gram-negative bacteria from the intestinal flora is more effective than immunosuppression with CyP and steroids in reducing the level of natural anti-Gal antibodies, although there is no discernible effect on IgM antibodies.

Structure of sulfated O-specific polysaccharide of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232.

A sulfated O-specific polysaccharide containing D-mannose, L-rhamnose, and the sulfate group was obtained by mild acid hydrolysis of lipopolysaccharide (S-form) of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232. Based on analysis of methylation and 13C-NMR spectroscopy of native and desulfated polysaccharides, the following structure of disaccharide repeat unit in the O-specific polysaccharide has been established: [scheme]. This is the first report of a sulfated O-specific polysaccharide isolated from gram-negative bacteria.

Structure of the capsular polysaccharide from Alteromonas nigrifaciens IAM 13010T containing 2-acetamido-2,6-dideoxy-L-talose and 3-deoxy-D-manno-octulosonic acid.

A capsular polysaccharide was obtained from Alteromonas nigrifaciens IAM 13010T by saline extraction. On the basis of 1H and 13C NMR spectroscopy, including one-dimensional (1D) NOE spectroscopy, 2D rotating-frame NOE spectroscopy (ROESY), and 1H-detected heteronuclear 1H,13C multiple-quantum coherence (HMQC), it was concluded that the polysaccharide contained inter alia an acidic sugar, 3-deoxy-D-manno-octulosonic acid (Kdo), and a rare amino sugar, 2-acetamido-2,6-dideoxy-L-talose (L-6dTalNAc, N-acetylpneumosamine), and has a pentasaccharide repeating unit of the following structure: [equation: see text]

Identification of a functionally important population in phenol-digesting activated sludge with antisera raised against isolated bacterial strains.

Antisera were raised against nine strains which had been isolated from phenol-acclimated oil refinery activated sludge. Although several antisera reacted significantly with the activated sludge during a period of adaptation to phenol, only an antiserum against one of the isolates, Alcaligenes sp. E2, reacted with the activated sludge after the adaptation period. A kinetic pattern of phenol-oxygenating activity of the activated sludge after the adaptation period was similar to that of strain E2. These results suggest that a functionally important population in the phenol-digesting activated sludge was serologically identified.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serine-glyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

Use of monoclonal antibodies against dibenzo-p-dioxin degrading Sphingomonas sp. strain RW1.

A monoclonal antibody prepared against surface antigen of Sphingomonas sp. strain RW1 was used for the direct detection of RW1-like organisms in environmental samples by epifluorescence microscopy and subsequent confirmation by Western blot. Of the 76 samples collected from various sources and probed using epifluorescence, only one sample, effluent from paper and pulp processing, gave a positive result. The effluent was cultured and yielded an organism which, by Western blot analysis, was shown to contain the 28 kDa protein recognized by the monoclonal antibody.

Immunoelectrophoretic characterization and cross-reactivity of Rochalimaea henselae, Rochalimaea quintana and Afipia felis.

The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed-line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species.

Antibody response in rabbits infected with Rochalimaea henselae, Rochalimaea quintana and Afipia felis.

Antibody responses in three pairs of rabbits inoculated with live Rochalimaea henselae, Rochalimaea quintana and Afipia felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross-reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens.

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.

Major antigens in Methylobacterium species and their location in cells using immuno-electron microscopic methods.

This work is a first step in the development of a specific probe for the study of the distribution and colonization of leaf surfaces by pink-pigmented, facultatively methylotrophic (PPFM) bacteria of the genus Methylobacterium. A polyclonal antiserum was produced in rabbits against whole cells of PPFM strain PC1, isolated from surfaces of white clover leaves. Major heat labile antigens were found in extracts of sonicated cells using the Ouchterlony double diffusion method. Very small amounts of a heat stable antigen were also observed. The major antigens were found in extracts of each of fifteen PPFM strains tested but were not found in extracts of other clover heterotrophs nor in extracts of other methylotrophs tested. The distribution of antigens in ultrathin sections of PPFM cells was investigated using PC1 antisera and gold labelled protein A. Gold particles were seen mainly in the outermost layer of the homologous strain, but isolated and washed cell envelopes of strain PC1 like other strains retained very little antigen. Sections of other PPFM strains showed the major antigens were located mainly in the cytoplasm.

Antitumor activity of Sphaerotilus natans and its slime fraction in mice.

Antitumor activity of the whole cell and slime of an aquatic sheathed bacterium, Sphaerotilus natans IAM 12068, against ascites form of Ehrlich carcinoma in ddY mice was investigated. Intraperitoneal injection of whole cells and the slime fraction showed remarkable antitumor activity against mice inoculated with 10(4) to 10(5) tumor cells, the slime fraction being more effective. To examine the chemical nature of the active principle in the slime fraction, separation by Sepharose 4B gel filtration was carried out and two fractions designated as GF-P-1 and GF-P-2, which are mainly composed of protein, carbohydrate, and lipid, were obtained. GF-P-1 fraction, which contains large amounts of fucose and unidentified sugar as neutral sugar, showed marked antitumor activity at half the dose of the slime fraction, whereas the antitumor activity of GF-P-2, which is composed mainly of protein, was weak. This finding indicates that GF-P-1 fraction of S. natans slime may be a main active principle. The consistently demonstrable antitumor activity of GF-P-1 was abrogated by treatment of mice with silica, an anti-macrophage agent, suggesting that the antitumor activity of GF-P-1 depends on the activation of macrophages.

Lipid A antibody determinations using ELISA on patients at a children's hospital: a preliminary report.

Using the enzyme-linked immunosorbent assay (ELISA), we studied the IgG and IgM antibody titers in various groups of pediatric patients (n = 81) infected with gram-negative organisms. Unlike the control group (n = 12), IgG antibodies were detected in only five (all under four months of age) of 19 children with sepsis. We assume that either the IgG antibodies are used up during the infection, or the lack of IgG antibodies results in a disposition to sepsis; the latter is more probable. Seventeen of 18 patients with urinary tract infections and proven renal involvement were IgM-positive. This indicates a permanent antigen stimulus, possibly in the form of a fixed antigen complex. Because of the heterogeneity of the groups studied, no overall statements can be made for the 93 children studied, some of whom were studied repeatedly. These children included 17 with tracheal colonization, 17 with recurrent urinary tract infections without proven renal changes and six with wound infections.