Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system.

Lipopolysaccharide (LPS) is a key inflammatory mediator. Due to its ability to potently activate host inflammatory and innate defense responses, it has been proposed to function as an important molecule that alerts the host of potential bacterial infection. However, although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, causes a highly unusual host innate host response. It is an agonist for human monocytes and an antagonist for human endothelial cells. Correspondingly, although it activates p38 MAP kinase in human monocytes, P. gingivalis LPS does not activate p38 nor ERK MAP kinase in endothelial cells. In fact, P. gingivalis LPS is an effective inhibitor of Escherichia coli LPS induced p38 phosphorylation. These data show that P. gingivalis LPS modulates host defenses in endothelial cells by interfering with MAP kinase activation. In addition, P. gingivalis LPS is unusual in that it engages TLR-2 but not TLR-4 when examined in stably transfected CHO cell lines. We propose that, since LPS is a key ligand for the human innate host defense system, these unusual properties of P. gingivalis LPS are associated with the bacterium's role in the pathogenesis of periodontitis.

Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier.

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the beta-defensin family, human beta-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-alpha and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower ( approximately 10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-alpha as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-alpha that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli and F. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Mutation of cytochrome bd quinol oxidase results in reduced stationary phase survival, iron deprivation, metal toxicity and oxidative stress in Azotobacter vinelandii.

Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity.

Antigenic variation and cross-reactivity in Bacteroides forsythus clinical isolates detected by western blot.

Bacteroides forsythus is one of the etiologic agents of destructive periodontal diseases. Determining which antigenic components of the bacterium are recognized in the immune response of periodontitis patients is an important step in assessing strategies for vaccine development. The aim of this study was to identify the major strain-variable and cross-reactive antigens of B. forsythus clinical isolates recognized by serum IgG from patients with early-onset rapidly progressive periodontitis. Ten patient sera with measurable IgG against antigenic components of the species were identified by Western blot. Positive sera were tested by checkerboard ELISA to identify those most responsive to strain-variable antigens in nine clinical isolates and ATCC strain 43037. Correlation analysis of the ELISA data suggested that different subsets of isolates were preferentially recognized by different sera. Western blots revealed that certain sera also recognized major shared components across all the isolates, but preferential recognition of different isolate subsets by different patients was clearly confirmed. To determine if the variable antigens recognized were nonprotein, proteinase K-digested isolates were compared to undigested controls by Western blot. The main strain-variable antigens were proteinase resistant, while proteins at 200 and 210 kDa were identified as the major shared components. Two-dimensional SDS-PAGE revealed that these proteins are the quantitatively dominant heat-modifiable components of the cell envelope. Even though variable antigens are prominent in the immune response of patients, a cross-protective vaccine based on the shared envelope proteins of B. forsythus seems feasible in light of these observations.

Serum antibodies to Porphyromonas gingivalis block the prostaglandin E2 response to lipopolysaccharide by mononuclear cells.

The ability of rabbit and monkey immune sera to neutralize prostaglandin E2 (PGE2) production by human monocytes stimulated with lipopolysaccharide (LPS) was examined. CD14-dependent LPS activation of PGE2 was examined under assay conditions which allowed the comparison of preimmune and immune sera. Serum obtained from rabbits immunized with formalin-fixed Porphyromonas gingivalis cells dramatically reduced the amount of PGE2 produced in response to LPS obtained from three different strains of P. gingivalis but not that from Escherichia coli or Bacteroides fragilis. In addition, a significant reduction in the mean PGE2 level was observed in the presence of sera from immunized but not control monkeys employed in a vaccine trial. Immune serum samples from five of nine immunized monkeys were able to reduce LPS-induced production of PGE2 by greater than 50% compared to that in the corresponding preimmune sera. Immune monkey serum, similar to immune rabbit serum, blocked PGE2 production in response to P. gingivalis LPS but not E. coli LPS. These data demonstrate that immunization with P. gingivalis whole cells can elicit an antibody response that is able to block the PGE2 response to LPS. Neutralization of LPS-mediated inflammatory mediator production may account in part for the observed suppression of alveolar bone loss in immunized monkeys.

Choline: its role in the growth of filamentous fungi and the regulation of mycelial morphology.

Choline is an essential metabolite for the growth of filamentous fungi. It occurs most notably as a component of the major membrane phospholipid, phosphatidyl choline (lecithin), and fulfills a major role in sulphate metabolism in the form of choline-o-sulphate in many species. Choline is usually synthesised endogenously, but exogenous choline can also be taken up, either to compensate for metabolic deficiencies in choline-requiring mutants such as those of Aspergillus nidulans and Neurospora crassa, or as a normal function by species such as Fusarium graminearum which do not require added choline for growth. F. graminearum has a highly specific constitutive uptake system for this purpose. Recent studies have begun to indicate that choline also plays an important role in hyphal and mycelial morphology. Over a wide range of concentrations, choline influences mycelial morphology, apparently by controlling branch initiation. At high concentrations of added choline, branching is inhibited but specific growth rate is unaffected, leading to the production of rapidly extending, sparsely branched mycelia. Reduction of choline concentration allows a progressive increase in branching. Additionally, in choline-requiring mutants which have a very reduced content of choline, multiple tip-formation and apical branching occurs. Just prior to cessation of growth in choline-starved cultures of A. nidulans choline-requiring mutants, hyphal morphology changes due to a brief phase of unpolarised growth to produce spherical swellings called balloons, at or near hyphal apices. The precise mechanism by which choline affects fungal morphology is not yet known, although in A. nidulans it appears to be at least partially due to the influence of membrane composition on the synthesis of the hyphal wall polymer chitin. Several hypotheses for the possible mode of action of choline in affecting fungal morphology are discussed here.

Effect of restrictive conditions on the growth and morphology of a temperature-sensitive mannose-requiring mutant of Aspergillus nidulans.

When incubated at 45 degrees C in the absence of added mannose, pregrown hyphae of a temperature-sensitive, mannose-relief mutant (mnrA455) of Aspergillus nidulans grew normally for a short time (4-5 h) before exhibiting an abnormal morphology consisting of the production by hyphae of discrete spherical swellings called balloons. These swellings could be up to 10 microns in diameter and were produced either at or behind the hyphal apex. Often only one swelling was produced in association with each hyphal tip, but in a significant minority of cases (approximately 19.6%) a second balloon was produced in close association with the first. Hyphal tip extension slowed before and during balloon formation, but growth at individual tips did not usually stop when a balloon began to be formed in the same hypha. All tip extension ceased after approximately 8 h in cultures maintained at 45 degrees C. However, normal growth resumed 45-60 min after transfer of such a culture to the permissive temperature of 37 degrees C even after 48 h at 45 degrees C. Electron microscopic examination indicated that balloons consistently had thicker walls than the surrounding hyphae but that no accumulation of cytoplasmic vesicles was apparent within them. This indicates that a modification of wall structure, probably including deposition of new wall material, was caused by a mannose deficiency, but that this altered wall synthesis and attendant hyphal swelling was not due to diversion of the normal vesicle-mediated tip-extension system to the side walls of hyphae.

Improved methods for the detection of beta-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate: VBzTM-gal (2-(2-(4-(beta-D-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3- methylbenzothiazolium toluene-4-sulphonate).

The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 microns) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4 degrees C for several days, and this identified the expression of beta-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water.

Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans.

Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi.

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.

Genetic transformation and cell morphology of Bacillus subtilis grown in Mg+(+)-limited chemostat culture.

The rate and frequency of genetic transformation of Bacillus subtilis grown in Mg+(+)-limited chemostat culture are dependent on the dilution rate (D) of the system and achieved maximum values at D = 0.23 h-1. Mg+(+)-limitation induced a morphological change in the cells from their normal rod shape to extended helices. Although this change in shape was a transient phenomenon, under some conditions it persisted for several days and resulted in an apparent increase in the transformation frequency.