Development of a proteoliposome model to probe transmembrane electron-transfer reactions.

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decahaem cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains Methyl Viologen as an internalized electron carrier and valinomycin as a membrane-associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.

The DNA protection during starvation protein (Dps) influences attachment of Escherichia coli to abiotic surfaces.

The attachment of bacterial species such as Escherichia coli to abiotic materials is of concern to the food industry. This study investigated the role of DNA protection during starvation protein (Dps) in cell surface hydrophobicity and attachment of E. coli to glass, stainless steel, and Teflon surfaces. The Dps was not found to influence hydrophobicity, but did have a putative role in attachment in a strain- and substrate-dependent manner.

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate.

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.

Irsogladine maleate abolishes the increase in interleukin-8 levels caused by outer membrane protein 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans through the ERK pathway in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Irsogladine maleate (IM) suppresses the increase in interleukin (IL)-8 production induced by outer membrane protein (OMP) 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans in cultures of human gingival epithelial cells (HGEC). However, how IM suppresses the OMP29-induced increase in IL-8 expression remains unknown. In this study, we focused on intracellular signaling pathways to elucidate the mechanism behind the suppression. MATERIAL AND METHODS: HGEC, which had been pretreated with inhibitors of intracellular signaling molecules, were exposed to OMP29 (1 microg/mL) with or without IM (1 microM). IL-8 expression at the mRNA and protein levels was examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Extracellular signal-regulated kinase (ERK) activity was measured with a p44/42 mitogen-activated protein kinase assay kit. RESULTS: An ERK inhibitor, PD98059, as well as IM, obviated the OMP29-induced increase in IL-8 levels in HGEC. A Jun kinase inhibitor, SP600125, and a nuclear factor kappaB inhibitor, PDTC, did not influence the OMP29-induced increase in IL-8 mRNA expression. The OMP29 stimulated phosphorylation of ERK in HGEC. Irsogladine maleate inhibited the phosphorylation. CONCLUSION: The suppression of the phosphorylation of ERK by IM in HGEC culminates in inhibition of the OMP29-induced increase in IL-8.

dye (arc) Mutants: insights into an unexplained phenotype and its suppression by the synthesis of poly (3-hydroxybutyrate) in Escherichia coli recombinants.

arcA codes for a central regulator in Escherichia coli that responds to redox conditions of growth. Mutations in this gene, originally named dye, confer sensitivity to toluidine blue and other redox dyes. However, the molecular basis for the dye-sensitive phenotype has not been elucidated. In this work, we show that toluidine blue redirects electrons to O2 and causes an increase in the generation of reactive O2 species (ROS). We also demonstrate that synthesis of poly (3-hydroxybutyrate) suppresses the Dye phenotype in E. coli recombinants, as the capacity to synthesize the polymer reduces sensitivity to toluidine blue, O2 consumption and ROS production levels.

Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1-1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages (Vo=15 mV) than the other (Vo=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less (n=1.3) than that observed for the other process or for the wild-type channel (n=1.5-1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiological function.

Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein.

A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties.

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola.

Treponema denticola, frequently isolated from the human oral cavity, is thought to be a major pathogen of human periodontal disease. Recent developments in molecular analysis have clarified the surface structure of this microorganism and the characteristics of its pathogenic factors. Structural analysis of the outer sheath showed T. denticola to have a new type of outer membrane lipid. Limited exposure of the major outer sheath protein is suggested by electron-microscopic analysis. A protease-deficient mutant has revealed the roles of the protease in the organization of the outer sheath material and in T. denticola pathogenicity. The surface features that contribute to the pathogenicity of T. denticola in periodontal disease are gradually being elucidated, and are reviewed.

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes.

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

Biotechnology and biomimetic with crystalline bacterial cell surface layers (S-layers).

Crystalline bacterial cell surface layers (S-layers) are the outermost cell envelope component of many eubacteria and archaeobacteria. S-layers are composed of a single protein or glycoprotein species and exhibit oblique, square or hexagonal lattice symmetry. Pores passing through these monomolecular arrays show identical size and morphology, and functional groups are aligned in well-defined positions and orientations. Due to these unique features, S-layers have broad application potential in biotechnology including functioning as biomimetic membranes. Presently, S-layers are used (i) for the production of isoporous ultrafiltration membranes with very well defined molecular sieving and adsorption properties, (ii) as matrices for the controlled immobilization of biologically active macromolecules (e.g., enzymes, antibodies, ligands) as required for biosensors, affinity membranes and affinity microparticles as well as for solid phase assays, (iii) as stabilizing structures for Langmuir-Blodgett films and liposomes and (iv) as carriers and adjuvants for weakly immunogenic antigens and haptens.

Streptococcus mutans major adhesion surface protein, P1 (I/II), does not contribute to attachment to valvular vegetations or to the development of endocarditis in a rat model.

Streptococcus mutans P1 antigen functions as an adhesion factor for binding to salivary pellicle on tooth surfaces. It induces increased antibody titres in patients with Strep. mutans endocarditis. A mutant of Strep. mutans deficient in the function of the gene (spa P) encoding the surface antigen P1, and its isogenic parental strain, were used in a rat endocarditis experiment. Absence of P1 did not decrease adhesion to vegetations determined l h after intravenous infection. The number of bacteria recovered from valvular vegetations after 48 h from animals with manifest endocarditis did not differ between the strains. Consequently, the Pl antigen appears to be unimportant both for adhesion and virulence in endocarditis caused by Strep. mutans.

Bacteria-mucin interaction in the upper aerodigestive tract shows striking heterogeneity: implications in otitis media, rhinosinusitis, and pneumonia.

The mucociliary system of the upper and lower respiratory tracts is a critical nonspecific pathway for the elimination of bacteria and other particulate matter. The interaction between bacteria and purified mucin of the upper and lower respiratory tracts has been a major focus of our laboratory for the past decade. We have previously demonstrated that nontypable Haemophilus influenzae and Moraxella catarrhalis adhere to human purified nasopharyngeal mucin and human middle ear mucin by a very limited number of specific outer membrane proteins. There have been no previous studies on the interaction of Streptococcus pneumoniae and purified mucin. Such information would be of extreme importance in identifying specific mechanisms of preventing colonization of this important pathogen to nasopharyngeal mucin. Using an overlay technique of purified radiolabeled mucins of the upper and lower respiratory tracts in a solid phase assay with 4 predominant pathogens of the upper and lower respiratory tracts, we found a striking heterogeneity of bacteria-mucin interaction. The implications of these interactions in the development of otitis media, rhinosinusitis, and lower respiratory infections are briefly discussed.

Porphyromonas gingivalis HmuY-induced production of interleukin-6 and IL-6 polymorphism in chronic periodontitis.

BACKGROUND: In chronic periodontitis (CP), the gene polymorphism of interleukin-6 (IL-6) to 174C/G has been associated with the altered production of this cytokine. The aim of this pilot study is to compare the allelic and genotypic frequencies in patients with CP with control individuals without periodontitis (NP) and to measure the production of IL-6 by whole blood cells stimulated with Porphyromonas gingivalis HmuY protein. METHODS: DNA was isolated from peripheral blood cells of 49 patients with CP and 60 control individuals classified as NP, and genotyping was performed by polymerase chain reaction using sequence-specific primers. Whole blood cells from 29 patients with CP and 30 control individuals were stimulated for 48 hours with HmuY, and IL-6 levels were measured using enzyme-linked immunosorbent assay. RESULTS: The proportion of individuals carrying the G allele at position -174 of the IL-6 gene was higher in the group with CP (85.7%) than in the normal control group (73.3%; P <0.03). P. gingivalis HmuY-induced production of IL-6 was higher in the group with CP (P <0.05). CONCLUSIONS: Our findings suggest that P. gingivalis HmuY may be associated with increased IL-6 production during CP. Furthermore, patients with periodontitis and individuals with higher HmuY-induced production of IL-6 show a high frequency of the G allele at position -174.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1ß. Some bacterial species can alter their physiological properties as a result of sensing IL-1ß. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1ß sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1ß through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1ß than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1ß, but this binding was not specific, as a control protein for IL-1ß also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1ß-binding surface-exposed lipoprotein that may be part of the bacterial IL-1ß-sensing system.

Virulence factors of the oral spirochete Treponema denticola.

There is compelling evidence that treponemes are involved in the etiology of several chronic diseases, including chronic periodontitis as well as other forms of periodontal disease. There are interesting parallels with other chronic diseases caused by treponemes that may indicate similar virulence characteristics. Chronic periodontitis is a polymicrobial disease, and recent animal studies indicate that co-infection of Treponema denticola with other periodontal pathogens can enhance alveolar bone resorption. The bacterium has a suite of molecular determinants that could enable it to cause tissue damage and subvert the host immune response. In addition to this, it has several non-classic virulence determinants that enable it to interact with other pathogenic bacteria and the host in ways that are likely to promote disease progression. Recent advances, especially in molecular-based methodologies, have greatly improved our knowledge of this bacterium and its role in disease.

Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement.

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

Further evidence that major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis stabilize bacterial cells.

INTRODUCTION: Porphyromonas gingivalis is one of the most important bacteria in the progression of chronic periodontal disease. We hypothesized that the major outer membrane proteins Pgm6/7, which are homologous to the OmpA protein in Escherichia coli, might contribute to the stabilization of the cell surface. In this study, the effects of Pgm6/7 on the cell surface were examined morphologically. METHODS: Deletion mutants of Pgm6/7 (Delta694, Delta695 and Delta695-694) were constructed using the polymerase chain reaction-based overlap extension method. Wild-type ATCC 33277 and Pgm6/7 mutants were grown under anaerobic conditions. Whole cells and thin sections of fixed cells were stained and examined by transmission electron microscopy. RESULTS: Compared with the wild-type, numerous vesicles released from cells were observed in each deletion mutant. The outer membrane appeared wavy and irregular. Increased numbers of vesicles were confirmed after their preparation from the culture supernatant. Total gingipain activity in vesicles was increased five- to 10-fold in the deletion mutants. CONCLUSION: This report provides further evidence that Pgm6/7 proteins in P. gingivalis play an important role in the maintenance of bacterial outer membrane integrity.

Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa.

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83.

The authors have shown previously that the vimA gene, which is part of the bcp-recA-vimA operon, plays an important role in protease activation in Porphyromonas gingivalis. The gingipain RgpB proenzyme is secreted in the vimA-defective mutant P. gingivalis FLL92. An important question that is raised is whether the vimA gene product could directly interact with the proteases for their activation or regulate a pathway responsible for protease activation. To further study the mechanism(s) of VimA-dependent protease activation, the vimA gene product was further characterized. A 39 kDa protein consistent with the size of the predicted VimA protein was purified. In protein-protein interaction studies, the VimA protein was shown to interact with gingipains RgpA, RgpB and Kgp. Immune sera from mice immunized with P. gingivalis immunoreacted with the purified VimA protein. Taken together, these data suggest an interaction of VimA with the gingipains and further confirm the role of this protein in their regulation or maturation.