Local and Systemic Effects of <i>Porphyromonas gingivalis</i> Infection

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors

Screening for donor and acceptor substrates.

<p>Change of absorbance at 557 nm with time for <b>A</b>. UDP-galactose as donor and <b>B</b>. UDP- glucose as donor was plotted using various sugars as acceptors. Lysates (100 µl) of <i>E. coli</i> expressing rVimF was used as enzyme source and, lysates of <i>E. coli</i> Top 10 cells was used for negative control. The reaction mix contained 2 mM phosphate, pH 8, 0.01 mM phenol red, 0.1 mM MnCl₂, and 10 mM acceptor sugars. A lower OD₅₅₇ value was observed when UDP-galactose was used as the donor.</p

Comparison of biofilm formation, autoaggregation, hemagglutination and invasion assay.

<p><b>A.</b> Biofilm formation of ATCC 33277, FLL476 and FLL476C’ were compared. Biofilm assay was done by staining adherent cells of overnight cultures grown in microtiter plates with 0.5% (w/v) crystal violet. Blank contained only media. Biofilm forming ability corresponded to OD₅₉₅. <b>B.</b> Autoaggregation of 33277, FLL476 and FLL476C’ corresponded to change in OD₆₀₀ monitored for about three hours after cells were washed and suspended in PBS. A representative sample is shown. <b>C.</b> Hemagglutination activities of ATCC 33277, FLL476 and FLL476C’ were assessed by serially diluting cells in PBS and incubating with sheep RBCs for 3 h at 4°C. Dilutions are listed above and last dilution showing matt formation was taken as the titer. The blank contained only media. <b>D.</b> Antibiotic Protection Assay was used to quantify invasion. <i>P. gingivalis</i> cells that were able to invade HeLa cell monolayers were released by lysis and cultured on BA plates. Infectivity was taken as the percentage of cells recovered. (* = p<0.05).</p

Electron micrograph showing changes in surface structures of <i>P. gingivalis</i> ATCC 33277 and W83.

<p>Bacterial cells grown to the log phase (OD₆₀₀ of 0.7–0.9) were processed for electron microscopic examination using formvar-carbon coated grids (500 mesh) and were examined using Philips Tecnai 12 TEM. Fimbriae were lacking in the <i>vimF</i> mutant FLL476 when compared with the wild ATCC33277 and the complemented strain FLL476C’. A thick glycocalyx along with vesicles and a well-defined outer membrane was observed in W83. FLL95 showed hazy outer membrane with reduced visicles. In the complemented strain FLL95C’ the outer membrane morphology was restored.</p

Plasmid and bacterial strains used in this study.

<p>Plasmid and bacterial strains used in this study.</p

Comparison of growth and gingipain activities of wild-type, <i>vimF</i> mutant and complemented strains of W83 and ATCC 33277.

<p>Growth rate of <i>P. gingivalis</i> ATCC 33277 (<b>A</b>) and W83 (<b>C</b>) were compared with their respective <i>vimF</i>-defective isogenic mutants (FLL476 and FLL95 ) and complemented strains (FLL476C’ and FLL95C’). The data shown is an average of three independent replicates. Error bars represent the SD. Gingipain activity of W83 (<b>D</b>) and ATCC 33277 (<b>B</b>) were compared with respective mutants and complemented strains. The activities were normalized to W83 and ATCC 33277 being 100% and the mutants reported as a percentage thereof. Error bars represent SD.</p

1D and 2D SDS-PAGE of rVimF.

<p>Purified rVimF was denatured in an LDS-containing buffer with DTT and heated for 10 min, and subjected to SDS-PAGE analysis. <b>A</b>. Simply Blue Safe stain of rVimF at 2 different concentrations: lane 1–0.4 µg and lane 2–1.2 µg. <b>B.</b> Western blot using anti-rVimF antibody against purified rVimF showed reacting bands at 50, 100 and 200 kDa. <b>C.</b> Simply blue safe stain of rVimF with horseradish peroxidase as positive (PC) and soybean trypsin inhibitor as negative (NC) controls for glycoproteins. <b>D.</b> Identical gel in panel C stained by periodic acid-Schiff (PAS) method for glycoproteins. <b>E.</b> Western blot using anti-rVimF showed a 47 kDa reactive band only when total proteins of W83 were deglycosylated and not with native (glycosylated) forms.</p

Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501.

International audienceThe capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture