Role of VimF in Gingipain Maturation in Porphyromonas gingivalis

Gingipain activity in Porphyromonas gingivalis, the major etiological agent in adult periodontitis, is post-translationally regulated by unique Vim proteins including VimF, a putative glycosyltransferase. To ascertain the VimF mediated phenotype we first inactivated the vimF gene in P. gingivalis ATCC 33277 (FLL476), a less virulent fimbriated strain. We observed that the vimF-defective mutant (FLL476) showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 background. While hemagglutination was not detected and autoaggregationn was reduced, biofilm formation was increased in FLL476. Also, invasive capacity decreased for this mutant. Furthermore, fimbrial structures were missing in FLL476, suggesting the role of VimF in fimbrial processing. We were able to restore the wildtype phenotype by complementing the defect. Secondly, we cloned, expressed and purified the vimF gene and demonstrated its ability to glycosylate gingipains. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. Further, in the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified my mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb IB5 antibody. Finally, the polyclonal antibody raised against rVimF that did not react well with native VimF from P. gingilvalis, reacted with a 47 kDa protein when the fractions were first deglycosylated before probing with the antibody. Galactose is vital for growing glycan chain leading to maturation/activation of gingipains. Taken together, these results suggest that VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation

Neuraxial anaesthesia is associated with improved outcomes and reduced postoperative complications in patients undergoing aseptic revision total hip arthroplasty

© The Author(s) 2020. Background: As the incidence of primary total hip arthroplasty (THA) continues to increase, revision THA (rTHA) is becoming an increasingly common procedure. rTHA is widely regarded as a more challenging procedure, with higher complication rates and increased medical, social and economic burdens when compared to its primary counterpart. Given the complexity of rTHA and the projected increase in incidence of these procedures, patient optimisation is becoming of interest to improve outcomes. Anaesthetic choice has been extensively studied in primary THA as a modifiable risk factor for postoperative outcomes, showing favourable results for neuraxial anaesthesia compared to general anaesthesia. The impact of anaesthetic choice in rTHA has not been studied previously. Methods: A retrospective study was performed using the American College of Surgeons National Surgical Quality Improvement Program database. Patients who underwent rTHA between 2014 and 2017 were divided into 3 anaesthesia cohorts: general anaesthesia, neuraxial anaesthesia, and combined general-regional (neuraxial and/or peripheral nerve block) anaesthesia. Univariate and multivariate analyses were used to analyse patient characteristics and 30-day postoperative outcomes. Bonferroni correction was applied for post-hoc analysis. Results: In total, 5759 patients were identified. Of these, 3551 (61.7%) patients underwent general anaesthesia, 1513 (26.3%) patients underwent neuraxial anaesthesia, and 695 (12.1%) patients underwent combined general-regional anaesthesia. On multivariate analysis, neuraxial anaesthesia was associated with decreased odds for any-one complication (OR 0.635; p \u3c 0.001), perioperative blood transfusion (OR 0.641; p \u3c 0.001), and extended length of stay (OR 0.005; p = 0.005) compared to general anaesthesia. Conclusions: Relative to those receiving general anaesthesia, patients undergoing neuraxial anaesthesia are at decreased risk for postoperative complications, perioperative blood transfusions, and extended length of stay. Prospective controlled trials should be conducted to verify these findings

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

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 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

Plasmid and bacterial strains used in this study.

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

Association of Anesthesia Type with Postoperative Outcome and Complications in Patients Undergoing Revision Total Knee Arthroplasty.

© by Thieme Medical Publishers, Inc. Revision total knee arthroplasty (TKA) is an increasingly common procedure and is effective in treating knee osteoarthritis, but it has higher complication rates than primary TKA. Anesthetic choice poses perioperative risk that has been extensively studied in primary TKA, showing favorable results for regional anesthesia compared with general anesthesia. The impact of anesthetic choice in revision TKAs is not well studied. A retrospective cohort study was conducted using the American College of Surgeons National Surgical Quality Improvement Program database. Patients who underwent revision TKAs between 2014 and 2017 were divided into three anesthesia cohorts: (1) general anesthesia, (2) regional anesthesia, and (3) combined general-regional anesthesia. Univariate and multivariate analyses were used to analyze patient characteristics and 30-day postoperative outcomes. Bonferroni correction was applied for post hoc analysis. In total, 8,820 patients were identified. Of whom, 3,192 patients underwent general anesthesia, 3,474 patients underwent regional anesthesia, and 2,154 patients underwent combined anesthesia. After multivariate analyses, regional anesthesia was associated with decreased odds for any complication (p = 0.008), perioperative blood transfusion (p \u3c 0.001), and extended length of stay (p \u3c 0.001) compared with general anesthesia. In addition, regional anesthesia was associated with decreased odds for perioperative blood transfusion (p \u3c 0.001) and extended length of stay (p = 0.006) compared with combined anesthesia. However, following multivariate analysis, regional anesthesia was not associated with decreased odds of wound, pulmonary, renal, urinary tract, thromboembolic, and cardiac complications, and was not associated with return to operating room, extended length of stay, minor and major complications, and mortality. Retrospective analysis of a large surgical database suggests that patients receiving general anesthesia have increased likelihood for developing adverse postoperative outcomes relative to patients receiving regional anesthesia. Prospective and controlled trials should be conducted to verify these findings