scholarly journals Porphyromonas gingivalis Gingipains and Adhesion to Epithelial Cells

2001 ◽  
Vol 69 (5) ◽  
pp. 3048-3056 ◽  
Author(s):  
Tsute Chen ◽  
Koji Nakayama ◽  
Lynn Belliveau ◽  
Margaret J. Duncan
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Surface Proteins ◽  
Heat Denaturation ◽  
Wild Type ◽  
Bacterial Surface ◽  
Catalytic Activities ◽  
Mutant Strains ◽  
Cell Fractions ◽  
High Level

ABSTRACT Porphyromonas gingivalis is one of the principal organisms associated with adult periodontitis. Bacterial surface proteins such as fimbriae and gingipain hemagglutinin domains have been implicated as adhesins that actuate colonization of epithelium lining the gingival sulcus. We investigated the genetics of P. gingivalis adhesion to monolayers of epithelial cells using wild-type and gingipain mutant strains. These experiments suggested that arginine-specific gingipain (Rgp) catalytic activity modulated adhesion. From the data obtained with rgp mutants, we constructed a working hypothesis predicting that attachment and detachment of P. gingivalis to epithelial cells were mediated by gingipain adhesin and Rgp catalytic domains, respectively. A membrane-based epithelial cell binding assay, used to locate adhesins in extracellular fractions of wild-type and mutant strains, recognized gingipain peptides as adhesins rather than fimbriae. We developed a capture assay that demonstrated the binding of gingipain adhesin peptides to oral epithelial cells. The adherence of fimbrillin to epithelial cells was detected after heat denaturation of cell fractions. The prediction that Rgp catalytic activities mediated detachment was substantiated when the high level of attachment of anrgp mutant was reduced in the presence of wild-type cell fractions that contained gingipain catalytic activities.

scholarly journals Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2

2019 ◽  
Vol 98 (10) ◽  
pp. 1150-1158 ◽  
Author(s):  
W. Chen ◽  
A. Alshaikh ◽  
S. Kim ◽  
J. Kim ◽  
C. Chun ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Oral Mucosa ◽  
Porphyromonas Gingivalis ◽  
Oral Bacteria ◽  
Conditional Knockout ◽  
Epithelial Barrier ◽  
Wild Type ◽  
Oral Epithelial ◽  
Junction Proteins

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis ( Pg)–induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides ( Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β- proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

scholarly journals Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3052-3064 ◽  
Author(s):  
S. Suwannakul ◽  
G. P. Stafford ◽  
S. A. Whawell ◽  
C. W. I. Douglas
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Cell Invasion ◽  
Protease Activity ◽  
Periodontal Pathogen ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Specific Protease ◽  
Oral Epithelial ◽  
First Time

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10–30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12–16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

scholarly journals Enhancing the natural toxicity of Salmonella Typhimurium towards tumours

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Najla Alfaqeer ◽  
Donal Wall
Keyword(s):  
Food Poisoning ◽  
Public Health Problem ◽  
Effector Proteins ◽  
Major Public Health Problem ◽  
Wild Type ◽  
Level Of Invasion ◽  
Mutant Strains ◽  
High Level ◽  
Initial Results ◽  
Number Of Bacteria

Background and objective: Salmonella is the underlying cause of foodborne diseases and poses a major public health problem worldwide. Research has developed a method to efficiently treat cancer using some of the same bacteria behind food poisoning, one of these bacteria is Salmonella which targets and penetrats tumours specifically by being attracted to the compounds produced by tumour cells and accumulating at the tumour site and inducing inflammation. In this project we aim to investigate the mechanism of Salmonella Typhimurium which has a tremendous ability to invade, replicate and compete to survive inside the cells by virtue of effector proteins such as: sipA, sipB, and AvrA which it possesses. Method: The S.Typhimurium strains used are wild-type SL1344 ( ΔsipA, ΔsipB, ΔavrA and VV341) and attenuated strain of SL7207. Bacteria were cultured in LB broth in a 37 °C shaker overnight to reach a stationary phase before using it to infect B16F10 (mouse melanoma). Results: The initial results show that the infection of B16F10 with wild-type SL1344 had a high level of invasion compared to the low number of bacteria with the deletion of sipB which impaired its entry into the cell. Similarly, the mutant strains ΔsipA and ΔavrA show an increasing number of intracellular bacteria, like the wild-type strain. We will be investigating further on the innate mechanisms of Salmonella in disrupting tumour growth and progression, that might help maximize the potential of using these bacteria in monotherapy or in tandem with other useful therapies.

scholarly journals Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin

2008 ◽  
Vol 52 (6) ◽  
pp. 2027-2034 ◽  
Author(s):  
Hassan Safi ◽  
Brendan Sayers ◽  
Manzour H. Hazbón ◽  
David Alland
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Single Mutation ◽  
Variable Degree ◽  
Wild Type ◽  
Growth Property ◽  
Major Mechanism ◽  
Mutant Strains ◽  
High Level ◽  
Competition Assays

ABSTRACT Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 μg/ml to 7, 7, 8.5, and 14 μg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 μg/ml or 28 μg/ml, respectively, to 3 μg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.

scholarly journals Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2180-2193 ◽  
Author(s):  
Devon O. Osbourne ◽  
Wilson Aruni ◽  
Francis Roy ◽  
Christopher Perry ◽  
Lawrence Sandberg ◽  
...  
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phenotypic Expression ◽  
Surface Proteins ◽  
Wild Type ◽  
In The Wild

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

scholarly journals Porphyromonas gingivalis Lipid A Phosphatase Activity Is Critical for Colonization and Increasing the Commensal Load in the Rabbit Ligature Model

2013 ◽  
Vol 82 (2) ◽  
pp. 650-659 ◽  
Author(s):  
Camille Zenobia ◽  
Hatice Hasturk ◽  
Daniel Nguyen ◽  
Thomas E. Van Dyke ◽  
Alpdogan Kantarci ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Cellular Responses ◽  
Structural Heterogeneity ◽  
Microbial Load ◽  
Wild Type ◽  
Microbial Composition ◽  
Content Type ◽  
Mutant Strains ◽  
Repeated Applications

ABSTRACTPeriodontitis is a disease of polymicrobial etiology characterized by inflammation, degradation of host tissue, and bone that irreversibly destroys the supporting apparatus of teeth.Porphyromonas gingivaliscontains lipid A with structural heterogeneity that has been postulated to contribute to the initiation of dysbiosis in oral communities by modulating the host response, thereby creating a permissive environment for its growth. We examined twoP. gingivalislipid A phosphatase mutants which contain different “locked” lipid A structures that induce different host cellular responses for their ability to induce dysbiosis and periodontitis in rabbits. Lipopolysaccharide (LPS) preparations obtained from these strains were also examined. After repeated applications of all strains and their respective LPS preparations,P. gingivaliswild type, but not the lipid A mutants, had a significant impact on both the oral commensal microbial load and composition. In contrast, in rabbits exposed to the mutant strains or the LPS preparations, the microbial load did not increase, and yet significant changes in the oral microbial composition were observed. All strains and their respective LPS preparations induced periodontitis. Therefore, the ability to alter the lipid A composition in response to environmental conditions by lipid A phosphatases is required for both colonization of the rabbit and increases in the microbial load. Furthermore, the data demonstrate that multiple dysbiotic oral microbial communities can elicit periodontitis.

scholarly journals The Helicobacter pylori Urease B Subunit Binds to CD74 on Gastric Epithelial Cells and Induces NF-κB Activation and Interleukin-8 Production

2006 ◽  
Vol 74 (2) ◽  
pp. 1148-1155 ◽  
Author(s):  
Ellen J. Beswick ◽  
Irina V. Pinchuk ◽  
Kyle Minch ◽  
Giovanni Suarez ◽  
Johanna C. Sierra ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Fibroblast Cell ◽  
Surface Proteins ◽  
Interleukin 8 ◽  
Gastric Epithelial Cells ◽  
Bacterial Surface ◽  
B Subunit ◽  
H Pylori ◽  
Urease B Subunit

ABSTRACT The pathogenesis associated with Helicobacter pylori infection is the result of both bacterial factors and the host response. We have previously shown that H. pylori binds to CD74 on gastric epithelial cells. In this study, we sought to identify the bacterial protein responsible for this interaction. H. pylori urease from a pool of bacterial surface proteins was found to coprecipitate with CD74. To determine how urease binds to CD74, we used recombinant urease A and B subunits. Recombinant urease B was found to bind directly to CD74 in immunoprecipitation and flow cytometry studies. By utilizing both recombinant urease subunits and urease B knockout bacteria, the urease B-CD74 interaction was shown to induce NF-κB activation and interleukin-8 (IL-8) production. This response was decreased by blocking CD74 with monoclonal antibodies. Further confirmation of the interaction of urease B with CD74 was obtained using a fibroblast cell line transfected with CD74 that also responded with NF-κB activation and IL-8 production. The binding of the H. pylori urease B subunit to CD74 expressed on gastric epithelial cells presents a novel insight into a previously unrecognized H. pylori interaction that may contribute to the proinflammatory immune response seen during infection.

Attachment of Streptococcus Pneumoniae to Human Pharyngeal Epithelial Cells in Vitro—Mechanism of Binding

1984 ◽  
Vol 92 (3) ◽  
pp. 266-269 ◽  
Author(s):  
B Andersson ◽  
A Fogh ◽  
F. JØRgensen ◽  
S Larsson ◽  
H Leffler ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Otitis Media ◽  
Binding Capacity ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Bacterial Surface

To colonize mucosal surfaces and to invade underlying tissues, bacteria need to bind to components of the mucosa. Unattached bacteria are transported away from the surface with the fluid flow. By binding to the nasopharyngeal mucosa, Streptococcus pneumoniae causing otitis media may persist at the site of infection. High binding capacity of the bacterium and increased receptivity of the epithelial cells for attaching bacteria may both contribute to the susceptibility of patients prone to otitis. Thus, epithelial cells from children with frequent episodes of otitis bind attaching bacteria more readily than do cells from age-matched controls. The binding mechanism probably involves bacterial surface proteins and epithelial cell surface glycoconjugate receptors. Evidence is presented that phosphorylcholine, a component of the bacterial surface, as well as epithelial cell receptor analogues, that is, natural or synthetic saccharides analogous to the lactoneoseries of glycolipids, inhibits pneumococcal attachment. Inhibition of bacterial binding in vivo may be a new approach to prophylaxis against otitis media.

scholarly journals Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

2002 ◽  
Vol 70 (2) ◽  
pp. 928-937 ◽  
Author(s):  
Yi Wu ◽  
Seok-Woo Lee ◽  
Jeffrey D. Hillman ◽  
Ann Progulske-Fox
Keyword(s):  
Mutant Strain ◽  
Porphyromonas Gingivalis ◽  
Type Strain ◽  
Virulence Genes ◽  
Wild Type Strain ◽  
Receptor Protein ◽  
Wild Type ◽  
Kb Cells ◽  
Mutant Strains

ABSTRACT An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Δivi10 and Δivi11, which indicated that mutant strains Δivi10 and Δivi11 are less able to survive in this model than the wild-type strain, while Δivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Δivi8, Δivi10, and Δivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Δivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.

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