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1.
Fig 9

Fig 9. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

India ink staining for the detection of capsule. The samples were stained with fuchsine and India ink as described in Materials and Methods. Images were taken under a Zeiss Imager A2 microscope (magnification, ×1,000).

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
2.
Fig 6

Fig 6. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Growth curves of Pg83 and the ΔPG352 mutant. (A) P. gingivalis strains were cultured in normal TSB medium or in this medium supplemented with Neu5Ac (S30 represents 30 μg/ml). (B) P. gingivalis strains were cultured in dextrose-free TSB medium (dTSB) or in dTSB medium supplemented with Neu5Ac (30 μg/ml).

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
3.
Fig 11

Fig 11. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Hematoxylin and eosin staining of lung and liver tissues from mice infected with Pg83 and the ΔPG352 mutant. (A and C) Lung and liver tissues from a mouse infected with Pg83. (B and D) Lung and liver tissues from a mouse infected with the ΔPG352 mutant. In Pg83-infected mouse, the lung and liver both show significant inflammation (arrows indicate granulocytes), while in the mutant-infected mouse, there is minimal to no inflammation.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
4.
Fig 2

Fig 2. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

PG0352 (SiaPg) exhibits neuraminidase activity. (A) Preparation of recombinant PG0352 protein (rSiaPg). (B) Filter paper spot test of rSiaPg. The assay was conducted using 4-MUNANA as the substrate, and the images were processed using the ChemiDoc XRS system (Bio-Rad) with an excitation wavelength of 302 nm and an emission wavelength of 548 nm. Neu5Ac2en is an inhibitor of neuraminidase.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
5.
Fig 4

Fig 4. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Transcriptional analysis of the PG0352 gene. (A) Upstream region of PG0352. The underlined sequences are the −10 and −35 regions of the identified P0352 promoter, and the boldface italic sequence represents the translation start codon of PG0352. (B) 5′-RLM-RACE analysis of the PG0352 transcript. The arrow shows the sequencing direction, and an asterisk (*) indicates the transcriptional start site of the PG0352 transcript.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
6.
Fig 5

Fig 5. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Characterization of the ΔPG352 mutant. (A) RT-PCR analysis of the ΔPG352 mutant. The transcripts of PG0352 and 16S rRNA (positive control) genes in Pg83 and the ΔPG352 mutant were detected by RT-PCR. (B) Western blotting analysis of the ΔPG352 mutant. The same amount of Pg83 and ΔPG352 whole lysates were analyzed by SDS-PAGE and then probed with a specific antiserum against PG0352. (C) Filter paper spot assay using the whole-cell lysates of Pg83 and the ΔPG352 mutant.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
7.
Fig 10

Fig 10. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Survival rates of Pg83 and the ΔPG352 mutant. Two strains were coincubated with 25% fresh serum or heat-inactivated serum for 1 and 3 h. The survival rates were calculated as follows: the total numbers of colonies present in the samples treated with 25% serum divided by the total numbers of colonies in the samples treated with heat-inactivated serum. The results are expressed as the average survival rates of triplicates. The data were statistically analyzed by Student t test at P < 0.01.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
8.
Fig 7

Fig 7. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Biofilm formation of Pg83 and the ΔPG352 mutant. The assay was carried out in 96-well microtiter plates as previously described (, ). The upper panel shows representative wells of Pg83 and the ΔPG352 mutant, and the lower panel gives the average absorbances of Pg83 and the mutant. The error bars represent the standard deviation. The numbers represent the concentration of Neu5Ac in the medium. The data were statistically analyzed by one-way ANOVA, followed by Tukey's multiple comparison at P < 0.01.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
9.
Fig 1

Fig 1. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Sequence alignment of neuraminidases. The underlined sequences represent the conserved domains identified in neuraminidases, including a RIP motif and three “Asp-box” motifs (S/T-X-D-[X]-G-X-T-W/F). Only a part of the alignment is presented. The aligned proteins include: Salmonella enterica serovar Typhimurium strain LT2 NanH (NP_459905), T. forsythia ATCC 43037 NanH (TF0035) (http://www.oralgen.lanl.gov/index.html), and P. gingivalis W83 (PG0352). The alignment was conducted using the program CLUSTAL W2.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
10.
Fig 3

Fig 3. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Lectin blot analysis of rSiaPg. The assay was carried out as previously described () using a DIG glycan differentiation kit (Roche). The DIG-labeled SNA, MAA, and DSA lectins were used to detect terminal α-(2-6)-linked sialic acid, α-(2-3)-linked sialic acid, and galactose linked to the GlcNAc of human α-1 acid glycoprotein (AGP), respectively. The recombinant neuraminidase (rSiaCp) of C. perfringens was used as a positive control for detecting the enzymatic activity (lane 3), and PBS was used as a negative control (lane 1). Arrows point to the products detected by three lectins.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.
11.
Fig 8

Fig 8. From: Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis.

Cellular architectures of Pg83 and the ΔPG352 mutant revealed by cryo-EM and cryo-ET. One central slice of a tomographic reconstruction from Pg83 and ΔPG352 is shown in panels A and B, respectively. (C and D) Corresponding surface views of the reconstructions from Pg83 (A) and ΔPG352 (B), respectively. The prominent structural features include the outer membrane (OM), inner membrane (IM), peptidoglycan layer (PG), and capsular polysaccharide (CPS). Noticeably, the density corresponding to CPS is significantly weaker in ΔPG352. (E) Proteinase K treatment (200 μg/ml for 40 min at 37°C) does not remove the thin CPS layer of the ΔPG352 mutant CPS. (F) Supplementation with Neu5Ac (30 μg/ml) restored the CPS formation in the ΔPG352 mutant. The thickness of the CPS layer in the mutant is ∼30 nm, which is similar to that of Pg83 (G). The black dots are golden particles (diameter, 15 nm) which were used to calibrate the measurements of CPS thickness. The numbers represent the thickness of CPS.

Chen Li, et al. Infect Immun. 2012 Jan;80(1):3-13.

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