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. 2015 Oct 5;10(10):e0139454.
doi: 10.1371/journal.pone.0139454. eCollection 2015.

Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas Gingivalis

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Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas Gingivalis

Ryota Ikai et al. PLoS One. .
Free PMC article

Abstract

Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The mfa4 gene product in P. gingivalis.
(A) Schematic diagram of the mfa gene cluster in P. gingivalis ATCC 33277, based on sequence NC_010729 deposited in NCBI. The cluster contains five genes, mfa1-5, in the same transcriptional orientation. (B) Mfa4 expression. Whole-cell lysates from P. gingivalis JI-1 (lane 1), FMFA4 (lane 2), and FMFA4C (lane 3) were separated by SDS-PAGE, immunoblotted, and probed with Mfa4 antiserum. (C) Subcellular localization. A culture of P. gingivalis JI-1 was harvested by centrifugation. The culture supernatant (CS) was collected and proteins were concentrated by ammonium sulfate precipitation. Whole-cell lysates (W), obtained by lysing cells through French press, were fractionated into soluble (Sol), envelope (Env), inner membrane (IM), and outer membrane (OM) fractions. Mfa1 fimbriae (F) were purified from the soluble fraction and used as positive control. Samples were separated by SDS-PAGE and subjected to immunoblotting using anti-Mfa4 as probe.
Fig 2
Fig 2. Polymerization and expression of Mfa1.
(A) Immunoblot analysis using antiserum against Mfa1 fimbriae. Whole-cell lysates were obtained from P. gingivalis JI-1 (lane 1), FMFA4 (lane 2), and FMFA4C (lane 3). mfa1-deficient SMF-fimA (lane 4) was used as negative control. Samples were denatured at 100°C for 5 min, 80°C for 10 min, or 60°C for 10 min, separated by SDS-PAGE, and analyzed by immunoblotting using antiserum against Mfa1 fimbriae purified from JI-1. Incomplete disassembly of Mfa1 polymers at 60–80°C generates a ladder of oligomers. (B) Filtration ELISA of intact cells. P. gingivalis ATCC 33277, JI-1, FMFA4, FMFA4C, SMF-fimA, and rgpA/B-deficient KDP112 were applied over filters in a filtration plate at 5×106 cells/well. Bacterial cells were probed with antibodies against Mfa1-only fimbriae purified from FMFA5, and then with peroxidase-conjugated goat anti-rabbit IgG at a dilution of 1:1000. Data are mean ± SD from two experiments with triplicates. *, statistically significant difference from JI-1 based on Dunnett’s test (p < 0.01).
Fig 3
Fig 3. Mfa3 expression and localization in P. gingivalis JI-1, FMFA4, and FMFA4C.
Proteins in the culture supernatant (CS) were concentrated by ammonium sulfate precipitation, while whole-cell lysates (W) were fractionated into soluble (Sol), envelope (Env), inner membrane (IM), and outer membrane (OM) fractions. Samples were separated by SDS-PAGE and probed with Mfa3 antibodies by immunoblotting.
Fig 4
Fig 4. Mfa5 expression and localization in P. gingivalis JI-1, FMFA4, and FMFA4C.
Culture supernatants (CS) and whole-cell lysates (W) from JI-1, FMFA4, and FMFA4C were separated by SDS-PAGE and probed with Mfa5 antibodies by immunoblotting. Structurally intact Mfa1 fimbriae purified from JI-1 (F), which contains Mfa5, were used as positive control. Possible Mfa5 degradation products with molecular weight 100 and 78 kDa are marked with *.
Fig 5
Fig 5. Components of purified Mfa1 fimbriae.
(A-C) Mfa1 fimbriae purified from JI-1 (lane 1) and FMFA4 (lane 2) were examined by SDS-PAGE and stained with Coomassie brilliant blue R-250 (A), or analyzed by immunoblotting using anti-Mfa3 (B) and anti-Mfa5 (C). (D) The N-terminus of Mfa4. Amino acids in boldface were identified by Edman degradation of mature Mfa4 incorporated into JI-1 fimbriae.
Fig 6
Fig 6. Auto-aggregation and biofilm formation.
(A) Relative turbidity, expressed as % of the initial optical density at 660 nm, is plotted as a function of time. Data points are mean ± SD from triplicate experiments. (B) Biofilm formation by ATCC 33277, JI-1, SFM4, SMF4C, and SMF-fimA, as measured by the crystal violet method. Data are means ± SD (n = 8). *, p < 0.01 on Dunnett’s test against JI-1.
Fig 7
Fig 7. Processing of Mfa1-4 in P. gingivalis KDP112.
Whole-cell lysates from JI-1 (lane 1) and KDP112 (lane 2) were separated by SDS-PAGE and immunoblotted using antibodies against Mfa1 (A), Mfa2 (B), Mfa3 (C), or Mfa4 (D). Lane 3 in each panel is a negative control containing whole-cell lysates from SMF-fimA, JI-12, FMFA3, or FMFA4, respectively. Possible unprocessed, immature forms are marked with *.

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