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. 2009 Oct;155(Pt 10):3333-3347.
doi: 10.1099/mic.0.028928-0. Epub 2009 Jul 9.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

Yoshiaki Hasegawa  1 , Jun Iwami  2   1 , Keiko Sato  1 , Yoonsuk Park  3 , Kiyoshi Nishikawa  1 , Tatsuo Atsumi  4   1 , Keiichi Moriguchi  5 , Yukitaka Murakami  1 , Richard J Lamont  3 , Hiroshi Nakamura  2 , Norikazu Ohno  5 , Fuminobu Yoshimura  1
Affiliations

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

Yoshiaki Hasegawa et al. Microbiology (Reading). 2009 Oct.

Abstract

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

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Figures

Fig. 1.
Fig. 1.
Mfa1 fimbriae-associated genes in P. gingivalis ATCC 33277. Mfa1 fimbrial genes in 33277 are shown in parallel with those in W83. The mfa1 gene has been previously sequenced in ATCC 33277 and 381 and deposited as a 67 kDa fimbrilin and a cell surface protein (AB016284 and D28770), respectively (Hamada et al., 2002; Ogawa et al., 1994), although the corresponding gene of W83 is split into pg0176 and pg0178 by insertion of pg0177 (ISPg4, transposase) (Park et al., 2005). W83 produces neither FimA nor Mfa1 fimbriae, presumably due to disruption of fimS (Hayashi et al., 2000) and mfa1, respectively. The mfa2 gene (annotated as pgn0288 in 33277 or pg0179 in W83, indicated by the grey bars with pointed ends) in 33277 was sequenced in this study, and the 33277 genome database was utilized for the sequence of genes downstream of mfa2. The gene products of pgn0289, pgn0290 and pg0291 were identified as minor components of pure Mfa1 fimbriae in JI-1 (ΔfimA) by MALDI-TOF MS, as described in the text (and also shown in Fig. 3, Table 3).
Fig. 2.
Fig. 2.
Unusual distribution of filaments of the Mfa1 fimbriae in the mfa2 mutant. (a) Partial protein profile of the parental 33277 (lane 1), mfa2 mutant JI-2 (lane 2) and complemented mutant strain JI-3 (lane 3). Whole cultures (Cells+Sup), cells and culture supernatants (Sup) were treated with cold TCA in order to recover all proteins and to destroy intrinsic, strong proteolytic activities of this organism (Nishiyama et al., 2007). The TCA precipitates were neutralized and separated by SDS-PAGE, followed by CBB staining. (b) Western immunoblotting of panel (a) using anti-Mfa1 serum. A band in lane 2 in Sup of (a), indicated by an arrow, was identified as Mfa1 by antiserum [lane 2 in Sup of (b)].
Fig. 3.
Fig. 3.
Identification of the minor components of the Mfa1 fimbriae. A typical SDS-PAGE pattern of highly purified P. gingivalis Mfa1 fimbriae is shown together with that of FimA fimbriae. Both FimA (lane FimA) and Mfa1 fimbriae (lane Mfa1) were purified without using any detergent from the mutants SMF1 (Δmfa1) and JI-1 (ΔfimA), respectively. Protein bands are numbered from 1 to 10 together with arrows to identify them by MALDI-TOF MS (see also Table 3).
Fig. 4.
Fig. 4.
Localization of Mfa2. (a) Localization of Mfa2 in the P. gingivalis cell. P. gingivalis whole-cell lysate (WCL), prepared by physical lysis, was fractionated into envelope (Env) and soluble (Sol) fractions, and Mfa2 was detected by Western blotting as described above. Lanes: 1, parental strain 33277; 2, JI-2; 3, JI-3. The band immunoreactive to anti-Mfa2 antiserum, which has a molecular mass of 24 kDa, appeared to cross-react with the antibody, but is unrelated to Mfa2 as it was present in the mfa2 mutant. (b) Localization of Mfa2 in the outer membrane fraction. The envelope fractions were separated into outer membrane (OM) and inner membrane (IM) fractions, and Western blotting using Mfa2 antiserum was performed. The lanes are the same as for (a). (c) Absence of Mfa2 in the purified Mfa1 fimbriae. Mfa2 was detected by Western blotting. Lane WCL, whole-cell lysate of JI-1 (1 μg protein); lane Mfa1 fimbriae, the purified Mfa1 fimbriae from JI-1 (5 μg protein). (d) Immunogold electron microscopy. Ultra-thin sections of JI-1 (ΔfimA) and JI-12 (ΔfimA and Δmfa2) cells were incubated with anti-Mfa2 serum, followed by labelling with 20 nm colloidal gold-labelled goat anti-rabbit serum. Bars, 200 nm.
Fig. 5.
Fig. 5.
Physical association and co-localization between Mfa1 and Mfa2. Mfa1 or Mfa2 was immunoprecipitated from whole-cell lysate of 33277 with anti-Mfa1 or anti-Mfa2 serum. The precipitates were separated by SDS-PAGE (a), followed by Western blotting using either anti-Mfa1 (b) or anti-Mfa2 serum (c). Lanes: 1, 33277 precipitate with anti-Mfa1; 2, 33277 precipitate with anti-Mfa2; 3, 33277 precipitate with anti-OmpA (Pgm6/7) as a negative control. The positions of Mfa1, Mfa2 and molecular mass markers are indicated. (d) Single-labelled, immunogold electron microscopy. Ultra-thin sections of JI-1 (ΔfimA) were reacted with chicken anti-Mfa1, followed by 6 nm gold-labelled anti-chicken Ig. Bar, 100 nm. (e) Double-labelling immunogold microscopy. The sections were first reacted with chicken anti-Mfa1 and rabbit anti-Mfa2 serum, followed by incubation with 6 nm gold-labelled anti-chicken Ig and 20 nm gold-labelled anti-rabbit IgG. Bar, 50 nm.
Fig. 6.
Fig. 6.
Electron micrographs of the cell and cell surface of the parental and mutant strains. A part of the cell surface of each strain is enlarged in the lower panel. (a) ATCC 33277, (b) JI-1 (ΔfimA), (c) JI-12 (ΔfimA and Δmfa2), (d) complemented strain JI-4. All strains were negatively stained with 1 % (w/v) ammonium molybdate. Bars, 100 nm.
Fig. 7.
Fig. 7.
Effect of mfa2 on the Mfa1 fimbriae length. (a) Electron micrograph of the purified Mfa1 fimbriae from JI-12 (ΔfimA and Δmfa2) negatively stained with 1 % (w/v) ammonium molybdate. The samples were observed under a Carl-Zeiss LEO LIBRA120 electron microscope. Bar, 100 nm. (b) Electron micrograph of the purified Mfa1 fimbriae from JI-1 (ΔfimA). The sample was negatively stained with 1 % (w/v) ammonium molybdate and observed under a JEM-1200EX (JEOL). Bar, 100 nm.
Fig. 8.
Fig. 8.
Time-course of autoaggregation of the wild-type and various mutants. The OD600 value of each cell suspension was measured at the indicated time points. Relative turbidities, defined as the relative OD600 value (as a percentage) normalized to the initial value of each suspension, are plotted against incubation time. All assays were performed in triplicate and the means±sd are shown. ○, Wild-type strain ATCC 33277; •, JI-1 (ΔfimA); □, JI-2 (Δmfa2); ▪, JI-12 (ΔfimA and Δmfa2); ▵, JI-3 (JI-2 containing pTCBex-mfa2, complemented strain); ▴, JI-4 (JI-12 containing pTCBex-mfa2, complemented strain).
Fig. 9.
Fig. 9.
Transcriptional analysis of the genes for Mfa1 fimbrial components. (a) The predicted PCR fragments from mfa1 to pgn0291 are indicated as thin lines at the right side of the numbers (1–11). The possible operon is shaded. Gene annotation corresponds to the 33277 genome database. (b) RT-PCR of 33277 mRNAs from mfa1 to pgn0291. RT-PCR amplification of intragenic and intergenic segments within the mfa1 gene cluster was carried out with primer pairs listed in Table 2. Results labelled as DNA indicate that PCR was carried out using chromosomal DNA as a template, showing that each primer set was appropriate, and those labelled as RNA indicate that RT-PCR was carried out without reverse transcriptase as negative controls, showing that the RNA preparation was pure, without contamination of DNA. Therefore, results labelled as cDNA indicate the presence or absence of mRNA. The lane numbers correspond to those in panel (a). Lanes: 1, primers for mfa1; 2, primers for mfa2; 3, primers for pgn0289; 4, primers for pgn0290; 5, primers for pgn0291; 6, primers for a region spanning mfa1 and mfa2; 7, primers for a region spanning mfa1 and pgn0289; 8, primers for a region spanning mfa2 and pgn0289; 9, primers for a region spanning mfa2 and pgn0290; 10, primers for a region spanning pgn0289 and pgn0290; 11, primers for a region spanning pgn0290 and pgn0291.
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