Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans

Abstract

Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.

Fig. 1.

Partial organization of the tad operon. Protein-coding genes are represented by arrows. Horizontal lines represent intergenic regions. Vertical lines in flp1 represent premature stop codons. Symbols below each gene indicate the detection (+) or absence ( − ) of mRNA or protein in A. actinomycetemcomitans VT1169. igr, Intergenic region; nd, not determined.

Fig. 2.

A. actinomycetemcomitans colony morphology. Bacteria were grown on solid TSBYE medium for 3 days at 37 °C in a humidified 10 % CO₂ atmosphere. Images were taken using a Leica MZ16F stereomicroscope. (a) WT (VT1169); (b) morC mutant (VT1650); (c) WT/pKM2/flp1-tadV (KM611); (d) morC mutant/pKM2/flp1-tadV (KM612); (e) fimbriated clinical isolate (VT1257). Bar, 1 mm.

Fig. 3.

Transmission electron microscopy of A. actinomycetemcomitans strains. Whole-mount negatively stained preparations of A. actinomycetemcomitans strains grown on solid medium were analysed by transmission electron microscopy. (a) Fimbriated clinical isolate (VT1257); (b) WT (VT1169); (c) morC mutant (VT1650); (d, e) WT (VT1169)/pKM2/flp1-tadV (KM611); (f) morC mutant/pKM2/flp1-tadV (KM612). The arrow indicates a fimbrial stub. Bar, 100 nm.

Fig. 4.

Transmission electron microscopy of fimbriae. Whole-mount negatively stained preparations of A. actinomycetemcomitans strains grown on solid medium were analysed by transmission electron microscopy and inter-bacterial regions containing fimbriae were extracted. (a) Fimbriated clinical isolate (VT1257); (b, c) WT (VT1169)/pKM2/flp1-tadV (KM611). Bar, 50 nm.

Fig. 5.

Quantification and confocal microscopy of biofilm formation of strains of A. actinomycetemcomitans. (a) Quantification of biofilm mass using a crystal violet assay. A. actinomycetemcomitans strains were grown as biofilms in glass-bottomed dishes and stained with SYTO 9. A series of Z-stack images was generated with a Zeiss LSM 510 META confocal microscope, and biofilm area was quantified using ImageJ. Asterisks indicate significant difference from the WT fimbriated strain (ANOVA with Dunnett's post-test, P < 0.05). A, Fimbriated clinical isolate (VT1257); B, WT (VT1169); C, morC mutant (VT1650); D, WT/pKM2/flp1-tadV (KM611); E, morC mutant/pKM2/flp1-tadV (KM612). Results are shown as means ± sd. (b) Representative fields of confocal microscopy of biofilms formed by the strains in (a). Upper left: Fimbriated clinical isolate (VT1257); middle: WT (VT1169; upper right: morC mutant (VT1650); lower left: WT/pKM2/flp1-tadV (KM611); lower right: morC mutant/pKM2/flp1-tadV (KM612). Bar, 20 μm.

Fig. 6.

Transmission electron micrographs of A. actinomycetemcomitans strains. Whole-mount negatively stained preparations of A. actinomycetemcomitans strains grown on solid medium were analysed by transmission electron microscopy and showed whole bacteria and inter-bacterial regions of WT (VT1257) (a) and the morC mutant (KM700) (b, c). Bar, 0.5 μm.

Fig. 7.

Transmission electron micrographs and quantification of fimbriae production of clinical and isogenic morC mutant strains of A. actinomycetemcomitans. (a, b) Whole-mount negatively stained preparations of A. actinomycetemcomitans strains grown on solid medium were analysed by transmission electron microscopy for WT (VT1257) (a) and the morC mutant (KM700) (b) Bar, 100 nm. (c) Quantification of surface-associated fimbriae assessed by ELISA. Asterisks indicate a significant difference from the WT strain (t-test, ***P < 0.001).

Fig. 8.

Confocal microscopy of biofilms generated by clinical strains of A. actinomycetemcomitans. A. actinomycetemcomitans strains were grown as biofilms in glass-bottomed dishes and stained with SYTO 9. A series of Z-stack images was generated with a Zeiss LSM 510 META confocal microscope. (a) Fimbriated clinical isolate (VT1257); (b) morC mutant (KM700). Images are representative of three separate experiments. Bar, 20 μm.