Identification of a novel protein promoting the colonization and survival of Finegoldia magna, a bacterial commensal and opportunistic pathogen

Abstract

Anaerobic bacteria dominate the human normal microbiota, but strikingly little is known about these commensals. Finegoldia magna is a Gram-positive anaerobe found in the skin and at other non-sterile body surfaces, but it is also an opportunistic pathogen. This study describes a novel protein designated FAF (F. magna adhesion factor) and expressed by more than 90% of F. magna isolates. The protein is present in substantial quantities at the F. magna surface but is also released from the surface. FAF forms large protein aggregates in solution and surface-associated FAF causes bacterial clumping. In skin F. magna bacteria were localized to the epidermis, where they adhere to basement membranes. FAF was found to mediate this adhesion via interactions with BM-40, a basement membrane protein. The biological significance of FAF is further underlined by the observation that it blocks the activity of LL-37, a major human antibacterial peptide. Altogether, the data demonstrate that FAF plays an important role in colonization and survival of F. magna in the human host.

Fig. 1

Analysis of F. magna aggregation. A. F. magna strains ALB8 (▪) and 505 (□) were grown under strict anaerobic conditions at 37°C for 3 days in TH broth supplemented with 0.5% Tween-80. Bacteria were resuspended and left to settle at 4°C. The optical density (620 nm) in the upper half of the test tubes was measured at various time points. B. Left panel: scanning electron micrographs showing ALB8 and 505 bacteria from 3 days cultures in TH broth. The bar represents 10 μm. Right panel: Transmission electron micrographs of the same cultures. The bar represents 0.5 μm.

Fig. 2

Protein FAF self-associates. A. Solubilized surface proteins from F. magna ALB8 bacteria were separated by SDS-PAGE. Two identical gels (10%) were run simultaneously; one was stained with Coomassie blue (STAIN), and one was blotted onto a PVDF membrane and probed with ¹²⁵I-labelled recombinant FAF (BLOT). Lane 1: proteins released with CNBr; lane 2: proteins shedded from the surface of ALB8 bacteria and purified as described (see Experimental procedures); lane 3: recombinant FAF; lane 4: recombinant protein PAB. Bands indicated in the figure with a star were excised and subjected to N-terminal amino acid sequencing. B. Electron micrographs after negative staining of untreated and CNBr-treated ALB8 bacteria. The bar represents 100 nm. C. Schematic representation of FAF. The signal sequence (Ss), the alanine rich region (A), the methionine residue (Met), the wall spanning (W) and the membrane spanning (M) regions are indicated. The fragment obtained by CNBr treatment and the recombinantly expressed fragments of FAF are shown below. Numbers refer to amino acid residue positions. D. Different amounts of FAF, fragments of FAF, protein PAB and GST were applied onto a PVDF membrane. The membrane was incubated with ¹²⁵I-labelled FAF, and binding was detected using the Fuji Imaging System. E. The binding of ¹²⁵I-labelled FAF to ALB8 bacteria at a concentration of 2 × 10⁹ cfu ml⁻¹ was inhibited with various amounts of unlabelled intact FAF (□), fragment I (◊), fragment II (○) or fragment III (▵) of FAF (see 2C) or protein PAB ().

Fig. 3

Secondary structure of protein FAF. A. Recombinant intact FAF at a concentration of 0.2 mg ml⁻¹ was analysed by CD spectroscopy. The far UV spectral region at 20°C is shown. B. Coiled-coil prediction of FAF according to (Lupas et al., 1991) using the Macstripe program showing the probability on the y-axis and amino acid residue numbers on the x-axis. C. Electron micrographs after negative staining of FAF shedded from ALB8 bacteria (ALB8 extract) or recombinant FAF (rFAF). The bar represents 100 nm (left panel). Right panel: selected particles shown at higher magnification (bar = 50 nm).

Fig. 4

Analysis of FAF in seven F. magna strains. Proteins released by CNBr from the isolates were separated by SDS-PAGE. Two identical gels (10%) were run simultaneously; one was stained with Coomassie blue (STAIN), and one was blotted onto a PVDF membrane and probed with antibodies against FAF. Lane 1: strain 505; lane 2: strain ALB8; lane 3: strain L3410; lane 4: strain 1462; lane 5: strain 2133; lane 6: strain 1766; lane 7: strain ELTI. Bands indicated in the figure with a star were excised and subjected to N-terminal amino acid sequencing.

Fig. 5

F. magna bacteria adhere to basement membranes. Human skin biopsies were incubated with ALB8 or 505 bacteria for 1 h at room temperature. Non-adherent bacteria were removed by washing in PBS, and the biopsies were incubated anaerobically at 37°C for 48 h, washed with PBST, fixed and prepared for SEM. A. ALB8 bacteria; B. 505 bacteria. The arrows point at ALB8 colonies and the arrowheads point at the basement membrane covering the junction between epidermis and dermis. The bar represents 10 μm.

Fig. 6

FAF interacts with BM-40. A. Surface plasmon resonance spectroscopy showing the affinity between the basement membrane component BM-40 and FAF. BM-40 was immobilized onto a CM5 sensorchip, and protein FAF was injected at concentrations from 31 to 250 nM at a flow rate of 50 μl min⁻¹. B. Intact FAF, fragments of FAF (see Fig. 2C), protein PAB and GST were applied onto PVDF membranes. Membranes were probed with ¹²⁵I-labelled BM-40, and binding was detected using the Fuji Imaging System. C. Electron micrographs after negative staining of FAF in complex with BM-40. The globular N-terminal GST-tag of recombinant FAF is indicated by arrows. BM-40 binds to the C-terminal part of FAF (arrowheads). At the concentrations used (10 nM) no FAF dimer formation was observed. The bar represents 25 nm.

Fig. 7

FAF-expressing F. magna bacteria are found at the basement membrane in human epidermis and colocalize with BM-40. Human skin biopsies were anaerobically incubated with F. magna ALB8 bacteria for 48 h at 37°C and prepared for ultrathin sectioning/transmission electron microscopy. A. An overview showing the epidermis, the basement membrane at the epidermal/dermal junction (B) and dermis. Arrows point at ALB8 bacteria. The bar represents 1 μm. B. The square in A indicates the selected area shown at higher magnification here. FAF-expressing ALB8 bacteria colocalize with BM-40 at the basement membrane (B). Arrows point at anti-FAF antibodies (10 nm gold) and arrowheads point at anti-BM-40 antibodies (5 nm gold). The bar represents 100 nm. C. Scanning electron micrograph of a thin section of a skin biopsy from a healthy donor. Colonies of cocci (arrows) are found at the basement membrane (B) at the epidermal/dermal junction. D. Interpretation of C in pseudocolours. The scale bar represents 10 μm. E. Ultrathin sectioning and transmission electron microscopy of a skin biopsy from a healthy donor after immuno-labelling of protein FAF. Bacteria labelled with colloidal gold are seen at the basement membrane (B). The bar represents 100 nm. F. A selected area indicated by the square in E at higher resolution is shown. Fibrillar protrusions labelled with colloidal gold (arrows) extend from the bacterial surface and interact with the basement membrane (B). The bar represents 50 nm.

Fig. 8

Protein FAF interferes with the antibacterial activity of LL-37. A. Various amounts of the antibacterial peptides LL-37 and α-defensin and BSA were applied to a PVDF membrane. The membrane was incubated with ¹²⁵I-labelled FAF, and binding was detected using the Fuji Imaging System. B. Left panel: F. magna strains ALB8 and 505 (2 × 10⁶ cfu ml⁻¹) were incubated with the antibacterial peptide LL-37 at various concentrations for 1 h at 37°C, and the cfu were determined. The bars represent the mean ± standard error of the mean of at least three experiments. Right panel: The bactericidal activity of LL-37 at 2.2 μM against 505 bacteria was inhibited with recombinant FAF. Experiments were repeated at least three times and a representative experiment is shown. C. ALB8 and 505 bacteria were grown under strict anaerobic conditions at 37°C for 3 days in TH broth supplemented with 0.5% Tween-80. Bacteria were washed and incubated with LL-37 and anti-FAF antibodies labelled with colloidal gold for 30 min at room temperature (LL-37, 5 nm gold; anti-FAF IgG, 20 nm gold). Following negative staining, samples were analysed by electron microscopy. The bar represents 100 nm. A selected area indicated by the square is shown at higher resolution; bar represents 50 nm.