Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

Abstract

Background: Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.

Results: Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these.

Conclusions: Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

Figure 1

Immuno-fluorescence microscopy of C. diphtheriae wild-type and mutant strains. An antiserum directed against the surface proteome of C. diphtheriae was used as primary antibody; Alexa Fluor 488 goat anti-rabbit was used as secondary antibody. A: ISS3319, B: Lilo1, C: ISS4060, D: Lilo2.

Figure 2

LIVE/DEAD staining of C. diphtheriae wild-type and mutant strains. Green fluorescent bacteria have a functional cytoplasmic membrane and are stained green, red propidium iodide staining indicates non-viable cells. A: ISS3319, B-C: Lilo1, D: ISS4060, E-F: Lilo2, C and F: cells subjected to 5 min of vigorous vortexing.

Figure 3

Analysis of surface proteins. Surface proteins were isolated from C. diphtheriae wild-type and mutant strains and subjected to SDS-PAGE (A), Western blotting (B), and 2-D PAGE (C). For SDS-PAGE 25 μg of protein prepared from strains ISS3319 (lane 2), Lilo1 (lane 3), ISS4060 (lane 4), and Lilo2 (lane 5) were applied per lane on a 10% polyacrylamide gel and silver-stained after electrophoresis. Molecular weight of marker proteins (lane 1, from top to bottom): 250, 130, 95, 72, 55, 36, 28, 17, 11 kDa. Western blotting was carried out after SDS-PAGE using a polyclonal antiserum directed against C. diphtheriae DSM44123 surface proteins. For 2-D PAGE surface protein preparations were separated according to their isoelectric point and molecular mass using a pH range of 3-10 for isoelectric focussing and 12.5% polyacrylamide gels for SDS-PAGE. Gels were stained with Coomassie Brilliant Blue. Molecular weight of marker proteins (from top to bottom): 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15 kDa.

Figure 4

Ultrastructural analysis of the cell surface of C. diphtheriae strains. (A) ISS3319, (B) Lilo1; red boxes in the low magnification images on the left hand side mark three areas shown with a higher magnification on the right hand side (upper row: topography/height, lower row: phase). Colour scale bars at the right hand side give height and phase magnitudes.