Fig 1.
The cwp84 mutant strain forms a robust biofilm.
cwp84 mutant grown in BHIS + glucose for 72 hours in liquid culture forms macroscopic structures, whereas the 630Δerm parent and the complemented cwp84 mutant strains do not (Panel A). Insets below the centrifuge tubes are magnified views of the box depicted on the tubes (the scale bar corresponds to 0.1 cm). Panel B shows biofilms of the parental 630Δerm strain, the cwp84 mutant and the complemented cwp84 mutant strain depicted before (Bi) or after (Bii) crystal violet staining. Panel C depicts biofilm quantitation. Data are representative of at least three independent experiments, each performed in triplicate. The error bars represent standard deviation. Panel D depicts enumeration of biofilm-associated bacteria generated by the various genotypes shown. Significantly different (p < 0.05) ratios are indicated by asterisks (Wilcoxon test for the comparison of Δerm 630/cwp84 and Student t tests for the others).
Fig 2.
C. difficile biofilms are visualized by CLSM and electron microscopy.
Biofilms elaborated by the 630Δerm parent strain (A) and the cognate cwp84 mutant and complemented strains (B and C respectively) were visualized by CLSM. The bar corresponds to a height of 50μm, captured via z-axis scans. Biofilm thickness averaged 39.2± 6.9μm for the cwp84 mutant strain and was significantly higher (p<0.01; paired Student t test) than that of the parent and complemented strains (17.7±5.8μm and 18.5±2.8 μm respectively). A top-down view using electron microscopy is shown for the parental and cwp84 mutant strains, in lower magnification (D and E respectively) or in higher magnification (F and G respectively). A side-view is shown for the parental and cwp84 mutant strains at lower magnification (H and I respectively). The cwp84 mutant biofilm (60.3±4 μm thick) is significantly larger than the biofilm formed by the parental strain (11.5±1.6 μm) (p<0.001; paired Student t test). Black bars, 5μm.
Fig 3.
S-layer profiling of C. difficile strains.
630Δerm (lane 1), cwp84 mutant (lane 2), cwp84 mutant+cwp84 (lane 3), cwp84 mutant+cwp84* (lane 4) grown in planktonic culture before protein extraction. S-layer proteins were analyzed using SDS-PAGE (Panel A) or via immunoblots using antisera specifically directed against Cwp84 (Panel B) or the LMW-SlpA P36 (Panel C). Equivalent amounts of proteins were loaded in each well.
Table 1.
Ribotypes and SlpA accession numbers (Genbank, http://www.ncbi.nlm.nih.gov/genbank) used in the study.
Fig 4.
Biofilm-forming ability is independent of SlpA primary sequence.
Biofilm propensity of different strains of C. difficile (X-axis) was compared using crystal violet staining (Y-axis; filled green bars). SlpA primary amino acid sequence was also determined for these same strains, and percentage identity (filled red dot) compared with that of the 1064 comparator strain. The strains evaluated were P30, 4684/08, 3457, 95–1078, R20291, VPI11186, CD196, 79685, CD4, 630Δerm, IT1106 and 95–1578 (see Table 1 for details). Biofilms formed by 1064, P30, 4684/08 and 3457 (group 1, OD570>4.6), 95–1078, R20291, VPI11186, CD196 and 79685 (group 2; 1.5 <OD570< 2.65) and CD4, 630Δerm, IT1106 and 96–1578 (group 3; OD570< 0.95) are significantly different (p<0.01; Student t test). However, amino acid sequences between groups are not significantly different.
Table 2.
Distribution of surface, matrix and released (secretome) proteins of the 630Δerm parental strain.
Table 3.
Proteins increased in abundance in the cwp84 mutant biofilm proteome compared with the 630Δerm parental strain biofilm proteome.
Table 4.
Proteins increased in abundance in the 630Δerm parental strain biofilm proteome compared with the cwp84 mutant biofilm proteome.
Fig 5.
C. difficile biofilm development.
Schematic representation of the C. difficile plate-grown biofilm. Matrix-associated and supernatant (planktonic) bacteria are shown. Specific individual biofilm fractions were harvested for proteomics as described in the Methods.
Fig 6.
Parental and cwp84 mutant strains display similar adhesion to abiotic surfaces.
Percentage initial adhesion of 630Δerm (black bars), cwp84 mutant (white bars), cwp84 mutant +cwp84 (grey bars) strains to a polypropylene matrix for 15–120 minutes. Error bars represent standard deviation. Data are representative of four independent experiments each performed in triplicate. The observed differences are not significant (Student t test).
Fig 7.
cwp84 mutant displays a growth defect only in planktonic culture.
The 630Δerm (●) and cwp84 mutant (□) strains were grown in competition in agitated planktonic culture (Panel A) or recovered from biofilm supernatants (non-settled/escaped bacteria; Panel B), and colony forming units (CFU) enumerated in three independent experiments. The planktonic cultures of both strains are significantly different (p<0.05; Student t test) whereas there is no significant difference for bacteria grown in biofilm, except for bacteria from the biofilm supernatant at 24h. Panel C, for biofilm-associated bacteria, colony counts (CFU), biomass after competition (grey bars, in vitro competition 630Δerm and cwp84 mutant strains) or biomass after individual growth (630Δerm, black bars; cwp84 mutant, white bars) were determined. Error bars represent standard deviation of the mean. Biofilms of strains in competition were significantly different to those formed by individual strains (p <0.05; Student t test). Significantly different ratios are indicated by asterisks.
Fig 8.
In vivo competition of parental and cwp84 strains.
The 630Δerm parent strain (●) and cwp84 mutant (□) were grown in competition in axenic mice for up to 5 days. In vivo analyses of bacterial counts were performed using feces or via cecal enumeration as described in the Methods. Error bars represent standard deviation. Significantly different (p < 0.05, Student t test) ratios are indicated by asterisks.