Limits...
The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

Pantaléon V, Soavelomandroso AP, Bouttier S, Briandet R, Roxas B, Chu M, Collignon A, Janoir C, Vedantam G, Candela T - PLoS ONE (2015)

Bottom Line: Of note, the toxin TcdA was found in the biofilm matrix.In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo.Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

View Article: PubMed Central - PubMed

Affiliation: EA4043, Faculté de Pharmacie, Université Paris Sud, Châtenay-Malabry, France.

ABSTRACT
Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

No MeSH data available.


Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414356&req=5

pone.0124971.g006: 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).

Mentions: We assessed cell-surface biophysical impact on the cwp84 mutant since the surface proteome differed from that of the parental strain, SlpA was not cleaved, and specific proteins predicted to be involved in cell-surface remodeling (CD630_18980; cell-wall hydrolase) were differentially abundant. Standard bacterial hydrophobicity assays were performed using the alkane hydrocarbon hexadecane. The parental 630Δerm strain as well as the complemented cwp84 mutant were weakly hydrophobic (3.91% ± 0.6% and 1.02% ±0.96% respectively), whereas the cwp84 mutant was significantly highly hydrophobic (44.27% ± 4.59; p<0.005; paired Student t test) in comparison with the parental strain. In other species, such as Lactococcus lactis, adhesion correlates with hydrophobicity [18]. We therefore assessed if altered hydrophobicity impacted bacterial adherence to abiotic surfaces, theorizing that initial attachment may have downstream consequences on biofilm formation. Parental, mutant and the complemented strains were analyzed. Initial adhesion levels were very low (ranging from 0.1%- 0.5%), and no significant differences were observed between strains (Fig 6) suggesting that Cwp84 was not involved in initial adhesion of C. difficile on the tested surface, the first biofilm step.


The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

Pantaléon V, Soavelomandroso AP, Bouttier S, Briandet R, Roxas B, Chu M, Collignon A, Janoir C, Vedantam G, Candela T - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414356&req=5

pone.0124971.g006: 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).
Mentions: We assessed cell-surface biophysical impact on the cwp84 mutant since the surface proteome differed from that of the parental strain, SlpA was not cleaved, and specific proteins predicted to be involved in cell-surface remodeling (CD630_18980; cell-wall hydrolase) were differentially abundant. Standard bacterial hydrophobicity assays were performed using the alkane hydrocarbon hexadecane. The parental 630Δerm strain as well as the complemented cwp84 mutant were weakly hydrophobic (3.91% ± 0.6% and 1.02% ±0.96% respectively), whereas the cwp84 mutant was significantly highly hydrophobic (44.27% ± 4.59; p<0.005; paired Student t test) in comparison with the parental strain. In other species, such as Lactococcus lactis, adhesion correlates with hydrophobicity [18]. We therefore assessed if altered hydrophobicity impacted bacterial adherence to abiotic surfaces, theorizing that initial attachment may have downstream consequences on biofilm formation. Parental, mutant and the complemented strains were analyzed. Initial adhesion levels were very low (ranging from 0.1%- 0.5%), and no significant differences were observed between strains (Fig 6) suggesting that Cwp84 was not involved in initial adhesion of C. difficile on the tested surface, the first biofilm step.

Bottom Line: Of note, the toxin TcdA was found in the biofilm matrix.In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo.Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

View Article: PubMed Central - PubMed

Affiliation: EA4043, Faculté de Pharmacie, Université Paris Sud, Châtenay-Malabry, France.

ABSTRACT
Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

No MeSH data available.


Related in: MedlinePlus