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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

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.
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pone.0124971.g004: 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.

Mentions: Since Cwp84 processes SlpA, and, as demonstrated above, that its proteolytic activity impacts biofilm formation, it is formally possible that the cwp84 mutant phenotype is manifested via uncleaved SlpA, suggesting that SlpA may be of importance in the biofilm formation in C. difficile. To date, slpA has not been disrupted in C. difficile, likely because it is essential [17]. Despite multiple attempts, we were also not able to inactivate it (data not shown). Therefore, to investigate if SlpA impacts biofilm formation in C. difficile, 13 strains expressing different slpA alleles were tested for their biofilm-forming abilities (Fig 4). The 1064, P30, 4684/08 and 3457 strains produced robust biofilms, whereas CD4, 630, IT1106 and 96–1578 were weak biofilm producers. Three strains forming the most robust biofilms belong to the ribotype 014/020 (Table 1). However, SlpA from the P30 strain is different from the 1064 and the 4684 strains, suggesting that the ability to form a biofilm is not strictly due to SlpA primary amino acid sequence. Overall, no correlation was evident between the SlpA amino acid sequence and the ability of the strains to produce biofilms (Fig 4).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414356&req=5

pone.0124971.g004: 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.
Mentions: Since Cwp84 processes SlpA, and, as demonstrated above, that its proteolytic activity impacts biofilm formation, it is formally possible that the cwp84 mutant phenotype is manifested via uncleaved SlpA, suggesting that SlpA may be of importance in the biofilm formation in C. difficile. To date, slpA has not been disrupted in C. difficile, likely because it is essential [17]. Despite multiple attempts, we were also not able to inactivate it (data not shown). Therefore, to investigate if SlpA impacts biofilm formation in C. difficile, 13 strains expressing different slpA alleles were tested for their biofilm-forming abilities (Fig 4). The 1064, P30, 4684/08 and 3457 strains produced robust biofilms, whereas CD4, 630, IT1106 and 96–1578 were weak biofilm producers. Three strains forming the most robust biofilms belong to the ribotype 014/020 (Table 1). However, SlpA from the P30 strain is different from the 1064 and the 4684 strains, suggesting that the ability to form a biofilm is not strictly due to SlpA primary amino acid sequence. Overall, no correlation was evident between the SlpA amino acid sequence and the ability of the strains to produce biofilms (Fig 4).

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