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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

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.
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pone.0124971.g005: 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.

Mentions: Due to the vastly different architecture of the parental and cwp84 mutant biofilms, and previously identified alterations in surface protein composition of the cwp84 mutant [9], we performed proteomic analysis on three fractions prepared from bacterial biofilms: the surface proteome of biofilm-associated bacteria, the biofilm matrix fraction, and the biofilm supernatant (Fig 5). This latter compartment contained proteins released from both the biofilm as well as planktonic bacteria growing outside the biofilm or released from the biofilm. All detected proteins are presented in S1 Table, S2 Table and S3 Table. Among these, distribution of surface, matrix and released proteins of the 630Δerm are presented Table 2. Both parental and cwp84 mutant strains were assayed and compared in Tables 3 and 4. In the biofilm formed by the 630Δerm parental strain, ~33% of identified molecules were Cwp proteins (17 out of 59). Except for Cwp13 and Cwp22, most of the Cwp proteins were found in the biofilm matrix and/or in the biofilm supernatant. Moreover, Cwp2, Cwp6, Cwp12, Cwp16, Cwp19, Cwp25 and CwpV were found mostly in the supernatant fraction. Interestingly, we observed accumulation of the glucosyltransferase toxin TcdA as well as the putative iron transporter FeoB1 (CD630_14790) in the biofilm matrix fraction. Overall, we detected up to 10 proteins putatively involved in the cell wall modeling (such as CD630_01830) and up to 4 involved in the stress response (such as CD630_08270) that were concentrated in one biofilm compartment, namely, the cell surface, the matrix, or the supernatant (Table 2). Comparison of the above results with those obtained for the cwp84 mutant strain revealed some specific differences (Tables 3 and 4). In general, the proteomes of bacterial surface or released proteins were largely similar between parental and mutant strains except for a few molecules (Cwp2, Cwp6 or CD630_18980). SlpA was found to be increased in abundance in the cwp84 mutant biofilm matrix. In contrast, Cwp16 and Cwp17 were only detected in the matrix of the parental strain. In all, and using the stringent analysis (see Materials and Methods) that we employed for this study, only six bacterial surface-associated proteins were quantitatively significantly different between the cwp84 mutant and parental strains, and seven proteins were similarly changed in quantity in the biofilm supernatant. Taken together, these results suggest that alterations in abundance of only a few proteins significantly impacts biofilm properties of the cwp84 mutant.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124971.g005: 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.
Mentions: Due to the vastly different architecture of the parental and cwp84 mutant biofilms, and previously identified alterations in surface protein composition of the cwp84 mutant [9], we performed proteomic analysis on three fractions prepared from bacterial biofilms: the surface proteome of biofilm-associated bacteria, the biofilm matrix fraction, and the biofilm supernatant (Fig 5). This latter compartment contained proteins released from both the biofilm as well as planktonic bacteria growing outside the biofilm or released from the biofilm. All detected proteins are presented in S1 Table, S2 Table and S3 Table. Among these, distribution of surface, matrix and released proteins of the 630Δerm are presented Table 2. Both parental and cwp84 mutant strains were assayed and compared in Tables 3 and 4. In the biofilm formed by the 630Δerm parental strain, ~33% of identified molecules were Cwp proteins (17 out of 59). Except for Cwp13 and Cwp22, most of the Cwp proteins were found in the biofilm matrix and/or in the biofilm supernatant. Moreover, Cwp2, Cwp6, Cwp12, Cwp16, Cwp19, Cwp25 and CwpV were found mostly in the supernatant fraction. Interestingly, we observed accumulation of the glucosyltransferase toxin TcdA as well as the putative iron transporter FeoB1 (CD630_14790) in the biofilm matrix fraction. Overall, we detected up to 10 proteins putatively involved in the cell wall modeling (such as CD630_01830) and up to 4 involved in the stress response (such as CD630_08270) that were concentrated in one biofilm compartment, namely, the cell surface, the matrix, or the supernatant (Table 2). Comparison of the above results with those obtained for the cwp84 mutant strain revealed some specific differences (Tables 3 and 4). In general, the proteomes of bacterial surface or released proteins were largely similar between parental and mutant strains except for a few molecules (Cwp2, Cwp6 or CD630_18980). SlpA was found to be increased in abundance in the cwp84 mutant biofilm matrix. In contrast, Cwp16 and Cwp17 were only detected in the matrix of the parental strain. In all, and using the stringent analysis (see Materials and Methods) that we employed for this study, only six bacterial surface-associated proteins were quantitatively significantly different between the cwp84 mutant and parental strains, and seven proteins were similarly changed in quantity in the biofilm supernatant. Taken together, these results suggest that alterations in abundance of only a few proteins significantly impacts biofilm properties of the cwp84 mutant.

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