Binding of the extracellular matrix laminin-1 to <i>Clostridioides difficile</i> strains

Santos, Mayara Gil de Castro; Trindade, Camilla Nunes dos Reis; Vommaro, Rossiane Cláudia; Domingues, Regina Maria Calvalcanti Pilotto; Ferreira, Eliane de Oliveira

doi:10.1590/0074-02760220035

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RESEARCH ARTICLE • Mem. Inst. Oswaldo Cruz 117 • 2022 • https://doi.org/10.1590/0074-02760220035 linkcopy

Binding of the extracellular matrix laminin-1 to Clostridioides difficile strains

SCIMAGO INSTITUTIONS RANKINGS

Abstract

BACKGROUND Clostridioides difficile is the most common cause of nosocomial diarrhea associated with antibiotic use. The disease’s symptoms are caused by enterotoxins, but other surface adhesion factors also play a role in the pathogenesis. These adhesins will bind to components of extracellular matrix.

OBJECTIVE There is a lack of knowledge on MSCRAMM, this work set-out to determine the adhesive properties of several C. difficile ribotypes (027, 133, 135, 014, 012) towards laminin-1 (LMN-1).

METHODS A binding experiment revealed that different ribotypes have distinct adhesion capabilities. To identify this adhesin, an affinity chromatography column containing LMN-1 was prepared and total protein extracts were analysed using mass spectrometry.

FINDINGS Strains from ribotypes 012 and 027 had the best adhesion when incubated with glucose supplementations (0.2%, 0.5%, and 1%), while RT135 had a poor adherence. The criteria were not met by RT014 and RT133. In the absence of glucose, there was no adhesion for any ribotype, implying that glucose is required and plays a significant role in adhesion.

MAIN CONCLUSIONS These findings show that in the presence of glucose, each C. difficile ribotype interacts differently with LMN-1, and the adhesin responsible for recognition could be SlpA protein.

Key words:
Clostridioides difficile; adhesion; laminin-1; virulence

About the authors

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Mayara Gil de Castro Santos

school

Universidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil

About the authors

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Camilla Nunes dos Reis Trindade

school

Universidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil

About the authors

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Rossiane Cláudia Vommaro

school

About the authors

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Regina Maria Calvalcanti Pilotto Domingues

school

Universidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil

About the authors

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Eliane de Oliveira Ferreira

school

Universidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil

0000-0002-9977-6460

SCImago Institutions Rankings

Universidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho e Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho e Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, Brasil

Figures | Tables

Figures (7)
Tables (1)

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Fig. 1: adherence test of Clostridioides difficile ribotypes (24 h growth) to laminin-1 by fluorescence microscopy using different concentrations of glucose (0.2%, 0.5% and 1%) and live/dead staining. Green spots represent active, membrane-intact bacteria. A, B, C and D correspond to the adherence assay of RT012 (CD630 strain); E, F, G and H the adherence assay of RT027 (BI/NAP1) and I, J, K and L the assay of RT135 (SJ1 strain). WG: without supplementation of glucose.

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Fig. 2: quantitative adhesion assay of Clostridioides difficile ribotypes (24 h growth) to laminin-1 at different glucose concentrations (0.2%, 0.5% and 1%). All tests were performed in triplicate and the bars of the standard deviation corresponded to the mean of the three technical replicates. Results were considered significant when p < 0.05 (Student’s t unpaired test). Bovine serum albumin (BSA) 2% was used as a negative control (C-).

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Fig. 3: adhesion test of ribotype RT012 (CD630) and RT027 of Clostridioides difficile strains to laminin-1 with 0.5% glucose and at different growth times (24 h, 48 h and 72 h). Bovine serum albumin (BSA) 2% was used as negative control (C-), without LMN-1. Results were considered significant when p < 0.05.

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Fig. 4: inhibition assay ribotype 012 (CD630) of Clostridioides difficile strains after chemical and enzymatic treatments. A - Proteinase K (15 µg/mL); B - Trypsin (20 µg/mL); C - sodium periodate (MPS; 100 mM); D - Bacteria without treatment; E - proteinase K (15 µg/mL) follow by MPS (100 mM); F - Negative control Bovine serum albumin (BSA) 2%, without LMN-1. All experiments were performed in triplicate. Magnification: 1000x.

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Fig. 5: quantification of the adhesion assay of Clostridioides difficile RT012 (CD630) against laminin-1 after chemical and enzymatic treatment. As negative control (C-) Bovine serum albumin (BSA - 2%) inoculated with strain CD630, without laminin, was used. The bars represent the standard deviations (*) of the means of two technical and two biological experiments. MPS: sodium metaperiodate. The results were considered significant when p < 0.05.

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Fig. 6: scanning electron microscopy (SEM) showing Clostridioides difficile RT012 (CD630) on glass coverslips at 24 h with 0.5% glucose. A - micrograph showing an extracellular fibrous-biofilm matrix (star) produced by C. difficile; B - micrograph showing C. difficile aggregation by the biofilm; C - Detail of the biofilm produced by C. difficile connecting the cell to the glass support (*). Scale bars: A - 20 μm; B - 10 μm; C - 2 μm.

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Fig. 7: immunoelectron microscopy of RT012 (CD630) from Clostridioides difficile. Cells with 24 h growth with 0.5% of glucose were incubated with laminin-1. Micrographs A and B show C. difficile cells incubated with LMN-1, treated with the anti-LMN-1 antibody and anti-IgG conjugated to colloidal gold beads. Arrows indicate recognition on the surface of bacterial cells. Scale bar: 2 μm.

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TABLE Clostridioides difficile strains used in this study

imageFig. 1: adherence test of Clostridioides difficile ribotypes (24 h growth) to laminin-1 by fluorescence microscopy using different concentrations of glucose (0.2%, 0.5% and 1%) and live/dead staining. Green spots represent active, membrane-intact bacteria. A, B, C and D correspond to the adherence assay of RT012 (CD630 strain); E, F, G and H the adherence assay of RT027 (BI/NAP1) and I, J, K and L the assay of RT135 (SJ1 strain). WG: without supplementation of glucose. open_in_new

imageFig. 2: quantitative adhesion assay of Clostridioides difficile ribotypes (24 h growth) to laminin-1 at different glucose concentrations (0.2%, 0.5% and 1%). All tests were performed in triplicate and the bars of the standard deviation corresponded to the mean of the three technical replicates. Results were considered significant when p < 0.05 (Student’s t unpaired test). Bovine serum albumin (BSA) 2% was used as a negative control (C-). open_in_new

imageFig. 3: adhesion test of ribotype RT012 (CD630) and RT027 of Clostridioides difficile strains to laminin-1 with 0.5% glucose and at different growth times (24 h, 48 h and 72 h). Bovine serum albumin (BSA) 2% was used as negative control (C-), without LMN-1. Results were considered significant when p < 0.05. open_in_new

imageFig. 4: inhibition assay ribotype 012 (CD630) of Clostridioides difficile strains after chemical and enzymatic treatments. A - Proteinase K (15 µg/mL); B - Trypsin (20 µg/mL); C - sodium periodate (MPS; 100 mM); D - Bacteria without treatment; E - proteinase K (15 µg/mL) follow by MPS (100 mM); F - Negative control Bovine serum albumin (BSA) 2%, without LMN-1. All experiments were performed in triplicate. Magnification: 1000x. open_in_new

imageFig. 5: quantification of the adhesion assay of Clostridioides difficile RT012 (CD630) against laminin-1 after chemical and enzymatic treatment. As negative control (C-) Bovine serum albumin (BSA - 2%) inoculated with strain CD630, without laminin, was used. The bars represent the standard deviations (*) of the means of two technical and two biological experiments. MPS: sodium metaperiodate. The results were considered significant when p < 0.05. open_in_new

imageFig. 6: scanning electron microscopy (SEM) showing Clostridioides difficile RT012 (CD630) on glass coverslips at 24 h with 0.5% glucose. A - micrograph showing an extracellular fibrous-biofilm matrix (star) produced by C. difficile; B - micrograph showing C. difficile aggregation by the biofilm; C - Detail of the biofilm produced by C. difficile connecting the cell to the glass support (*). Scale bars: A - 20 μm; B - 10 μm; C - 2 μm. open_in_new

imageFig. 7: immunoelectron microscopy of RT012 (CD630) from Clostridioides difficile. Cells with 24 h growth with 0.5% of glucose were incubated with laminin-1. Micrographs A and B show C. difficile cells incubated with LMN-1, treated with the anti-LMN-1 antibody and anti-IgG conjugated to colloidal gold beads. Arrows indicate recognition on the surface of bacterial cells. Scale bar: 2 μm. open_in_new

table_chartTABLE Clostridioides difficile strains used in this study

Strains	Ribotypes	Toxigenic profile^b	Clinical conditions	References
SJ1	135^a	tcdA⁺/tcdB⁺/CDT^-	SH, DM, hemorrhagic EVA	²³
HU09	133^a	tcdA⁺/tcdB⁺/CDT^-	Crohn’s disease	²⁴
1598	014	tcdA⁺/tcdB⁺/CDT^-	HIV⁺	²⁵
BI/NAP1^c	027	tcdA⁺/tcdB⁺ /CDT⁺	-	²⁶
630	012	tcdA⁺/tcdB⁺/CDT^-	Pseudomembranous colitis	²⁷

a: ribotypes of C. difficile exclusive from Brazil; b: polymerase chain reaction (PCR) was used to the detection of the toxin genes; c: BI/NAP1/027 strain (ANA #2004016) was kindly provided by Dr Angela Thompson from the CDC, Atlanta, USA; tcdA⁺ and tcdB⁺ (positive for the presence of toxins TcdA and TcdB); CDT: (+) positive or (-) negative for the presence of binary toxin genes; SH: system hypertension; DM: diabetes mellitus; EVA: haemorrhagic encephalic vascular accident; HIV+: positive for the human immunodeficiency virus.

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