Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis

Abstract

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.

Figure 1

Binding of recombinant CD2831 full-length protein and subdomains to collagen. (A) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. (B) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. (C) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. (D) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by HRP-conjugated-secondary antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.

Figure 2

CD2831 overexpression increases C. difficile binding to collagens and human fibroblasts. (A) (Left panel) Overnight cultures of C. difficile overexpressing CD2831 (pCwp2-2831) and control (empty pCwp2 vector) strains were centrifuged and proteins released in the supernatant (Sn) were TCA precipitated. The cell wall (CW) was isolated from cytoplasm and membranes (CM) and the three fractions were analyzed by western blot using rabbit anti-CD2831 serum. Full-length western blot showed in Figure S1. (Right panel) Flow-cytometry analysis of CD2831 surface exposure: in light grey is showed the wild type condition (empty pCwp2 vector), in dark gray is the overexpressing condition (pCwp2-2831). The negative black line represents the overexpressing strain stained only with the secondary antibody as negative control. (B) Adhesion of C. difficile overexpressing CD2831 and control strains to immobilized collagens type I, III and V (left panel) and to IMR90 cells (right panel) after 1-hour incubation in anaerobic condition. Adhering bacteria were stained by using anti-CD630Δerm serum followed by Alexa-fluor conjugated secondary antibody and revealed by a fluorescence plate reader. Adhesion of C. difficile to cells was normalized for the levels of collagens produced by cells in each well. (C). Confocal images of C. difficile control and CD2831-overexpressing strains adhering to the collagen-producing IMR90 cells, after 1-hour incubation at 37 °C in anaerobic conditions. Cells nuclei are stained with DAPI (blue), bacteria are showed in green and collagen in red.

Figure 3

CD2831 contribution to biofilm formation. (A) Flow-cytometry analysis of CD2831 expressed in C. difficile 630Δerm. Bacteria were cultured either in BHIS for 3 hours (light grey peak) or in BHIS + 2% (w/v) glucose on plastic plates for 3 days for biofilm formation (dark grey peak). (B) Overnight cultures of C. difficile constitutively expressing CD2831 and control strains were diluted in BHIS + 2% (w/v) glucose and allowed to form biofilm for 24 hours. Bacterial biofilm was stained with crystal violet, methanol-extracted dye was diluted 1:10 and absorbance was measured at 595 nm (left). Z-stack confocal microscopy of 3-day biofilm from C. difficile bacteria. Bacterial nuclei and extracellular DNA were stained using DAPI (right). (C) Western blot showing CD2831 expression in bacterial lysates and supernatants of C. difficile pTet-CD2831 after induction with 0, 25, 50 and 100 ng/mL of anhydrotetracycline (ATc) for 24 hours (left). Full-length western blot showed in Figure S2. C. difficile pTet-CD2831 strain 24-hour biofilm was measured by crystal violet staining after inducing CD2831 expression, using different concentration of ATc. Methanol-extracted dye was diluted 1:2 (right).

Figure 4

L. lactis as heterologous system to study CD2831 adhesive properties. (A) Flow-cytometry analysis showing surface exposure of CD2831 in L. lactis transformed with pAM401-CD2831. (B) L. lactis strains were allowed to adhere to ECM-producing IMR90 human fibroblasts for 1 hour at 37 °C. Data show the contribution of CD2831 in adhesion of L. lactis to cells and inhibition of L. lactis expressing CD2831 adhesion upon pre-incubation of bacteria with 0.1% (v/v) rabbit anti-CD2831 serum for 1 hour at 37 °C. (C) L. lactis strains were cultured as biofilm on abiotic surface for 24 hours. Bacterial biomass was measured by crystal violet, methanol-extracted dye was diluted 1:2 and absorbance at 595 nm was measured.

Figure 5

Recombinant CD2831 binding to human complement C1q. (A) ELISA plates coated with 10 µg/ml of purified human complement component C1q were incubated with several dilutions of recombinant CD2831 (15 nM-1 µM) for 1 hour. C. difficile recombinant TcdB and CWP25 were used as control. (B) ELISA plates coated with 10 µg/ml purified human IgM were incubated with 1% (v/v) Normal Human Serum (NHS) and different concentrations of recombinant CD2831. After 1 hour, deposition of human C1q, C1s and C1r was measured. 1% (v/v) C1q-depleted serum (C1q-dpl) was used as negative control.