PMC

Amino acid residues are numbered based on their positions in the alignment (including gaps). Identical residues are white on a blue background. Residues with similar properties throughout are white on a pink background. The catalytic domains of CD11 and CD27L are labeled in green and red boxes, respectively. The unboxed regions are the binding domains. The active sites of CD27L are highlighted with black asterisks, while the hydrogen bonding sites important for CD27L activity are marked with red asterisks.

(a) The activity of the catalytic domain and binding domain of CD11 (CD11CD and CD11BD, respectively) against mid-log phase C. difficile cells in PBS and growth medium. (b,c) Binding of CD11CD and CD11BD respectively to mid-log phase C. difficile cells. Abbreviations: PBS, phosphate buffered saline; M, growth medium; B, bound fraction (pellet); U, unbound fraction (supernatant); W, wash, loosely bound fraction; L, molecular weight ladder. Data represents means ± standard deviations of triplicate assays, and the highlighted band is CD11.

(a) The stabilization of wall teichoic acids (WTAs) in growth medium leads to a lack of cell binding or cell wall cleavage by CD11, and the cells are not killed by CD11. (b) The conformational change of WTAs in PBS allows for the access of CD11 to the cell wall, the digestion of the stem peptides from the peptidoglycan, and the lysis of C. difficile cells. (c) Partial removal of WTAs by tunicamycin increases the accessibility of the cell wall to CD11 in growth medium, and leads to cell wall degradation and cell lysis by CD11.

(a) Binding of CD11 to intact cell wall in live C. difficile cells. (b) Binding of CD11 to isolated cell wall materials. Abbreviations: PBS, phosphate buffered saline; M, growth medium; BHI, brain heart infusion; YE, yeast extract; P, peptone; P3, peptone No. 3; D, dextrose; C, L-cysteine; B, bound fraction (pellet); U, unbound fraction (supernatant); W, wash, loosely bound fraction; L, molecular weight ladder. The arrows indicate the band of CD11.

(a) Activity of CD11 against mid-log phase C. difficile cells cultured in different concentrations of tunicamycin. (b) The level of isolated WTAs from C. difficile cells treated with tunicamycin. (c) Binding of CD11 to mid-log phase, tunicamycin-treated C. difficile cells in growth medium. Abbreviations: PBS, phosphate buffered saline; M, growth medium; B, bound fraction (pellet); U, unbound fraction (supernatant); L, molecular weight ladder. Arrows indicate CD11. Data represents means ± standard deviations of triplicate experiments, and the p value is calculated by a two-tailed student t-test (p value = 0.004 and 0.00002 for 5 and 10 μg/mL tunicamycin, respectively).

(a) Activity of CD11 against C. difficile cells in buffer and growth medium. (b) Activity of CD11 against isolated C. difficile cell wall fragments. (c) Activity of CD11 against mid-log phase C. difficile cells in medium components. (d) Activity of CD11 against C. difficile cells in different media. Log kill represents the difference of log(CFU) between buffer-treated and enzyme-treated cells. Abbreviations: CFU (C), colony forming unit in no-enzyme control group; CFU (E), colony forming unit in enzyme-treated group; E, enzyme CD11; PBS, phosphate buffered saline; M, growth medium; BHI, brain-heart infusion; YE, yeast extract; P, peptone; P3, peptone No. 3; D, dextrose; C, L-cysteine; LB, Lysogeny Broth. Results are the means ± standard deviations of triplicate assays.