Fig. 3. Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease produce pro-invasive peptides. (A) To investigate the generation of pro-invasive peptides, a synthetic β-casein 33mer peptide (33mer) was incubated for 48 h in the presence of exhaustively washed bacteria and/or collagen type I. Peptides generated by proteolysis were sequenced by RP-HPLC-MS/MS and their pro-invasive activity measured in the collagen assay. Closed and open arrowheads indicate cleavage sites for a collagen-associated protease and a L.monocytogenes protease, respectively (left panel). The effect of synthetic β-casein peptides (33, 13, 10 and 7mer) on HCT-8/E11 cell invasion was also evaluated (right panel). Invasion indices (%) were determined as in Figure 1A. *Significantly different from 33mer at p < 0.005. (B) HCT-8/E11 cells were incubated, on top of collagen type I gels, with: CM of exhaustively washed bacteria (CM^CollListeria) prepared with TSB (CM^CollListeria+TSB), β-casein (CM^CollListeria+β-casein) or the synthetic 33mer peptide (CM^CollListeria+33mer), CM of such β-casein sources prepared in absence of bacteria (CM^CollTSB, CM^Collβ-casein, CM^Coll33mer), CM from tumour biopsies of colon cancer patients, prepared in the absence (CMTumour) or in the presence of the synthetic 33mer peptide with or without antibiotics (CMTumour+antibiotics+33mer or CMTumour+33mer, respectively). Invasion indices (%) were determined as in Figure 1A. *Significantly different from CM^CollTSB at p < 0.005. (C) HCT-8/E11 cells were incubated, on top of collagen type I gels, with CM^CollTSB, CM^CollListeria wild-type (CM^CollListeria/TSB), Δmpl (CM^CollListeriaΔmpl/TSB), ΔactA (CM^CollListeriaΔactA/TSB) or ΔplcB (CM^CollListeriaΔplcB/TSB) mutants, grown in TSB. Invasion indices (%) were determined as in Figure 1A. *Significantly different from CM^CollTSB at p < 0.005. (D) β-casein zymography reveals a trypsin-like serine protease with caseinolytic activity in collagen type I solution. Collagen solution was loaded on a SDS gel containing β-casein as substrate. After electrophoresis, gel was incubated in a serine-protease buffer with (+) or without (–) 1 µM TLCK. Arrow indicates a ∼21 kDa protein, revealed by Coomassie® staining.