The propeptide of PC-PLC is 24 amino acids long and serves two functions: it inhibits enzyme activity and influences the compartmentalization of PC-PLC. In the present study, we tested the hypothesis that different parts of the propeptide control PC-PLC activity and compartmentalization. Our results indicate that inhibition of PC-PLC activity requires the presence of only a single propeptide residue. The ability of PC-PLC to remain bacterium-associated is conferred by the N-terminus of the propeptide. Furthermore, four individual amino acid residues located between the third and ninth positions in the N-terminus of the propeptide influence the efficacy of Mpl-mediated maturation of PC-PLC, although a six-residue propeptide is sufficient for Mpl to mediate the proteolytic activation of PC-PLC.
The propeptide of PC-PLC serves to inhibit its enzymatic activity, as deletion of the entire propeptide generates an enzyme that is constitutively active [13
]. However, deletion of 23 out of 24 amino acids did not compromise the ability of the propeptide to inhibit enzyme activity as shown in . The crystal structure of the Bacillus cereus
orthologue to PC-PLC (PLCBC
) (PDB code 1AH7) indicates that the first N-terminal residue of the catalytic domain (P1′) is one of the nine zinc-co-ordinating residues, and that the following three residues (P2′–P4′) are located within the active site [21
]. PC-PLC and PLCBC
have identical P1′–P3′ residues and a similar P4′ residue [23
]. PC-PLC and PLCBC
are approx. 40% identical and the homology model of PC-PLC suggests that its structure is nearly identical with that of PLCBC
(H. Marquis, unpublished work). Therefore it is reasonable that a single propeptide amino acid residue is sufficient to inhibit PC-PLC activity either by steric hindrance within the active-site pocket or by preventing native folding of the catalytic domain.
During intracellular infection, bacteria maintain a pool of PC-PLC at the membrane-cell-wall interface [12
]. Upon cell-to-cell spread, bacteria sense a decrease in vacuolar pH and rapidly release this pool of bacterium-associated PC-PLC. The ability of PC-PLC to remain bacterium-associated is mediated by its propeptide. Our results indicate that deletion of the N-terminus of the propeptide, but not of the C-terminus, leads to an increase in protein translocation across the bacterial cell wall at physiological pH ( and ). Deleting the entire propeptide did not increase the efficacy of translocation above that of the N-terminal 12-amino-acid deletion (ΔC28–P39). It is not known how PC-PLC manages to remain bacterium-associated at physiological pH, because PC-PLC does not have a transmembrane domain or cell-wall-anchoring motif [24
]. One possible hypothesis is that the propeptide contributes to the formation of a complex that immobilizes PC-PLC at the membrane-cell-wall interface. Alternatively or concomitantly, the propeptide may interfere with protein folding, slowing down its rate of translocation as the rate of protein translocation across the cell wall is, for some proteins, directly proportional to the rate of folding [25
]. Either mechanism could be mediated by the N-terminus of the propeptide.
The most unexpected result from the present study was the importance of individual amino acid residues within the N-terminus of the propeptide in facilitating Mpl-mediated proteolytic maturation of PC-PLC. Deletion and substitution mutants encompassing Tyr32, Leu33 and Pro36 all showed a statistically significant PC-PLC maturation defect ( and ). In addition, the presence of a negatively charged residue, either Asp30 or Glu31, is important for Mpl-mediated maturation of PC-PLC, as the D30A/E31A substitution mutant was defective, whereas the C28A/C29A/D30A or the E31A substitution mutants were not. Considering that the residues influencing PC-PLC maturation are located between 22 and 16 residues upstream of the propeptide cleavage site, we propose that these residues interact with Mpl, leading to processing of the propeptide at Ser51. An additional deletion mutant (ΔH40–P47) that did not include Asp30, Glu31, Tyr32, Leu33 or Pro36 showed a major maturation defect. However, triple replacement mutants involving residues Pro39–Pro47 behaved like wild-type PC-PLC. It is possible that the defect associated with the ΔH40–P47 mutant indicates that the spatial location of the residues influencing PC-PLC maturation is imperative to the ability of PC-PLC to interact with Mpl.
Amino acid residues in proximity to the propeptide cleavage site are important for Mpl-mediated proteolytic maturation of PC-PLC. We had identified previously the cleavage-site mutant S51D/S53N whose maturation at pH 6.5 was greatly reduced [13
]. However, the requirement for a serine residue at position 51 is not essential because a S51G mutant that we generated in that study behaved like wild-type PC-PLC. In the present study, the L50A/S51A mutant did not show a maturation defect, whereas the H48A/K49A mutant showed a small but statistically significant decrease in PC-PLC maturation. This result indicates that residues located at the P4 and/or P3 position in relation to the propeptide cleavage site also influence the ability of Mpl to mediate PC-PLC maturation. Interestingly, deletion of 18 residues (ΔC28–K45) did not prevent Mpl-mediated proteolytic activation of PC-PLC, but deletion of 21 residues (ΔC28–H48) completely abolished Mpl-mediated activation of PC-PLC. Thus a six-residue propeptide is sufficient for Mpl to mediate the maturation of PC-PLC, but a three-residue propeptide is not.
The regulation of PC-PLC activity during intracellular infection has been characterized using mouse macrophage-like J774 cells [11
]. However, during infection, L. monocytogenes
appears to multiply primarily in non-professional phagocytic cells, such as epithelial cells, hepatocytes and trophoblasts [3
]. To address whether pH regulates PC-PLC activity in non-professional phagocytic cells, we used human epithelial HeLa cells as the infection host. In these cells, we observed that pH regulates PC-PLC compartmentalization and maturation in a manner similar to that observed in macrophages (Supplementary Figure S2
), suggesting that the activity of PC-PLC is pH-regulated in both professional and non-professional cell types.
The results from the present study are compatible with the following model. The first 12 residues of the PC-PLC propeptide retard translocation of the protein across the bacterial cell wall by interacting with the cell wall or a protein complex, and/or by possibly interfering with native folding of the catalytic domain. Upon a decrease in pH, Mpl interacts with the N-terminus of the propeptide destabilizing the interaction of PC-PLC with the cell wall or protein complex. PC-PLC residues Tyr32, Leu33 and Pro36, which are located at the fifth, sixth and ninth positions within the N-terminus of the propeptide, along with a negatively charged residue located at the third or fourth position of the propeptide, stabilize the Mpl-PC-PLC interaction and promote native folding of the PC-PLC catalytic domain leading to rapid translocation of PC-PLC across the cell wall. It is possible, but not necessary, that Mpl concomitantly mediates the proteolytic maturation of PC-PLC. Future studies will aim to test this model.