Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes.
The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28-Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane-cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1' (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.
Slepkov
Emily R
ER
Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, USA.
Pavinski Bitar
Alan
A
Marquis
Hélène
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals; D021381:Protein Transport; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D045726:Metalloproteases