title: Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog.
abstract: The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: W R
authors.initials: WR
authors.lastname: Zückert
authors.forename: H
authors.initials: H
authors.lastname: Marquis
authors.forename: H
authors.initials: H
authors.lastname: Goldfine
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence; D015638:Cytochalasin D / Q000494:pharmacology; D004847:Epithelial Cells / Q000382:microbiology; D006801:Humans; D008089:Listeria monocytogenes / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D008088:Listeriosis; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D015032:Zinc
chemical_list: D008667:Metalloproteins; D015638:Cytochalasin D; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D015032:Zinc