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Infection and Immunity, October 1998, p. 4823-4831, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Wolfram R. Zückert,1,dagger Hélène Marquis,1,2 and Howard Goldfine1,*

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104,1 and Department of Microbiology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 802622

Received 19 May 1998/Returned for modification 26 June 1998/Accepted 17 July 1998

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.

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, 255 Johnson Pavilion, Philadelphia, PA 19104-6076. Phone: (215) 898-6384. Fax: (215) 898-9557. E-mail: goldfinh{at}mail.med.upenn.edu.

dagger Present address: Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697.

Infection and Immunity, October 1998, p. 4823-4831, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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