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Infection and Immunity, January 1999, p. 182-186, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutagenesis of Active-Site Histidines of Listeria monocytogenes Phosphatidylinositol-Specific Phospholipase C: Effects on Enzyme Activity and Biological Function

Trudi Bannam,dagger and Howard Goldfine*

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076

Received 12 August 1998/Returned for modification 2 October 1998/Accepted 20 October 1998

Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, 301C Johnson Pavilion, Philadelphia, PA 19104-6076. Phone: (215) 898-6384. Fax: (215) 573-4856. E-mail: goldfinh{at}mail.med.upenn.edu.

dagger Present address: Department of Microbiology, Monash University, Clayton 3168, Australia.

Infection and Immunity, January 1999, p. 182-186, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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