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Journal of Bacteriology, June 2009, p. 3594-3603, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01168-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection{triangledown}

Heather S. O'Neil ,{dagger},{ddagger} Brian M. Forster,{dagger} Kari L. Roberts, Andrew J. Chambers, Alan Pavinski Bitar, and Hélène Marquis*

Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853

Received 18 August 2008/ Accepted 24 March 2009

Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (Mpl{Delta}pro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mpl{Delta}pro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, VMC Rm C5-169, Cornell University, Ithaca NY 14853-6401. Phone: (607) 253-3273. Fax: (607) 253-3384. E-mail: hm72{at}cornell.edu

{triangledown} Published ahead of print on 3 April 2009.

{dagger} H.S.O. and B.M.F. have contributed equally to this work.

{ddagger} Present address: Tetragenetics, Inc., Cornell Business and Technology Park, 95 Brown Road, Box 1010/Suite 220, Ithaca, NY 14850.

Journal of Bacteriology, June 2009, p. 3594-3603, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01168-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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