JOURNAL OF BACTERIOLOGY, June 2009, p. 3594–3603
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 11
The Propeptide of the Metalloprotease of Listeria monocytogenes
Controls Compartmentalization of the Zymogen
during Intracellular Infection?
Heather S. O’Neil,†‡ Brian M. Forster,† Kari L. Roberts, Andrew J. Chambers,
Alan Pavinski Bitar, and He ´le `ne 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?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 compartmental-
ization. This defect could not be rescued by providing the propeptide in trans to the mpl?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
Listeria monocytogenes is a gram-positive facultative intra-
cellular pathogen and the causative agent of the food-borne
disease listeriosis in humans and a variety of vertebrates (41).
The success of L. monocytogenes as a pathogen can be attrib-
uted largely to its ability to replicate in the host cell cytosol and
to spread from cell to cell without entering the extracellular
milieu (10, 39). Efficacy of escape from vacuoles formed upon
initial entry into a host cell or upon cell-to-cell spread is im-
perative to the virulence of L. monocytogenes (31, 39, 40).
There are multiple bacterial factors involved in escape from
double-membrane vacuoles, including the broad-range phos-
pholipase C (PC-PLC) (31, 40). PC-PLC is synthesized as an
inactive proenzyme, whose maturation into an active form
requires proteolytic cleavage of an N-terminal propeptide (23).
During infection, PC-PLC is stored as a proenzyme at the
interface of the bacterial membrane and cell wall (19, 33) and
is released as a bolus of mature protein upon a drop in pH,
such as is experienced in the host vacuole (18, 19). Maturation
and translocation of PC-PLC across the cell wall are depen-
dent on a decrease in pH and on the activity of the zinc
metalloprotease of Listeria, Mpl (18, 19, 24, 25).
ActA, an L. monocytogenes surface protein, also serves as a
substrate for Mpl. Similar to PC-PLC, ActA is cleaved by Mpl
upon a decrease in pH (26). ActA mediates the polymerization
of host actin filaments on the bacterial surface to generate
bacterial movement in the cytosol (13). Presumably, cleavage
of ActA upon exit from the spreading vacuole enables bacterial
replication before actin-based movement resumes (26).
Mpl is a member of the M4 family of metalloproteases,
represented by thermolysin from Bacillus thermoproteolyticus
(22). Mpl is translated as a preproenzyme with a 24-amino-acid
signal sequence that is removed upon secretion across the
bacterial membrane (21). The secreted Mpl zymogen matures
exclusively by intramolecular autocatalysis releasing a 176-amino-
acid propeptide and a 310-amino-acid mature protease (5).
Similar to Mpl, many proteases are synthesized as preproen-
zymes. Propeptides often function as intramolecular chaper-
ones catalyzing the folding of their covalently bound protease
(30). However, many propeptides remain functional as folding
catalysts when added in trans (17, 20, 37, 45). This folding
process is best studied in the serine proteases subtilisin and
?-lytic protease (34, 36, 43). The propeptide guides folding of
the catalytic domain through a nonnative folding intermediate
to an unprocessed native fold by lowering the free energy
required to achieve the native state (1, 27, 34). This is rapidly
followed by autocatalysis to cleave off the propeptide. The
propeptide is retained in a stable propeptide-protease complex
and serves as an inhibitor (2, 35, 43). The rate-determining
step in production of active protease is the degradation of the
inhibitory propeptide (36). Degradation of the propeptide by
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, VMC Rm C5-169, Cornell University, Ithaca
NY 14853-6401. Phone: (607) 253-3273. Fax: (607) 253-3384. E-mail:
† H.S.O. and B.M.F. have contributed equally to this work.
‡ Present address: Tetragenetics, Inc., Cornell Business and Tech-
nology Park, 95 Brown Road, Box 1010/Suite 220, Ithaca, NY 14850.
?Published ahead of print on 3 April 2009.
its cognate protease releases the active enzyme, increasing the
energy barrier and preventing unfolding (34).
Propeptides can serve functions other than as a folding cat-
alyst. They can slow protein folding, as observed in the in vitro
refolding of a denatured lipase from Rhizopus oryzae (3).
Propeptides can target their proteases to the proper cellular
compartment. This phenomenon was observed with the aspar-
tic acid protease cathepsin D, whose propeptide is required for
trafficking from the endoplasmic reticulum to the lysosome (9,
38). Conversely, propeptides can retard trafficking of the pro-
tein. For example, the propeptide of the human myeloperoxi-
dase retards protein exit from the endoplasmic reticulum,
probably to aid in heme incorporation, a requirement for fur-
ther processing (11). Similarly, the propeptide of L. monocy-
togenes PC-PLC retards translocation of the protein across the
cell wall, enabling the protein to accumulate at the membrane-
cell wall interface until it is released as a bolus to mediate
bacterial escape from acidifying vacuoles formed during cell-
to-cell spread (44). Propeptides can also influence secretion of
a protease. Secretion of the cell surface metalloprotease
ADAMTS9 depends on a covalently bonded propeptide that is
properly glycosylated. Cell surface-associated furin cleaves the
propeptide, enabling release of the protease from the cell
The present study examines the role of the Mpl propeptide.
Our results indicated that the propeptide of Mpl is not essen-
tial for activity when bacteria are grown on egg yolk agar
(EYA) plates, although it increases activity. However, we ob-
served that the propeptide serves to retain bacterium-associ-
ated Mpl and that the compartmentalization of Mpl is integral
to its ability to process its substrates during intracellular infec-
MATERIALS AND METHODS
Bacterial strains and growth conditions. The L. monocytogenes strains used in
this study are listed in Table 1, and plasmids are listed in Table 2. L. monocy-
togenes was routinely cultured in brain heart infusion (BHI) broth. Escherichia
coli DH5-? strains carrying pKSV7 (32)-derived plasmids were cultured in Luria-
Bertani (LB) broth supplemented with ampicillin (100 ?g/ml), whereas E. coli
strains carrying pAM401 (42) were cultured in LB broth supplemented with
chloramphenicol (10 ?g/ml). L. monocytogenes strains carrying pKSV7 or
pAM401 were cultured in BHI broth supplemented with chloramphenicol (10
?g/ml). In preparation for intracellular immunoprecipitation and immunofluo-
rescence assays, L. monocytogenes was cultured overnight in BHI broth with or
without chloramphenicol (10 ?g/ml) as appropriate, at 30°C without shaking.
Cultures used for Western immunoblots were grown in LB broth with 50 mM
morpholinepropanesulfonic acid (MOPS), adjusted to pH 7.3 and supplemented
with 0.2% activated charcoal and 20 mM glucose (LB-MOPS-Glc) (33), as well
as chloramphenicol (10 ?g/ml) if appropriate.
Construction of in-frame deletion mutants. A 528-bp gene segment coding for
the propeptide of Mpl was deleted to create mpl?pro by site-directed mutagen-
esis with overlap extension (SOEing) (12). Two sets of primers were designed to
amplify DNA from regions upstream and downstream of the segment to be
deleted. Amplification from L. monocytogenes 10403S chromosomal DNA of a
503-bp region encompassing the mpl 5?-untranslated region, promoter region,
and open reading frame coding for the signal sequence was performed by PCR
using forward primer Marq344 and reverse primer Marq345 (Table 3). A 368-bp
region coding for the N terminus of the Mpl mature form was also amplified by
PCR using forward primer Marq346 and reverse primer Marq347. These two
PCR products were used in a SOEing PCR with primers Marq344 and Marq347.
The resulting 824-bp product was digested with KpnI and EcoRI and ligated into
the shuttle vector pKSV7, creating plasmid pHSO849. The cloned fragment was
sequenced to ensure accuracy, and the plasmid was electroporated into 10403S
and NF-L943 to replace the wild-type mpl allele with mpl?pro by allelic exchange
(7), generating strains HEL-871 and HEL-927, respectively. Mutants were iden-
tified by PCR amplification of an 824-bp fragment with primers Marq344 and
Marq347 from chloramphenicol-sensitive clones.
A 276-bp internal in-frame deletion of plcB, the gene coding for PC-PLC, was
generated by allelic exchange in NF-L943 using the pKSV7-based plasmid DP-
1888 (31), generating strain HEL-925. Plasmid DP-1888 was originally used to
create DP-L1935, a 10403S derivative with an internal in-frame deletion in plcB.
Chloramphenicol-sensitive colonies were screened for the absence of PC-PLC
activity on EYA plates as described below.
Construction of mpl point mutants. Point mutations in the mpl gene were
generated by SOEing PCR as described above. The histidine residue at position
75 was replaced with a valine residue using primer pairs Marq313 and Marq314
and Marq315 and Marq316. Marq314 and Marq315 provide the necessary codon
change as well as a silent mutation to create a novel Eco0109I restriction site for
screening. A final PCR product of 1,002 bp was digested with PstI and SacI and
TABLE 1. L. monocytogenes strains used in this study
StrainGenotype and relevant features
Wild type (serotype 1/2a)
prfA(G155S) in 10403S background;
overexpresses PrfA-dependent genes,
including mpl and plcB
Internal in-frame deletion of plcB in
Deletion of mpl structural gene in
Internal in-frame deletion of mpl in
mpl(H95L) in 10403s background
mpl(H95L) in NF-L943 background
mpl(H75V) in 10403S background
mpl(H75V) in NF-L943 background
mpl-Flag in NF-L943 background
Deletion of mpl propeptide in 10403S
10403S ? pAM401
HEL-871 ? pAM401
DP-L2343 ? pAM401
HEL-871 ? mpl propeptide in trans
Internal in-frame deletion of plcB in
Deletion of mpl propeptide in NF-L943
HEL-927 ? mpl propeptide in trans
HEL-925 ? pAM401
NF-L943 ? pAM401
HEL-469 ? pAM401
HEL-927 ? pAM401
mpl?pro-Flag in NF-L943 background
mpl(H75V)-Flag in NF-L943 background
TABLE 2. Plasmids used in this study
pKSV7?plcB for internal in-frame deletion
pKSV7 mpl C terminus plus Flag tag
pAM401 with spac promoter
pHSO890 with mpl signal sequence and
Shuttle vector for allelic exchange
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