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J Bacteriol. 2009 June; 191(11): 3594–3603.
Published online 2009 April 3. doi:  10.1128/JB.01168-08
PMCID: PMC2681923

The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection[down-pointing small open triangle]

Abstract

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 (MplΔpro) 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 mplΔpro 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.

Listeria monocytogenes is a gram-positive facultative intracellular 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 attributed 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 imperative 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 phospholipase 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 dependent 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 preproenzymes. Propeptides often function as intramolecular chaperones 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 α-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 its cognate protease releases the active enzyme, increasing the energy barrier and preventing unfolding (34).

Propeptides can serve functions other than as a folding catalyst. 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 aspartic 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 protein. For example, the propeptide of the human myeloperoxidase retards protein exit from the endoplasmic reticulum, probably to aid in heme incorporation, a requirement for further processing (11). Similarly, the propeptide of L. monocytogenes 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 surface (14).

The present study examines the role of the Mpl propeptide. Our results indicated that the propeptide of Mpl is not essential for activity when bacteria are grown on egg yolk agar (EYA) plates, although it increases activity. However, we observed that the propeptide serves to retain bacterium-associated Mpl and that the compartmentalization of Mpl is integral to its ability to process its substrates during intracellular infection.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The L. monocytogenes strains used in this study are listed in Table Table1,1, and plasmids are listed in Table Table2.2. L. monocytogenes was routinely cultured in brain heart infusion (BHI) broth. Escherichia coli DH5-α strains carrying pKSV7 (32)-derived plasmids were cultured in Luria-Bertani (LB) broth supplemented with ampicillin (100 μg/ml), whereas E. coli strains carrying pAM401 (42) were cultured in LB broth supplemented with chloramphenicol (10 μg/ml). L. monocytogenes strains carrying pKSV7 or pAM401 were cultured in BHI broth supplemented with chloramphenicol (10 μg/ml). In preparation for intracellular immunoprecipitation and immunofluorescence assays, L. monocytogenes was cultured overnight in BHI broth with or without chloramphenicol (10 μg/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 μg/ml) if appropriate.

TABLE 1.
L. monocytogenes strains used in this study
TABLE 2.
Plasmids used in this study

Construction of in-frame deletion mutants.

A 528-bp gene segment coding for the propeptide of Mpl was deleted to create mplΔpro by site-directed mutagenesis 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 (Table3).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 mplΔpro by allelic exchange (7), generating strains HEL-871 and HEL-927, respectively. Mutants were identified by PCR amplification of an 824-bp fragment with primers Marq344 and Marq347 from chloramphenicol-sensitive clones.

TABLE 3.
Oligonucleotide primers used in this study

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 ligated into the shuttle vector pKSV7, creating plasmid pKR779. The histidine residue at position 95 was replaced with a leucine residue using primer pairs Marq313 and Marq317 and Marq318 and Marq316. Marq317 and Marq318 provide the necessary codon change and a silent mutation to generate a Bst1107I restriction site for screening. A final PCR product of 1,002 bp was digested with PstI and SacI and ligated into the shuttle vector pKSV7, creating plasmid pKR778. Point mutations and fidelity of sequence were verified by sequencing before electroporating the plasmids into 10403S and NF-L943 to replace the wild-type mpl allele by allelic exchange. Mutants were screened for chloramphenicol sensitivity and acquisition of a novel Eco01091 restriction site for the H75V mutation or Bst1107I restriction site for the H95L mutation. The mpl(H75V) mutant strains generated were identified as HEL-784 (10403S background) and HEL-786 (NF-L943 background). The mpl(H95L) mutant strains generated were identified as HEL-780 (10403S background) and HEL-782 (NF-L943 backgound).

Mpl propeptide complementation in trans.

The mplΔpro strains HEL-871 and HEL-927 were complemented in trans by cloning the mpl gene fragment coding for the signal sequence and propeptide downstream of a spac promoter on a multicopy plasmid. The spac promoter was amplified from pLiv (8) using Marq398 and Marq399. The product was digested with BamHI and SphI, ligated into pAM401, sequenced, and named pHSO890. The sequence coding for mpl signal sequence and propeptide was amplified from 10403S genomic DNA using Marq395 and Marq396. This 622-bp product was digested with SphI and ligated into pHSO890, creating pHSO909. Chloramphenicol-resistant colonies were screened by PCR using Marq398 and Marq396 to determine if the insert was present and in the proper orientation. Cloned constructs were verified by sequencing and then electroporated into HEL-871 and HEL-927. Chloramphenicol-resistant colonies were selected, generating HEL-908 and HEL-938. Control strains were electroporated with the vector pAM401.

Western immunoblotting.

L. monocytogenes strains with an NF-L943 background were used for the Western immunoblotting experiments. Bacterial cultures grown in LB-MOPS-Glc to an optical density at 600 nm (OD600) of ~1.0 were chilled on ice and centrifuged, and supernatants were collected. Trichloroacetic acid was added to a final concentration of 5%, and supernatants were incubated on ice for 1 h. Precipitated proteins were washed with acetone, dissolved in 2× sample buffer (125 mM Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol, 20 mM dithiothreitol), and heat denatured for 5 min. Extracted proteins equivalent to 1.0 ml of culture with an OD600 of 1.0 were resolved on 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane using a semidry electroblotting apparatus. The protein blot was reacted with rabbit immune serum to L. monocytogenes Mpl (kindly provided by Daniel Portnoy) at a dilution of 1/1,000 followed by goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (24 ng/ml) (Jackson ImmunoResearch Laboratories, Inc.). Enzymatic reactivity was detected with nitroblue tetrazolium (0.33 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.17 mg/ml).

Phospholipase activity on EYA.

L. monocytogenes strains with an NF-L943 background were used to determine phospholipase activity on egg yolk agar. PC-PLC activity was detected as previously described on LB-EYA supplemented with 25 mM glucose-1-phosphate (18). Strains not carrying a plasmid were spot inoculated on a single agar plate not supplemented with chloramphenicol, whereas strains carrying pAM401 or a pAM401-derived plasmid were inoculated on a second agar plate supplemented with chloramphenicol. Both plates were inoculated at the same time and scanned after 48 h of incubation at 37°C.

Metabolic labeling and immunoprecipitation experiments.

L. monocytogenes strains with a 10403S background were used for metabolic labeling and immunoprecipitation experiments. Briefly, infected mouse macrophage-like J774 cells were pulse-labeled with [35S]methionine/cysteine in the presence of host protein synthesis and proteasome inhibitors as described previously (33, 44). For detection of ActA and PC-PLC, bacterial cell-to-cell spread was blocked with cytochalasin D prior to metabolic labeling, and the intracellular pH of labeled infected cells was modified during a chase period of 5 or 10 min by incubating the cells in a potassium-based buffer supplemented with nigericin. For detection of ActA, pulse-chased infected cells were lysed in warm 2× sample buffer, incubated at 100°C for 5 min, frozen quickly on dry ice, heat treated for an additional 5 min, and then resolved on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. For immunoprecipitation of PC-PLC, infected cells were lysed in NP-40 buffer (33) and lysates were centrifuged to pellet bacteria. Bacteria were lysed using a Listeria-specific cell wall hydrolase (15). PC-PLC was immunoprecipitated independently from host cell and bacterial lysates using affinity-purified rabbit antibodies as described previously (33). For immunoprecipitation of Mpl, samples were processed as described above for PC-PLC, except that cells were pulse-labeled for 30 min and Mpl was immunoprecipitated from host cell lysates only with an anti-Mpl rabbit polyclonal serum. Labeled proteins were detected by autoradiography. For quantification, three sets of immunoprecipitates were prepared from experiments performed on 3 consecutive days. The first two sets were stored at −80°C immediately after immunoprecipitation. At the end of the third experiment, the three sets were resolved on a single SDS-polyacrylamide gel and labeled proteins were detected by phosphorimaging using a Storm 860 scanner. Quantification of band intensities for the pro and mature forms of Mpl was performed with ImageJ (rsbweb.nih.gov/ij/). After background subtraction, the results were divided by the number of methionine and cysteine residues in each form: the proform contains five methionine and four cysteine residues, whereas the mature form contains three methionine and three cysteine residues. The normalized set of data was analyzed by a two-tailed paired t test.

Addition of a C-terminal Flag tag to Mpl species.

A Flag tag sequence was fused in frame to the 3′ end of mpl in strains HEL-927 and HEL-786 by allelic exchange using the pKSV7-based shuttle vector pAB796 (5), generating strains HEL-943 (MplΔpro-Flag) and HEL-1000 [Mpl(H75V)-Flag), respectively.

Immunofluorescent staining.

Detection of bacterium-associated Mpl was performed by adapting a method developed for the detection of bacterium-associated PC-PLC (19). HeLa cells were seeded on 18- by 18-mm glass coverslips at a concentration of 4 × 105 per 35-mm tissue culture dish, infected at a multiplicity of infection of approximately 50 with either HEL-798 (Mpl-Flag), HEL-943 (MplΔpro-Flag), HEL-1000 [Mpl(Η75V)-Flag], or NF-L943 (Mpl) for 1 h, and then washed with phosphate-buffered saline (PBS; pH 7.1). At 1.5 h postinfection, gentamicin was added (10 μg/ml). At 3 h postinfection, coverslips were washed with PBS, fixed in acetone-methanol (1:1 [vol/vol]) for 2 min, and then washed with PBS and Tris-buffered saline (pH 8.0) with 0.1% Triton X-100 (TBS-TX). When indicated on the figure, samples were treated with 700 U of mutanolysin (Sigma) in sodium phosphate buffer (pH 6.0) for 15 min at 37°C. Coverslips were washed with TBS-TX and blocked for 30 min with TBS-TX plus 10% bovine serum albumin. Samples were reacted with anti-Flag M2 mouse monoclonal antibody (Sigma) at 1/1,000 (1.0 μg/ml) for 2 h and then with donkey anti-mouse conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, Inc.) at 1/1,000 (1.5 μg/ml) for 45 min. Bacteria were stained with Texas red-conjugated wheat germ agglutinin (WGA) (Molecular Probes) at 1/1,000 (1 μg/ml) for 30 min. Samples were extensively washed with TBS-TX followed by TBS, air dried, and mounted with Prolong Gold antifade with DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes) onto glass slides. Images were acquired with an Olympus BX51 fluorescent microscope equipped with an Olympus DP70 digital camera and software.

RESULTS

The absence of the propeptide does not affect Mpl stability or production.

The metalloprotease of L. monocytogenes, Mpl, is secreted as a 55-kDa zymogen that undergoes intramolecular autocatalysis, releasing a 20-kDa propeptide and a 35-kDa catalytic domain (5). In this study, our aim was to determine the function of the Mpl propeptide. Propeptides often serve as intramolecular chaperones, assisting in the folding of enzyme catalytic domains and contributing to the stability and activity of their protein partner (30). To assess the role of the Mpl propeptide, we deleted its chromosomal sequence by allelic exchange. The corresponding mutant strain (mplΔpro) synthesizes an Mpl molecule (MplΔpro) that is secreted as a mature enzyme upon cleavage of the signal sequence. This mutation was generated in two L. monocytogenes background strains: the clinical isolate 10403S (4) and its isogenic mutant, NF-L943 (29). NF-L943 contains a point mutation in the positive transcriptional regulator PrfA [PrfA(G155S)] causing the overexpression of PrfA-dependent genes, including mpl and plcB. NF-L943-derived mutants were used as a matter of convenience for non-tissue culture assays, whereas 10403S-derived mutants were used for most tissue culture assays as the expression of PrfA-dependent genes is upregulated intracellularly.

Stability of MplΔpro was assessed in broth culture. Secreted proteins were trichloroacetic acid precipitated from the supernatant of broth-grown bacteria, and Mpl was detected by Western immunoblotting (Fig. (Fig.1).1). Four species of Mpl are typically identified by Western immunoblotting: a 55-kDa species corresponding to the proform or zymogen; the mature form, which runs aberrantly as a protein of approximately 40 kDa; a degradation product of 32 kDa; and the 20-kDa propeptide (5) (Fig. (Fig.1A,1A, lanes 1 and 5). An Mpl species comigrating with the mature form of Mpl was detected in the lane corresponding to the mplΔpro mutant (Fig. (Fig.1A,1A, lane 2), indicating that absence of the propeptide did not influence Mpl secretion or stability in broth-grown bacteria. Providing the propeptide in trans did not affect the secretion and stability of mature Mpl by the mplΔpro mutant strain (Fig. (Fig.1A,1A, lane 3). Also, addition of a C-terminal Flag tag did not affect the secretion and stability of Mpl synthesized in the presence or absence of its propeptide (Fig. (Fig.1B,1B, lanes 2 and 3). These results indicated that the propeptide is dispensable for Mpl to reach a stable conformation.

FIG. 1.
Detection of Mpl from culture supernatants by Western immunoblotting. Strains were grown in LB-MOPS-Glc, supplemented with chloramphenicol for strains carrying pAM401-derived plasmids. The equivalent of 1.0 ml of culture to an OD600 of 1.0 was loaded ...

The propeptide is not required for Mpl activity in vitro.

Proteolytic activation of PC-PLC is mediated by active Mpl (5, 18, 25). To determine if the propeptide is integral to the generation of active Mpl, PC-PLC activity was monitored by spot inoculating bacterial strains onto EYA. PC-PLC activity was detected around wild-type bacterial colonies as a zone of opacity resulting from phospholipid hydrolysis (Fig. (Fig.2,2, panels 1 and 6). This surrounding zone of opacity was not detected in the absence of PC-PLC or Mpl (Fig. (Fig.2,2, panels 4, 5, 10, and 14), confirming that the zone of opacity is generated by active PC-PLC and that activation of PC-PLC is Mpl dependent. A zone of opacity was detected around mplΔpro mutant colonies (Fig. (Fig.2,2, panels 2 and 7), indicating that Mpl synthesized in the absence of its propeptide is functional and capable of mediating PC-PLC maturation. Overall, these results demonstrated that the propeptide is not essential for Mpl to mediate PC-PLC maturation.

FIG. 2.
Detection of Mpl-dependent PC-PLC activity on LB-EYA. Agar was spot inoculated with strains constructed in the NF-L943 background. Mpl-mediated activation of PC-PLC results in hydrolysis of egg yolk phospholipids, creating a zone of opacity around the ...

The zones of opacity surrounding the mplΔpro mutant colonies were smaller than the zones of opacity surrounding colonies expressing wild-type mpl (Fig. (Fig.2,2, compare panels 7 to 6 and 2 to 1). Addition of a C-terminal Flag tag compromised further the ability of MplΔpro to mediate PC-PLC maturation (Fig. (Fig.2,2, compare panels 12 and 7), even though it did not affect the stability of the protein (Fig. (Fig.1B).1B). These results indicated that, although not essential, the propeptide of Mpl contributes to Mpl activity.

For the large majority of studied proteases, propeptides serve as intramolecular chaperones and are essential for their associated protease to reach native conformation. However, in many instances, native protein folding and protease activity can be rescued by providing the propeptide in trans (17, 20, 37, 45). Therefore, we sought to determine if providing the propeptide in trans could restore full Mpl activity. The sequence coding for the Mpl signal sequence and propeptide was cloned in a multicopy vector downstream of a constitutive spac promoter. As observed by Western immunoblotting, the propeptide is secreted normally and appears stable when synthesized in trans of its protease partner (Fig. (Fig.1A,1A, lane 3). On EYA, the zone of opacity surrounding the propeptide-complemented mplΔpro mutant strain was equivalent in size to the zone surrounding the noncomplemented mplΔpro mutant strain (Fig. (Fig.2,2, panels 2 and 3). This result indicated that the defect observed with the mplΔpro mutant cannot be complemented by providing the propeptide in trans. Overall these results indicated that the propeptide of Mpl is not essential for Mpl to mediate PC-PLC maturation. However, the propeptide enhances the production of active Mpl when present in cis, but not when provided in trans.

Mutations affecting propeptide stability do not prevent Mpl activity in vitro.

During an investigation to identify propeptide residues that are important for autocatalysis, we discovered that two mutants generated by site-directed mutagenesis, mpl(H75V) and mpl(H95L), have an unstable propeptide. Western immunoblot analysis of bacterial culture supernatants revealed the presence of a novel Mpl species below the proform around 48 kDa and the absence of the 20-kDa propeptide (Fig. (Fig.1A,1A, lanes 6 and 7). Presumably, the 48-kDa band results from an initial cleavage within the propeptide, as the size of the mature form was unaltered, whereas the propeptide was undetectable. Mpl is not responsible for this novel cleavage, since when the H75V and H95L point mutations were constructed in an Mpl background that is catalytically inactive [Mpl(E350Q)] (5), the 48-kDa product was still present (data not shown). However, the susceptibility of the propeptide to degradation suggests that the H75V and H95L mutations affect the conformation of the Mpl propeptide.

Fusion of a C-terminal Flag tag to Mpl(H75V) appeared to stabilize the proform of Mpl, as no degradation product was detected below the proform (Fig. (Fig.1B,1B, lane 4). In addition, Mpl(H75V)-Flag showed a decrease in autocatalytic activity, as a less mature form was generated (Fig. (Fig.1B,1B, compare lanes 2 and 4). Nevertheless, the total absence of propeptide suggested that, similar to Mpl(H75V), Mpl(H75V)-Flag has an unstable propeptide.

The ability of the two propeptide mutants to mediate PC-PLC maturation on EYA was examined. The results showed that both single-site mutations, mpl(H75V) and mpl(H95L), produce a zone of opacity slightly larger than the strain expressing MplΔpro but smaller than the zone produced by the wild-type strain (Fig. (Fig.2,2, compare panels 8 and 9 to 7 and 6). Similar to what we observed with the propeptide deletion mutants, addition of C-terminal Flag tag compromised further the ability of Mpl(H75V) to mediate PC-PLC maturation (Fig. (Fig.2,2, compare panels 13 and 8). Overall, these results indicated that a stable propeptide is not essential for generation of active Mpl, although propeptide stability influences the levels of Mpl activity.

The activity of Mpl is influenced by its propeptide during intracellular infection.

Mpl and PC-PLC contribute to the intracellular life cycle of L. monocytogenes by mediating bacterial escape from double-membrane vacuoles. Intracellular bacteria maintain a pool of PC-PLC in its inactive proform. Rapid maturation and translocation of PC-PLC across the cell wall are dependent on Mpl activity and a decrease in pH (44). To assess if mature Mpl synthesized in the absence of its propeptide is functional in a host cell environment, the behavior of PC-PLC was monitored in cells infected with the mplΔpro mutant strain. Infected J774 cells were pulse-labeled with [35S]methionine/cysteine, and the intracellular pH was manipulated during a 5-min chase period to mimic cytosolic or vacuolar pH. Infected cells were lysed under conditions that preserve bacterial integrity, enabling physical separation of bacterial cells from host cell lysates, followed by lysis of the bacterial cells. The compartmentalization of PC-PLC was determined by immunoprecipitation of the protein from bacteria and host cell lysate fractions. As previously observed, PC-PLC remains primarily bacterium associated and in its proform at pH 7.3 (Fig. 3A to C, lanes 1 and 2), whereas it is found primarily in the host cell fraction and in its mature form at pH 6.5 (Fig. 3A to C, lanes 3 and 4). In the absence of Mpl, a decrease in pH does not influence PC-PLC compartmentalization as it remains bacterium associated and in its proform at pH 6.5 (Fig. (Fig.3A,3A, lanes 13 and 14). The phenotype of the mplΔpro mutant was similar to that of the Δmpl mutant as PC-PLC remained primarily bacterium associated and in its proform upon a decrease in pH (Fig. (Fig.3A,3A, lanes 5 to 8). This defect could not be complemented by providing the propeptide in trans (Fig. (Fig.3A,3A, lanes 9 to 12). In addition, Mpl(H75V) and Mpl(H95L) were unable to mediate the rapid maturation or translocation of PC-PLC in response to a decrease in pH in infected J774 cells, similar to the behavior of the mplΔpro mutant strain (Fig. (Fig.3B,3B, lanes 5 to 8) (data not shown). These results indicated that Mpl synthesized in the absence of its propeptide, or with an unstable propeptide, is unable to mediate the rapid maturation and cell wall translocation of bacterium-associated PC-PLC upon a decrease in pH during intracellular infection, and synthesis of the propeptide in trans does not complement this defect.

FIG. 3.
Detection of Mpl-mediated and pH-dependent PC-PLC maturation and translocation across the bacterial cell wall in infected J774 cells. All strains are derivatives of 10403S. Infected cells were pulse-labeled with [35S]methionine/cysteine and chased for ...

Mpl is also responsible for the pH-dependent cleavage of the bacterial surface protein ActA (26). Mpl-mediated cleavage of ActA was monitored as a secondary assessment of the intracellular activity of Mpl synthesized in the absence of its propeptide. Infected J774 cells were pulse-labeled in the presence of host protein synthesis inhibitors, and the intracellular pH of the cells was manipulated during a 10-min chase period to mimic cytosolic or vacuolar pH as described above. ActA was extracted from the bacterial surface with sample buffer, resolved by SDS-PAGE, and detected by autoradiography. ActA is phosphorylated during intracellular infection and forms a characteristic triplet on a protein gel (6) (Fig. (Fig.4,4, lanes 1 to 3). As previously observed, membrane-associated ActA is lost upon a decrease in intracellular pH, and this phenomenon is dependent on Mpl activity (Fig. (Fig.4,4, lanes 4 and 6). Bacterium-associated ActA was stable at acid pH in cells infected with the mplΔpro mutant, similar to cells infected with the Δmpl mutant (Fig. (Fig.4,4, lanes 5 and 6). This result showed that the propeptide of Mpl is essential for Mpl-mediated cleavage of surface-associated ActA. Overall, the results indicated that Mpl is defective in processing its substrates, PC-PLC and ActA, during intracellular infection when its propeptide is absent (Fig. (Fig.33 and and4),4), and providing the propeptide in trans does not complement Mpl's ability to mediate PC-PLC maturation (Fig. (Fig.3).3). In addition, Mpl is defective in processing PC-PLC during intracellular infection when its propeptide is unstable (Fig. (Fig.33).

FIG. 4.
Detection of Mpl-mediated proteolytic cleavage of ActA during intracellular infection. J774 cells were infected with 10403S-derived strains. Cells were pulse-labeled with [35S]methionine and then chased for 10 min with a nigericin-containing medium at ...

The compartmentalization of Mpl is influenced by its propeptide during intracellular infection.

During intracellular infection, the compartmentalization of PC-PLC is regulated in part by its propeptide. When synthesized with its propeptide, PC-PLC remains largely bacterium associated until the bacteria become entrapped in acidifying vacuoles. However, PC-PLC synthesized in the absence of its propeptide is constitutively translocated across the bacterial cell wall in a manner independent of pH and Mpl (44). Therefore, we speculated that, similar to PC-PLC, the propeptide of Mpl influences its compartmentalization during intracellular infection. Mpl constructs with a C-terminal Flag tag were used to detect Mpl by immunofluorescence. As shown in Fig. Fig.1B,1B, addition of a C-terminal Flag tag did not decrease the stability of the proteins. Results from immunofluorescence staining revealed the presence of bacterium-associated Mpl-Flag in infected cells (Fig. (Fig.5B).5B). Cells infected with a strain expressing Mpl without a Flag tag served as a negative control for the staining (Fig. (Fig.5J).5J). Detection of bacterium-associated Mpl-Flag was increased upon digesting the cell wall of fixed samples with mutanolysin (compare Fig. 5B and D), indicating that bacterium-associated Mpl is not surface accessible, similar to bacterium-associated PC-PLC. However, Mpl was not detected with each bacterium in samples treated with mutanolysin (Fig. (Fig.5B),5B), presumably because of incomplete cell wall digestion. MplΔpro-Flag and Mpl(H75V)-Flag were not found to be bacterium associated in infected cells (Fig. 5F and H).

FIG. 5.
Detection of bacterium-associated Mpl in infected HeLa cells by fluorescence microscopy. HeLa cells were infected with NF-L943-derived strains. Fixed infected host cells were reacted with an anti-Flag (α-Flag) M2 antibody followed by a fluorescein ...

The influence of the Mpl propeptide on compartmentalization was also assessed by immunoprecipitating Mpl from cleared lysates of pulse-labeled infected cells. Contrary to what we observed in broth culture, the zymogen was the only Mpl species detected in cells infected with wild-type strain, the mpl(H75V) mutant, or the mpl(H95L) mutant (Fig. (Fig.6,6, lanes 1, 5, 7, and 8), presumably because Mpl autocatalysis is tightly regulated during intracellular infection. The mature form of Mpl was detected in cells infected with the mplΔpro mutant (Fig. (Fig.6,6, lanes 2, 3, and 6). Complementation of the mplΔpro strain with the propeptide in trans did not influence the behavior of MplΔpro (Fig. (Fig.6,6, lane 3).

FIG. 6.
Immunoprecipitation of secreted Mpl from infected J774 cells. All strains are derivatives of 10403S. Infected cells were pulse-labeled with [35S]methionine/cysteine. Secreted Mpl was immunoprecipitated from cleared host cell lysates and detected by autoradiography ...

The influence of the Mpl propeptide on compartmentalization of Mpl was quantified for wild-type Mpl and the MplΔpro and Mpl(H75V) mutants. Quantification of band intensities for the secreted pro and mature forms of Mpl was performed for three independent immunoprecipitation experiments. After correction for background and the number of methionine and cysteine residues contained in the pro and mature forms of Mpl, the average densities ± standard deviations were determined to be 77.1 ± 50.07 for wild-type Mpl, 234.5 ± 28.4 for MplΔpro, and 252.9 ± 61 for Mpl(H75V). Analysis of the data by a two-tailed paired t test indicated that MplΔpro and Mpl(H75V) were secreted in significantly larger amount than wild-type Mpl (P = 0.02) in infected J774 cells. Unfortunately, technical difficulties prevented us from immunoprecipitating bacterium-associated Mpl. Overall, these results suggested that intracellular bacteria maintain a pool of Mpl that is not surface accessible and that the compartmentalization of Mpl is dependent on the presence of its propeptide in cis, as Mpl molecules synthesized with an unstable propeptide or without a propeptide are found uniquely in the cytosol of infected cells.

DISCUSSION

L. monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of infected cells and spreads from cell-to-cell using an actin-based mechanism of motility (10, 39). Upon initial invasion of a host cell and during cell-to-cell spread, L. monocytogenes becomes entrapped in vacuoles that it must escape to perpetuate its intracellular cycle. Among the factors contributing to bacterial escape from double-membrane vacuoles are PC-PLC and Mpl (31, 40). Both enzymes are made as proproteins, which undergo maturation by cleavage of an N-terminal propeptide. The propeptide of PC-PLC serves to inhibit enzymatic activity and to retard PC-PLC translocation across the bacterial cell wall (18, 19, 33, 44). Rapid maturation and translocation of PC-PLC across the cell wall depend on a decrease in pH and on Mpl activity. In this study, we sought to determine the function of the Mpl propeptide. Our results indicate that the propeptide retains Mpl bacterium associated and that the compartmentalization of Mpl is integral to its ability to process its bacterium-associated substrates during intracellular infection.

Propeptides of bacterial proteases are normally important for catalyzing the folding of their covalently bound catalytic domains and can often perform this function either in cis or in trans (30). In this study, we first investigated whether the propeptide of Mpl serves as a folding catalyst by testing the behavior of an L. monocytogenes mutant lacking the sequence coding for the Mpl propeptide. Detection of Mpl from broth-grown bacteria by Western immunoblotting indicated that the propeptide and catalytic domain fold into a stable form when synthesized as independent monomers. Whether MplΔpro achieves native conformation was determined by assessing its enzymatic activity. The EYA activity assay demonstrated that active Mpl can be generated in the absence of the propeptide. Therefore, contrary to the large majority of studied proproteases, the propeptide of Mpl is dispensable for the production of active enzyme. This is not the first example of a bacterial metalloprotease whose propeptide is dispensable for activity, as a similar observation was made for the neutral protease of Bacillus stearothermophilus (16). Additional observations led us to consider that perhaps the propeptide of Mpl does not function as a folding catalyst. First, MplΔpro is less active than wild-type Mpl in the EYA activity assay, and this defect cannot be rescued by providing the propeptide in trans. Second, the propeptide appeared to be stable as it was detectable by Western immunoblotting after cleavage from the proenzyme and when provided in trans. Together, these two observations suggested that the propeptide of Mpl does not interact with its associated protease in trans, since according to the folding-catalyst model, it would be degraded by its protease partner once the native conformation is attained. Third, two destabilizing propeptide mutations, H75V and H95L, did not prevent the generation of an active mature form of Mpl. Alternatively, our observations could indicate that the propeptide of Mpl functions as a folding catalyst but that the free energy required for the transition from the intermediate to the native state is low, enabling the mature form to reach its native state in the absence of the propeptide although with slower kinetics. This hypothesis would explain the decreased activity of MplΔpro detected on EYA, but would not explain the propeptide stability or why it cannot complement this defect in trans. Biochemical and biophysical analyses of purified Mpl will be required to solve whether the propeptide functions as a folding catalyst. However, our data clearly indicate that Mpl is an atypical thermolysin-like protease, as its propeptide is not required to generate active enzyme.

PC-PLC and Mpl are virulence factors that contribute to the intracellular life cycle of L. monocytogenes. Therefore, we assessed Mpl activity during intracellular infection. Mpl mediates the rapid maturation and translocation of bacterium-associated PC-PLC and cleaves membrane-anchored ActA in acid-pH vacuoles. These two functions were tested by manipulating the intracellular pH of infected cells to synchronize the reaction for all intracellular bacteria. Interestingly, the MplΔpro protein did not demonstrate activity toward either of its two substrates during intracellular infection. In addition, the two propeptide single-site mutants, Mpl(H75V) and Mpl(H95L), did not mediate the rapid maturation and translocation of PC-PLC across the bacterial cell wall during intracellular infection. ActA proteolysis by Mpl was not assessed for these two mutants. Together, these data indicated that the propeptide of Mpl is integral to the intracellular function of Mpl.

The behavior of PC-PLC during intracellular infection suggests that Mpl and PC-PLC interaction occurs prior to translocation across the bacterial cell wall, in the confined interface between cell wall and membrane. Perhaps the propeptide of Mpl serves a function similar to the propeptide of PC-PLC, to retard translocation of the protein across the bacterial cell wall. To test this possibility, the compartmentalization of Mpl during intracellular infection was determined. By immunofluorescence microscopy, we observed that wild-type Mpl, but not MplΔpro or Mpl(H75V), associates with intracellular bacteria. Additionally, using an immunoprecipitation assay, we found three times more (P = 0.02) MplΔpro molecules than wild-type Mpl in the secreted fraction from intracellular bacteria. Mpl(H75V), whose propeptide appears to be misfolded and subject to degradation after autocatalysis, was also found in the secreted fraction of intracellular bacteria in a larger amount than wild-type Mpl (3.3-fold; P = 0.02). Together, these data support the hypothesis that the propeptide of Mpl serves a function similar to the propeptide of PC-PLC, to retard translocation of the protein across the bacterial cell wall. Conceivably, the proform of Mpl is sequestered at the membrane-cell wall interface by interacting with cell envelope components, preventing efficient translocation of the protein across the cell wall. Alternatively, the Mpl zymogen may adopt a conformation that is incompatible with cell wall translocation. In either case, it appears that bacterial association is a prerequisite for Mpl to mediate PC-PLC maturation during intracellular infection, but that prerequisite is less stringent when bacteria are grown on agar plates. In fact, when a plcB deletion mutant and an mpl deletion mutant were streaked on EYA perpendicular to each other without touching, a small zone of opacity developed between the two strains (data not shown), indicating that Mpl-mediated maturation of PC-PLC can occur in the extracellular milieu. However, when L. monocytogenes becomes entrapped in a vacuole, Mpl-mediated maturation of PC-PLC must occur very rapidly as the efficacy of bacterial escape from vacuoles is in part dependent on PC-PLC activity (31, 40). Therefore, the presence of PC-PLC and Mpl at the cell wall membrane interface may serve to increase the probabilities of interaction between these two proteins and consequently of bacterial escape from vacuoles.

In conclusion, this study revealed that the propeptide of Mpl serves to retain the bacterium-associated protease, and this function is integral to the ability of Mpl to mediate the rapid maturation and translocation of PC-PLC across the bacterial cell wall during intracellular infection. There are many similarities between Mpl and PC-PLC. Both enzymes are synthesized as a proprotein; both enzymes are found to be bacterium associated during intracellular infection; their individual propeptides serve to retard protein diffusion across the bacterial cell wall; neither propeptide contains a transmembrane domain or a cell wall anchoring motif; and, finally, maturation of PC-PLC is dependent on Mpl activity. Future studies will aim to determine the mechanism by which these propeptides interfere with protein translocation across the bacterial cell wall, a phenomenon that is critical to the pathogenesis of L. monocytogenes.

Results from this study also revealed that the propeptide of Mpl is not required for Mpl to fold into its native state, although there is evidence that the propeptide enhances the ability of Mpl to fold into an active form. Additional biochemical and biophysical studies will be required to determine if the propeptide of Mpl functions as a folding catalyst.

Acknowledgments

We thank Marci Scidmore for granting us the use of the fluorescence microscope and Emily R. Slepkov for helping with the quantification experiment.

This work was supported by Public Health Service grant AI52154 from NIAID to H.M.

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 April 2009.

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