In this study, we developed a new mariner-based transposition system for Listeria monocytogenes. The mariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis in L. monocytogenes.Listeria monocytogenes is a saprophytic gram-positive bacterial rod that is ubiquitous in nature and is an opportunistic foodborne pathogen of humans and a variety of other vertebrates (24). During infection, L. monocytogenes multiplies intracellularly in the cytosols of host cells (19,23). Intracellular survival of L. monocytogenes relies largely on its ability to subvert host functions by escaping phagocytic vacuoles and spreading from cell to cell without exiting the intracellular milieu. In the last two decades, multiple studies have aimed at identifying virulence factors and deciphering the mechanisms by which L. monocytogenes survives in a wide range of environments (9, 13).One of the most valuable genetic tools used to study bacteria is the transposon. Transposons can be used to perform high-throughput mutagenesis of an entire chromosome, generating banks of mutants that can be screened for identification of factors related to specific bacterial functions. The transposon delivery systems that are currently available for use with L. monocytogenes are not ideal for these types of studies (4,7,14). For example, the most commonly used transposon delivery system, Tn917-LTV3, is more than 22 kb in size and has a low efficiency of transposition and a high rate of vector retention (4). In this study, we aimed at designing a transposon delivery system that is more suitable to high-throughput mutagenesis.In recent years, Himar1 mariner has been used as the transposon of choice in performing high-throughput mutagenesis in many different bacterial species, including low-GC-content gram-positive species (1, 2, 18). Himar1 was originally isolated from the horn fly, Haematobia irritans, and is a member of the Tc1/mariner superfamily of transposable elements (21). The Tc1/mariners are the most-widespread transposons in nature. These elements require no factors for transposition other than their self-encoded transposases (16), a feature that makes them ideal candidates for development into generalized genetic tools. Moreover, the mariner requirement for insertion is the dinucleotide TA, which makes it perfect for transposition into low-GC-content organisms such as L. monocytogenes (39% GC). We reasoned that a mariner-based transposition * Corresponding author. Mailing address: Department of Microbiology and Immunology, VMC C5-169, Cornell University,
The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain of L. monocytogenes expressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.Listeria monocytogenes is a facultative intracellular bacterial pathogen that multiplies in the cytosol of host cells and uses an actin-based mechanism of motility to spread from cell to cell without exiting the intracellular milieu (26). Upon cell-to-cell spread, bacteria are temporarily located in double-membrane vacuoles from which they must exit to perpetuate the intracellular life cycle. Among the factors contributing to vacuolar lysis is a bacterial phospholipase C (PC-PLC) (24, 28). PC-PLC is made as a proenzyme whose activation requires cleavage of a 24-amino-acid propeptide. The metalloprotease of Listeria (Mpl) contributes to PC-PLC activation (21). Moreover, Mplmediated maturation of PC-PLC is regulated in a temporal and spatial manner during the intracellular life cycle of L. monocytogenes (15).Mpl possesses the HEXXH motif that is characteristic of the Zincins superfamily of metalloproteases. Within this family, Mpl is most closely related to thermolysin, which is the prototype member of the M4 family of metalloproteases (20). Thermolysin is made by Bacillus thermoproteolyticus, and all members of the thermolysin family originate from bacteria. The active site zinc ion of these enzymes is coordinated by a water molecule and three amino acid residues, including the two histidines present within the HEXXH motif and a glutamic acid located 20 residues downstream of this motif (2, 8). In addition, the glutamic acid residue located within the HEXXH motif and a histidine residue located 83 residues downstream of this motif interact with a water molecule at the active site and are required for catalysis (2, 3).Thermolysin and related metalloproteases are synthesized as preproenzymes. The prodomain, which accounts for ϳ40% of the proenzyme, serves as a chaperone and as an inhibitor of catalysis (17,23,29). Processing of the prodomain generates the mature active form of the protease. Autocatalysis has been suggested to be the mechanism of maturation of thermolysinlike proteases, as catalytic site mutants fail to generate mature proteases (10, 11, 13, 16). Furthermore, a mechanism of intramolecular autocatalysis was suggested for thermolysin itself, as purified active t...
Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.Listeria monocytogenes is a Gram-positive, facultative intracellular bacterial pathogen. It is the causative agent of the food-borne disease listeriosis, which has a high mortality rate (37). L. monocytogenes is able to invade host cells and spread from cell to cell using host actin (35). To escape the vacuoles formed upon initial entry into a cell or cell-to-cell spread, L. monocytogenes relies on multiple virulence factors. These factors include listeriolysin O (LLO) (7, 35), a phosphatidylinositol-specific phospholipase C (4), and a broad-range phospholipase C known as PC-PLC (phosphatidylcholine phospholipase C) (32). PC-PLC is synthesized as an inactive proenzyme and translocates across the cell membrane, where it accumulates at the membrane-cell wall interface (21, 34). A decrease in pH and the metalloprotease of L. monocytogenes (Mpl) are required for PC-PLC maturation, which coincides with the rapid secretion of mature PC-PLC across the bacterial cell wall (21, 31).Mpl is a member of the thermolysin family of metalloproteases which contains a Zn 2ϩ ion in the active site (11). Mpl is produced as a zymogen with an N-terminal propeptide (22). Similar to PC-PLC, Mpl translocates across the bacterial membrane and accumulates at the membrane-cell wall interface (24,34). This compartmentalization...
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 compartmentalization. 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 maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.
The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.
c Cutaneous leishmaniasis is a sand fly-transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain of Listeria monocytogenes as a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge with Leishmania major and sand fly saliva when vaccinated intravenously. However, this protection was short-lived. Importantly, groups of vaccinated mice were protected long term when challenged with infected sand flies. Protection correlated with smaller lesion size, fewer scars, and better parasite control be-
Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.
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