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Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France
Correspondence
Alain Charbit
charbit{at}necker.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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91–99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91–99 region in the production of a stable and functional LLO. LD50 determinations in the mouse model suggest a possible link between LLO stability and virulence.
A supplementary table of primer sequences is available with the online version of this paper.
These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Vazquez-Boland et al., 2001). This pathogen is a facultative intracellular micro-organism, capable of invading a wide variety of eukaryotic cells (Dramsi et al., 1995; Gaillard et al., 1986, 1996; Kuhn & Goebel, 1989), including endothelial cells (Drevets et al., 1995) and macrophages (Mackaness, 1962). Each step of intracellular parasitism by L. monocytogenes is dependent upon the production of virulence factors (Cossart, 2002; Goebel & Khun, 2000). The major virulence genes identified so far are clustered into two distinct loci on the chromosome and are controlled by a single pleiotropic regulatory activator, PrfA, which is required for virulence (Johansson et al., 2002; Renzoni et al., 1999). Among these virulence factors, listeriolysin O (LLO, encoded by the hly gene) plays a crucial role in the escape of bacteria from the phagosomal compartment to the cytoplasm of infected host cells. LLO-negative mutants remain trapped in the vacuole, do not grow intracellularly and are avirulent in the mouse model of infection (Gaillard et al., 1986; Portnoy et al., 1988).
LLO belongs to a family of cholesterol-dependent, pore-forming cytolysins (CDCs) (Alouf, 2000). The best-characterized members of the CDCs are perfringolysin O (PFO) and streptolysin O (SLO), which are both secreted by extracellular bacterial pathogens: Clostridium perfringens and Streptococcus pyogenes, respectively. The three-dimensional structure of LLO is currently unknown but that of monomeric PFO has been determined by X-ray crystallography (Rossjohn et al., 1997). Replacement of LLO with PFO in L. monocytogenes results in a strain that is able to escape from a vacuole, albeit at a reduced efficiency, but that kills the infected cells (Jones & Portnoy, 1994a). Thus, LLO is apparently unique among the CDCs in that it can disrupt a vacuolar membrane, but does not kill the host cell upon growth in the cytosol. The molecular mechanism of LLO-dependent phagosomal escape of intracellular L. monocytogenes remains unknown. It is believed that a rapid degradation of LLO in the cytosol, controlled by host-cell-mediated degradation, may ensure that the host cytoplasmic membrane remains intact during infection by L. monocytogenes (Bubert et al., 1999; Moors et al., 1999). Several motifs or residues that might control intracytosolic degradation of LLO have been proposed and/or studied. In particular, a region rich in proline, glutamate, serine and threonine residues (called the PEST motif) has been identified close to the N-terminus of the mature protein. PEST motifs are thought to target eukaryotic proteins for phosphorylation and/or rapid degradation by the proteasome (Decatur & Portnoy, 2000; Lety et al., 2002; and references therein).
A series of immunological studies demonstrated that the induction of acquired cellular immunity is crucial in the elimination of L. monocytogenes. In particular, the generation of antigen-specific cytotoxic T-cells leads to the killing of infected cells. Intracytosolic multiplication of L. monocytogenes induces, in mice, strong MHC class I-restricted cytotoxic T-lymphocyte (CTL) responses (Busch et al., 1997; Finelli et al., 1999; Sijts et al., 1996). One LLO epitope (residues 91–99) was shown to elicit a very large, dominant response (Busch & Pamer, 1998; Pamer, 1994; Pamer et al., 1997; Vijh & Pamer, 1997; Vijh et al., 1999; Villanueva et al., 1995). These studies provided the only direct evidence of a proteasome-mediated cytosolic degradation of LLO.
Correct cleavage of a CTL epitope by the proteasome is a crucial step in the formation of precursor peptides leading to MHC presentation, and a single residue exchange in the flanking region may abolish accurate proteasome-mediated cleavage (Beekman et al., 2000; Yellen-Shaw & Eisenlohr, 1997; Yellen-Shaw et al., 1997).
These observations led us to pursue the systematic analysis of the different motifs (or regions) comprised within the LLO sequence that may contribute to its biological activities. We addressed here the role of the CTL epitope region, focusing on the relation between LLO stability and haemolytic activity in broth; and phagosomal escape and cytotoxicity, in eukaryotic cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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hly is a derivative of EGD-e (serotype 1/2a), which contains an in-frame chromosomal deletion of 1080 bp in the hly gene (Guzman et al., 1995). EGDhly was transformed with the different recombinant plasmids by electroporation, as previously described (Park & Stewart, 1990). Bacteria were grown in Brain Heart Infusion (BHI) broth (Difco) at 37 °C without antibiotics, except for the pAT28-transformed strains, which were grown on BHI broth containing 60 µg spectinomycin (Spc) ml–1.
Construction of the recombinant cytolysins.
Chromosomal DNA, plasmid isolation, restriction enzyme analyses and amplification by PCR were performed according to standard protocols (Ausubel et al., 1990; Sambrook et al., 1989).
Seven LLO mutants were constructed by site-directed in vitro mutagenesis. Residues R89 and K90 were changed to glycine residues (denoted LLO-RK/GG), in the region upstream of the 91–99 epitope. Four mutants were constructed in the KKKK downstream region, substituting to alanine: one, two, three or all four K residues (mutations K103A; K103A, K104A; K103A, K104A, K105A; and K103A, K104A, K105A, K106A, respectively). For simplicity, the corresponding proteins were denoted LLO-K1/A1, LLO-K2/A2, LLO-K3/A3 and LLO-K4/A4, respectively. Two types of mutations were generated within the 91–99 epitope: an in-frame deletion of residues 91 to 99 (mutant protein LLO-91–99), and a substitution of the 91–99 epitope of LLO by the corresponding residues of PFO, which introduces four amino acid substitutions (Y92K, D94A, E97K and Y98F; denoted LLO-91–99P).
The LLO mutants LLO-RK/GG, LLO-K1/A1, LLO-K2/A2, LLO-K3/A3, LLO-K4/A4 and LLO-91–99 were constructed by using the GeneTailor Site-Directed Mutagenesis System (Invitrogen). Mutagenesis was performed on pAT28-phly-hly according to the manufacturer's recommendations.
The LLO mutant LLO-91–99P was generated by in vitro site-directed mutagenesis on M13mp18-phly-hly as described previously (Lety et al., 2001). The mutation was then transferred onto pAT28, a Gram-positive/Gram-negative shuttle vector, by restriction enzyme excision from the replicative form (BamHI–SalI) and insertion into the corresponding sites of pAT28. All the constructs were checked by PCR sequence analysis. Recombinant pAT28 plasmids were transferred into EGDhly by electroporation, as described previously (Lety et al., 2001). EGDhly expressing wild-type LLO (LLOwt) was used as a positive control and EGDhly as a negative control.
The mature form of LLO-K4/A4 protein was expressed in Escherichia coli with an N-terminal His-tag. For this, the portion of the mutant hly gene (carried on plasmid pAT28) encoding the mature form of the protein was amplified by PCR. The amplified product was cut with restriction enzymes BamHI and XhoI and inserted into the BamHI and XhoI sites of the expression vector pET28a (Novagen), downstream of the His-tag cloning sequence. The recombinant protein was denoted LLO-K4/A4 His-tag. The construct was transferred by transformation into the E. coli recipient strain BL21star (Invitrogen). Transformed bacteria were grown on LB medium containing ampicillin. Expression of the LLO-K4/A4 His-tag protein was induced by addition of IPTG (1 mM final concentration). After several hours of induction (up to 22 h), bacteria were harvested by centrifugation. The bacterial pellets were resuspended directly into SDS-PAGE loading buffer (typically a 2 ml culture was resuspended into 50 µl loading buffer) and the samples were boiled for 5 min before loading onto gels.
The primers used to create the different LLO mutants are listed in supplementary Table S1 (available as supplementary material with the online version of this paper).
Protein preparations and analyses.
Proteins were prepared from cell culture supernatants of bacteria grown in LB supplemented with 10 % BHI. Concentrated culture supernatants were prepared as described previously (Lety et al., 2003). Cell-free supernatants were filtered through a 0·22 mm pore size Millipore filter and concentrated by centrifugation through ultrafree Biomax units. The LLO-mutant proteins were identified by Western blot analysis, using polyclonal anti-LLO antibody. Identical amounts of each concentrated culture supernatant were loaded per well (except mutant LLO-91–99P, for which the concentrated supernatant was further concentrated by TCA precipitation).
Intracellular LLO analysis.
LLO produced by cytosolic bacteria was detected by immunoprecipitation with anti-LLO monoclonal antibodies SE1 and SE2, essentially as described previously (Lety et al., 2002). Briefly, monolayers of bone-marrow-derived macrophages (BMM) from BALB/c mice, seeded into 60 mm dishes, were infected at a bacterium : macrophage ratio of 10 : 1. After 30 min, monolayers were washed and reincubated for 2 h. The medium was then replaced with methionine-free RPMI minimal medium containing 10 % dialysed fetal calf serum and 225 µg cycloheximide ml–1. After 30 min, monolayers were pulsed-labelled for 1 h with 100 µCi (3·7 MBq) [35S]methionine (Amersham). Cells were then lysed and LLO was immunoprecipated with anti-LLO monoclonal antibodies. The immunoprecipitated material was finally subjected to SDS-PAGE and subsequent analysis on a PhosphorImager (Molecular Dynamics).
Anti-Listeria and anti-LLO antibodies.
The polyclonal anti-LLO serum was purchased from Diatheva. It was used in Western blot at a final dilution of 1/1000, according to the manufacturer's recommendation. The polyclonal rabbit anti-Listeria antibody (J. Rocourt, Institut Pasteur) was used in immunofluorescence at a final dilution of 1/200. Anti-LLO monoclonal antibodies SE1 and SE2 were kindly provided by Dr A. J. Ainsworth, Mississippi State University.
Infection of macrophages and microscopic analyses.
For confocal microscopy analyses, BMM from BALB/c mice were cultured and infected at a bacterium : macrophage ratio of 10–20 : 1 (Frehel et al., 2003). Growth in J774 macrophage-like cells was performed as described previously (Jones & Portnoy, 1994b) in the absence or the presence of gentamicin (50 µg ml–1 final concentration), at a bacterium : macrophage ratio of 50 : 1.
Processing for confocal microscopy.
Double fluorescence labelling of F-actin and bacteria was performed, using phalloidin coupled to Alexa 488 (Molecular Probes) and a rabbit anti-Listeria polyclonal antibody revealed with anti-IgG antibody coupled to Alexa 546 (Molecular Probes). Images were scanned on a Zeiss LSM 510 confocal microscope.
Cytotoxicity.
Monolayers of BMM from BALB/c mice, seeded into 60 mm dishes, were infected with the same ratios of bacteria per cell as those used for the immunofluorescence assays. Supernatants were removed from each well 4 and 6 h after infection and assayed for lactate dehydrogenase (LDH) activity by using the Cyto Tox 96 kit (Promega), as recommended by the manufacturer.
Two independent experiments were performed. In each experiment, three wells were counted per point. Percentage cytotoxicity=100x(experimental LDH release–spontaneous LDH release)/(maximal LDH release–spontaneous LDH release). Spontaneous LDH release was measured in supernatants of non-infected macrophages. Maximal LDH release corresponds to the macrophages lysed after treatment with 0·9 % Triton X-100 (final concentration), for 45 min at 37 °C. The percentage indicated in the text corresponds to the mean of three wells of a single experiment. For example, the background of spontaneous LDH release was 348±13 (mean±SD) and the value of maximal LDH release was 9530±339. With LLOwt, after 6 h, a value of 4460±155 was recorded. The percentage cytotoxicity thus corresponded to 100x(4460–335)/(9530–335)=44·7 %.
Invasion assays.
The human colon carcinoma cell line Caco-2 (ATCC HTB37) and the human hepatocellular carcinoma cell line HepG-2 (ATCC HB 8065, kindly provided by P. Cossart, Institut Pasteur) were propagated in Dulbecco's modified Eagle's medium (DMEM; 25 mM glucose; Gibco). Cells were seeded at 8x104 cells cm–2 in 24-well tissue culture plates (Falcon). Monolayers were used 24 h after seeding. The invasion assays were carried out essentially as described previously (Garandeau et al., 2002). Briefly, cells were inoculated with bacteria at an m.o.i. of 
50 bacteria per cell. They were incubated 1 h to allow the adherent bacteria to enter and were then washed three times with RPMI and overlaid with fresh DMEM containing gentamicin (10 µg ml–1) to kill extracellular bacteria. At selected intervals, cells were washed three times and processed for counting of infecting bacteria. Cells were lysed by adding cold distilled water. The titre of viable bacteria released from the cells was determined by spreading onto BHI plates. Each experiment was carried out in triplicate and repeated three times.
Infection of mice and virulence assays.
The virulence of the L. monocytogenes mutant strains was estimated by determining the LD50, in pathogen-free, 6–8-weeks-old female Swiss mice (Janvier). Groups of five mice were intravenously inoculated with bacterial suspension (0·5 ml in 0·15 M NaCl) in the lateral tail vein. Animals were pretreated with Spc (1 mg per mouse per day) in order to overcome in vivo instability of the recombinant plasmids, as described previously (Lety et al., 2002). The mortality was followed for 7–10 days. The virulence of the strains was estimated, using the graphic Probit method (Roth, 1961). For example, the LD50 of 105·5, recorded with the strain expressing LLOwt, corresponded to 100 % death at a dose of 106 bacteria per mouse (5/5 mice died within 1 week) and 0 % death at a dose of 105 bacteria per mouse; the LD50 of 107·5, recorded with the strain expressing LLO-RK/GG, corresponded to 100 % death at a dose of 108 bacteria per mouse and 0 % of death at a dose of 107 bacteria per mouse.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Residues R89 and K90 were changed to glycine residues (denoted LLO-RK/GG), in the region upstream of the 91–99 epitope. Four mutants were constructed in the KKKK downstream region, changing one, two, three or all four K residues to alanine (Fig. 1b). Two types of mutations were generated within the 91–99 epitope: an in-frame deletion of residues 91 to 99 (mutant protein LLO-91–99) and a substitution of the 91–99 epitope of LLO by the corresponding residues of PFO (see Methods for details).
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), LLO residues 10–36 comprise a putative PEST motif (Decatur & Portnoy, 2000; Lety et al., 2001) that has no counterpart in PFO. The 91–99 epitope is located 40 residues downstream of this motif. In the cytolysins secreted by two other members of the genus Listeria, ivanolysin O from Listeria ivanovii (ILO) and seeligeriolysin O from Listeria seeligeri (LSO), the 91–99 epitope sequence is highly conserved, with only one E97Q substitution in ILO and one N96S in LSO (as compared to six, six and four substitutions in the cytolysins secreted by S. pyogenes (SLO) and Streptococcus pneumoniae (PLY) and C. perfringens (PFO) respectively; Fig. 1c). The KKKK cluster downstream of the epitope is fully conserved in the three cytolysins of the genus Listeria. In the other cytolysins, in spite of sequence divergence, at least three positive charges are conserved. The RK sequence upstream is not conserved, but at least one positively charged residue is present in all cases.
On the predicted three-dimensional model of LLO folding (Dubail et al., 2001), the 91–99 region is located at the tip of domain 2 (Fig. 1d). The turn corresponds to G95, N96, and is conserved in the LLO, ILO and PFO sequences.
Immunodetection.
The two mutant proteins LLO-RK/GG and LLO-K1/A1 were efficiently secreted in the culture supernatant of L. monocytogenes grown in BHI (Fig. 2a). The amounts of LLO-RK/GG and LLO-K1/A1 detected by anti-LLO antibodies in Western blot (Fig. 2b) were comparable to that of wild-type LLO (LLOwt). LLO-91–99P, which was also secreted into the culture supernatant, appeared to be significantly more degraded. The band corresponding to the full-size protein was less abundant than that of LLOwt (or LLO-RK/GG and LLO-K1/A1) and several additional bands of lower molecular mass, most likely corresponding to degradation products, were detected by the anti-LLO antibody in the supernatant of bacteria grown to stationary phase (Fig. 2a). These low-molecular-mass bands were not detected in the supernatant of exponentially grown bacteria (Fig. 2c), suggesting that LLO-91–99P degradation occurred mainly during the stationary phase of growth.
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91–99, LLO-K2/A2, LLO-K3/A3 and LLO-K4/A4, were not detectable by the anti-LLO antibody (in exponential or stationary phase of growth). We tested whether LLO-K4/A4 could be expressed in E. coli with an N-terminal His-tag (see Methods for details). Whole-cell extracts from IPTG-induced cultures were loaded onto SDS-polyacrylamide gels (data not shown). Both the anti-LLO antibody and the Ni-NTA conjugate detected a band corresponding to the full-size recombinant protein. This experiment demonstrated that the recombinant protein LLO-K4/A4 could be stably expressed in E. coli, confirming the assumption that it is produced but rapidly degraded in L. monocytogenes.
Haemolytic activities.
The haemolytic activities of culture supernatants of the EGDhly derivatives were measured on horse erythrocytes (Lety et al., 2002). The activities recorded did not strictly correlate with the apparent stability of the LLO proteins in Western blot (Fig. 2d). Mutants LLO-K1/A1 and LLO-91–99P showed only a twofold lower haemolytic activity than LLOwt. In contrast, the activity recorded with mutant LLO-RK-GG was eightfold lower than that of LLOwt. No haemolytic activity was detected with the mutants LLO-91–99, LLO-K2/A2, LLO-K3/A3 and LLO-K4/A4, confirming the Western blot observations.
Phagosomal escape and intracellular survival
Phagosomal escape and intracellular multiplication was first studied in BMM from BALB/c mice, by confocal microscopy (Lety et al., 2001). Bacterial uptake was monitored up to 4 h after infection (Fig. 3a). The capacity of the mutant strains to promote phagosomal escape was determined by calculating the ratio between the number of bacteria surrounded by polymerized actin and the total number of bacteria in infected cells. These values were determined on an average of at least 50 infected cells. After 2 h infection, 33 % of the bacteria expressing LLOwt had reached the cytosol and were surrounded by polymerized actin (Fig. 3a, d). Phagosomal escape of the three mutant strains was even more efficient, with at least 61 % escape after 2 h. No bacterial multiplication and no actin polymerization were observed with the strain expressing LLO-91–99 or LLO-K4/A4 (Fig. 3e).
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). Bacterial uptake was monitored up to 6 h after infection. Phagosomal escape of the three mutant strains was also very efficient, confirming the BMM observations. After 2 h, the three mutant strains had already started to multiply. After 4 h, the percentage of bacteria surrounded by polymerized actin was roughly comparable with LLOwt and with the three LLO mutants tested, ranging between 49 and 71 %. After 6 h, the mean number of bacteria per infected cell was almost twofold higher with LLO-RK/GG and LLO-K1/A1 than with LLOwt and LLO-91–99P (with 8–9 and 5–6 bacteria per cell, respectively). Altogether, the confocal microscopy analyses showed that phagosomal escape and actin polymerization were not affected in LLO-RK/GG, LLO-K1/A1, or LLO-91–99P, in a phagocytic as well as in a non-phagocytic host cell.
We also measured the kinetics of bacterial invasion in the mouse macrophage-like cell line J774 (Fig. 4a) and in the enterocyte-like cell line Caco-2 (Fig. 4b). Cells were inoculated with bacteria, and, at selected intervals, cells were processed for counting of infecting bacteria (see Methods). In J774 cells, intracellular multiplication of LLO-K1/A1, LLO-RK/GG and LLO-91–99P was followed over an 8 h period, in the absence (not shown) as well as in the presence of gentamicin (50 µg ml–1 final concentration). If any of the LLO mutants had damaged the host cell membrane, intracellular entry of gentamicin should have led to an inhibition of bacterial multiplication. The three mutants showed normal growth properties, in the absence or presence of gentamicin (Fig. 4a), thus indicating their lack of cytotoxicity. In Caco-2 cells, the three mutants also showed a normal growth over the infection period tested (Fig. 4b). After 3 h, bacteria started to multiply actively and during the next 2 h the growth rates of the mutant strains were almost identical to that of the strain expressing wild-type LLO, reflecting normal phagosomal escape and intracytoplasmic survival in these cells.
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). In all cases, a single major band was detected (Fig. 2c). However, the intensity of the major band detected with the three LLO mutants was weaker than that of LLOwt (37 %, 10 % and 5 % of LLOwt for LLO-K1/A1, LLO-RK/GG and LLO-91–99P, respectively: Table 1).
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). Less than 1 % LDH release was recorded in macrophages infected with the LLO-negative strain EGDhly. In agreement with our earlier observations, 44·7 % LDH release was recorded with the strain expressing wild-type LLO after 6 h infection, reflecting cell death (Barsig & Kaufmann, 1997). A comparable value was obtained with mutant LLO-K1/A1 (53 % LDH release after 6 h). The values recorded with LLO-RK/GG and LLO-91–99P ranged between 24·3 % and 31 % LDH release (Table 1). These results indicated a lack of cytotoxicity of the three LLO mutants tested.
Finally, the virulence of the L. monocytogenes mutants was evaluated in the mouse infection model by determining LD50, using the graphic Probit method (Roth, 1961). Groups of outbred female Swiss mice were inoculated by intravenous injection with different doses, in tenfold dilutions. As previously shown, EGDhly is totally avirulent and expression of plasmid-encoded wild-type LLO restores virulence to the strain (LD50 105·5). The strains expressing LLO-RK/GG and LLO-K1/A1 showed a strong attenuation of virulence (
100-fold decrease) and the strain expressing LLO-91–99P was almost avirulent (LD50>108) (Table 1).
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DISCUSSION |
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; Glomski et al., 2002, 2003; Villanueva et al., 1995). The identification of a putative PEST-like sequence close to the N-terminus of the protein suggested that this region might control intracytosolic LLO degradation and therefore prevent cytotoxicity (Decatur & Portnoy, 2000; Lety et al., 2001). However, the direct role of this region in cytosolic degradation of LLO is not yet established. In order to identify additional LLO motifs involved in optimal intracellular adaptation of L. monocytogenes, we focused here on the immunologically well characterized 91–99 CTL epitope region. Out of the seven LLO mutants constructed, only three could be detected in the supernatant of L. monocytogenes, indicating that the 91–99 region is important for protein stability.
Earlier studies on integral membrane protein had led to the notion of permissive and non-permissive sites in proteins (Charbit et al., 1986 and references therein). A permissive site was defined as a site that accommodates local sequence modifications without complete loss of protein stability and/or functionality, whereas a non-permissive site does not tolerate changes without extensive protein degradation. According to this terminology, residues 91–99 can be considered as a non-permissive region of LLO. Due to the complex structural organization of the LLO molecule and to the conformational changes occurring upon interaction with a cholesterol-containing membrane (Schuerch et al., 2005 and references therein), probably many other sites or regions of the LLO molecule are also non-permissive.
The KKKK stretch downstream of the epitope deserves particular attention since only a single amino acid substitution was tolerated. One hypothesis to account for this result could be that the loss of two positive charges in this region is not acceptable to maintain a stable structure. Based on the sequence similarities between LLO and PFO, on the folded monomer, the tip of D2 is close to the autonomously folded domain D4. Mutations at the tip of D2 may thus alter the proper interaction between these domains. The fact that the mutant protein bearing all four K residues changed to alanine (LLO-K4/A4) could be produced in E. coli suggests that the protein produced in L. monocytogenes is abnormally folded and therefore rapidly degraded by proteases (this may also hold for the other proteins that were not detectable).
While secreted as monomers by bacteria, the CDCs form oligomers upon binding to cholesterol-containing membrane. In the case of LLO, complete pore-like structures have been visualized on erythrocyte membranes (Jacobs et al., 1998). However, the notion of pore formation in the acidic phagosomal compartment, where LLO expression is induced, is as yet purely speculative. The mechanisms of LLO-mediated phagosomal disruption are probably quite distinct from the pore-formation process observed on erythrocyte membranes and may not imply any oligomerization. Indeed, in spite of a two- to eightfold reduction of haemolytic activity, the three mutants LLO-K1/A1, LLO-RK/GG and LLO-91–99P allowed efficient phagosomal escape and intracellular survival of L. monocytogenes, in both phagocytic and non-phagocytocic cells.
In conclusion, the present work showed that three LLO mutants bearing mutations in the 91–99 region, LLO-K1/A1, LLO-RK/GG and LLO-91–99P, could grow efficiently in vitro (in different cell lines) but had a severe reduction of virulence in the mouse model. This novel phenotype contrasts with that of mutants in the PEST region we had generated previously (Lety et al., 2001, 2002). Indeed, all the LLO-PEST mutants were secreted by L. monocytogenes in normal amounts and were fully haemolytic but they were all impaired to different extents in phagosomal escape. At this stage, one may hypothesize that both the PEST and the CTL region would control cytosolic LLO stability, limiting the possible toxicity of the LLO molecule produced during intracytosolic multiplication of L. monocytogenes.
Mutations reducing the cytosolic stability of LLO without altering intracellular survival of L. monocytogenes were not expected to impair bacterial virulence. The drastic effect of the mutations in LLO on the LD50 of L. monocytogenes could be due for example to reduced haemolytic activity. Indeed, the development of the infection leading to brain invasion requires an initial multiplication in the infected target organs and a subsequent transitory bacteraemia (Berche, 1995). Other mechanisms may also be involved in vivo, requiring specific LLO properties distinct from those necessary for in vitro multiplication.
Finally, from an immunological point of view, it would be interesting to monitor quantitatively the capacity of the stable cytolysins LLO-RK/GG and LLO-K1/A1 to be processed by the proteasome to produce CTL epitopes as well as their ability to trigger a protective immune response.
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ACKNOWLEDGEMENTS |
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Received 9 December 2005;
revised 25 January 2006;
accepted 30 January 2006.
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, LLOwt; 
, LLO-RK; 
, LLO-K1; +, LLO-91–99P.
