Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 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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These authors contributed equally to this work.
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INTRODUCTION |
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Each step of the intracellular parasitism by L. monocytogenes and L. ivanovii is dependent upon the production of virulence factors (Goebel & Khun, 2000; Cossart & Bierne, 2001
). The major virulence gene cluster (prfAplcAhlymplactAplcB) of L. monocytogenes is present, with the same genetic structure and at an identical position, in the chromosome of L. ivanovii (Gouin et al., 1994
; Chakraborty et al., 2000
). This gene cluster is organized around hly, which encodes a pore-forming cytolysin : listeriolysin O (LLO) in L. monocytogenes and ivanolysin O (ILO) in L. ivanovii [see Bayley (1997)
and Alouf (2000)
]. The hly gene product plays a crucial role in the escape of bacteria from the phagosomal compartment. LLO-negative mutants of L. monocytogenes remain trapped in the vacuole, do not grow intracellularly and are avirulent in the mouse model of infection (Gaillard et al., 1986
; Kathariou et al., 1987
; Portnoy et al., 1988
). L. ivanovii is unique among members of the genus Listeria in possessing, in addition to the two phospholipase genes (plcA and plcB), a gene named smcL, which encodes a sphingomyelinase (SmcL; Gonzalez-Zorn et al., 1999
). This enzyme has been shown to participate in the disruption of the phagocytic vacuole and in subsequent intracellular proliferation of the pathogen.
LLO and ILO, which are highly similar at both the nucleotide and protein level, belong to a large family of cholesterol-dependent cytolysins (Alouf, 2000). This family of toxins comprises, to date, 23 members from different Gram-positive genera, including perfringolysin (PFO) whose X-ray structure has been solved (Rossjohn et al., 1997
), a cytolysin secreted by the extracellular pathogen Clostridium perfringens. Despite structural and functional similarities between LLO and PFO (44 % identity at the peptide level), Portnoy and co-workers (Jones & Portnoy, 1994
) have previously shown that the pfo gene, cloned under the control of the promoter phly, was unable to complement an hly mutation in L. monocytogenes for virulence in the mouse model of infection. The resulting strain was fully haemolytic and partially able to replicate in the mouse macrophage-like J774 cell line (PFO-mediated vacuolar escape at approx. 50 % of the efficiency of LLO). However, expression of PFO damaged the host cell, preventing further intracellular proliferation of the bacteria, suggesting that LLO has evolved structural features specifically adapted to the intracellular life cycle of L. monocytogenes.
Here, we tested whether ILO could replace LLO for efficient phagosomal escape of L. monocytogenes, intracellular multiplication and ultimately, for virulence in the mouse model of infection. For this, we expressed the ilo structural gene in a hly derivative of L. monocytogenes. We also constructed an artificial operon encoding both the smcL and the ilo gene. The properties of the recombinant L. monocytogenes strains were studied in vitro and in vivo. The implications on the role(s) of LLO and ILO in bacterial virulence are discussed.
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METHODS |
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Constructs.
Chromosomal DNA, plasmid isolation, restriction enzyme analyses and amplification by PCR were performed according to standard protocols (Sambrook et al., 1989; Ausubel et al., 1990
). All the constructs were carried on the shuttle vector pAT28 (Trieu-Cuot et al., 1990
), and the recombinant plasmids were transferred into EGD
hly by electroporation as described by Park & Stewart (1990)
. EGD
hly expressing wild-type LLO under the same conditions was used as a positive control (Dubail et al., 2000
; Lety et al., 2001
); EGD
hly was used as a negative control.
The recombinant plasmid pAT28-phly-ilo is a derivative of plasmid pAT28-phly-pfo (Lety et al., 2001), which carries the pfo gene encoding PFO under the control of the promoter phly. Briefly, plasmid pD1868 (kindly provided by Dr D. A. Portnoy, Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA; Jones & Portnoy, 1994
) was digested with SalI and XbaI, and the 1·6 kb SalIXbaI fragment comprising phlypfo was subcloned into the SalIXbaI sites of pAT28, yielding plasmid pAT28-phly-pfo.
ILO.
The ilo gene was amplified by PCR using primers 5'-CG GGA TCC AGG AGA GTG AAA CCC ATG AAA AAA ATA ATG CTA CTT TTA ATG ACA TTG TTA-3' and 5'-GC GAA TTC TTA CTT AAT TGG ATT ATC TAC AGT ATC ACT-3'. Plasmid pAT28-ilo was constructed by substitution of the BamHIEcoRI fragment carrying the pfo gene of pAT28-pfo by a BamHIEcoRI fragment carrying the ilo gene (the BamHI and EcoRI sites are underlined).
The 15 bases preceding the ATG start codon of the ilo gene were designed to correspond to those preceding the hly gene and thus, comprise the ShineDalgarno motif of hly.
SmcLILO.
Plasmid pAT28-phly-smcL-ilo was constructed by cloning a BglIIBglII fragment carrying smcL of L. ivanovii into the BamHI site of plasmid pAT28-phly-ilo in the same orientation as ilo (checked by PCR amplification with the appropriate pairs of primers). smcL was amplified by PCR using primers 5'-CG AGA TCT AGG AGA GTG AAA CCC ATG GAA AAA TTT AAA ATT ATA AAA ACA ATA CCC-3' and 5'-CG AGA TCT TTA GTT ATT ATC AGT GAA ACC AAC TAC AGG-3'. The 15 bases preceding the ATG start codon of smcL correspond to those preceding hly. A BglII site was created immediately upstream of the ShineDalgarno sequence; a second BglII site was created immediately downstream of the stop codon. We checked, by RT-PCR with appropriate primer pairs, that both the smcL and the ilo gene were transcribed in this construct (not shown).
Preparation of proteins and analyses.
Proteins were prepared from supernatants of strain EGDhly transformed with the different pAT28 derivatives. Concentrated culture supernatants from bacteria grown in LuriaBertani rich medium were prepared essentially as described previously (Lety et al., 2001
). Cell-free supernatants were filtered through a 0·22 µm pore-size Millipore filter. The filtered supernatants were concentrated by centrifugation through ultrafree Biomax units (cut-off 30 kDa). Cytolysins were identified by Western blot analysis with anti-pneumolysin mAb PLY-5, which was kindly provided by Dr De los Toyos, Area de Immunologia, Facultad de Medicina, Universidad de Oviedo, Spain (Jacobs et al., 1999
). mAb PLY-5 was used at a final dilution of 1/200, as described previously (Lety et al., 2001
). Identical volumes of each concentrated culture supernatant were loaded into each well.
Haemolysis.
Haemolytic phenotypes were visualized by spreading bacteria onto horse- or sheep-blood agar plates containing Columbia base medium (BioMérieux). CAMP-like tests were performed on horse- and sheep-blood agar plates, as described by Ripio et al. (1995). Haemolytic activity of bacterial culture supernatants was measured by lysis of horse or sheep erythrocytes, as described by Jones & Portnoy (1994)
. The values corresponding to the reciprocal of the dilution of culture supernatant required to lyse 50 % of horse erythrocytes were used to compare the haemolytic activities of the different supernatants.
Infection of mice and virulence assays.
The virulence of strains was estimated by determining the LD50 using the Probit method and by the bacterial survival in tissues. Specific pathogen-free, 6- to 8-week-old female Swiss mice (Janvier, Le Genest St Isle, France) were used. Bacteria were grown for 18 h in BHI broth, centrifuged, washed once, appropriately diluted in 0·15 M NaCl and then inoculated intravenously into the mice via the lateral tail vein (0·5 ml). Groups of five mice were challenged with various doses of bacteria (109, 108, 107 or 106 bacteria per mouse), and mortality was observed for 10 days.
Kinetics studies.
Twenty mice were inoculated per mutant. At days 1, 2, 3 and 6 after inoculation, groups of five mice were killed and the organs (spleen and liver) were aseptically removed and separately homogenized in 0·15 M NaCl. Bacterial numbers in organ homogenates were determined at various intervals on BHI plates containing spectinomycin. Five-hundred microlitres of bacterial suspension were injected into each mouse (containing 5x105 bacteria for EGDhly expressing LLO, or 108 bacteria for EGD
hly expressing ILO alone or co-expressing SmcL and ILO).
Assays were carried out on animals that had been pre-treated with spectinomycin (1 mg spectinomycin per mouse per day), in order to overcome in vivo instability of the recombinant plasmids (except for EGDhly alone).
Culture of cell lines and microscopy analyses
Cell cultures and infections.
Bone marrow (BM)-derived macrophages from BALB/c mice were cultured as described previously (De Chastellier & Berche, 1994) and then infected as follows. Overnight-grown bacteria were diluted in cell culture medium to give a bacterium-to-macrophage ratio of 10 : 1. Bacteria were allowed to adhere to cells by incubation on ice for 15 min, then to enter cells by placing the cells at 37 °C for 15 min. After thorough washings, infected cells were re-fed with fresh medium.
Human hepatocellular carcinoma cell line HepG-2 (ATCC HB 8065) was obtained from S. Dramsi and P. Cossart (Institut Pasteur, Paris, France). HepG-2 cells were cultured using RPMI 1640 medium supplemented with 10 % FCS at 37 °C under a 5 % CO2 atmosphere seeded at 2x104 cells per culture dish. Cells were infected 4872 h after seeding with overnight-grown bacteria, at a ratio of 50 : 1 for 1 h at 37 °C. As for macrophages, non-ingested bacteria were removed by washing, and infected cells were fed with fresh culture medium.
Processing for confocal microscopy.
At selected intervals after infection, cells were fixed and double fluorescence staining for F-actin and bacteria was performed, as described previously (Lety et al., 2001), using phalloidin coupled to Alexa 488 (Molecular Probes) and a rabbit anti-Listeria polyclonal antibody revealed with anti-IgG coupled to Alexa 546 (Molecular Probes). Images were scanned on a Zeiss LSM 510 confocal microscope.
Processing for electron microscopy.
At selected intervals following infection (between 0 and 8 h after infection), cells were fixed and processed as described previously (De Chastellier & Berche, 1994). Thin-sections were stained with 2 % uranyl acetate and lead citrate.
Cytotoxicity.
Monolayers of BM-derived macrophages 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. Four and 6 h after infection, supernatant was removed from each well and assayed for lactate dehydrogenase (LDH) activity by using the CytoTox 96 kit (Promega), as recommended by the manufacturer. Percentage cytotoxicity=100x times; (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 for 45 min at 37 °C (a 40-fold difference was observed between the minimal and maximal values). The numbers reported in Table 1 correspond to the mean of four wells of a single experiment and are representative of two independent experiments.
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RESULTS |
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On blood agar, L. ivanovii differs from L. monocytogenes in that it causes a bi-zonal haemolytic effect and when grown close to Rhodococcus equi, gives rise to a characteristic shovel-shaped patch of synergistic haemolysis on sheep-blood agar also called the CAMP-like phenomenon due to the expression of the sphingomyelinase (SmcL) encoded by smcL (Gonzalez-Zorn et al., 1999). As shown in Fig. 2(e), a
strong activity could be visualized on sheep-blood agar plates, demonstrating the production of SmcL by the L. monocytogenes derivative carrying both the smcL and the ilo gene.
Expression of ILO allows intracellular multiplication and restores in vivo survival
The ability of the ILO-expressing strain to disrupt phagosomal membranes and to multiply in the cytosol of infected cells was evaluated both in BM-derived macrophages and in hepatocytes. Virulence was studied by measuring the ability of the strain to multiply and persist in the target organs of infected Swiss mice after intravenous inoculation. The in vitro and in vivo properties of the ILO-expressing strain were then compared to those of a strain co-expressing SmcL and ILO.
In vitro properties.
The intracellular fate of EGDhly expressing ILO was examined by confocal microscopy in BM-derived macrophages from BALB/c mice, after double staining with an anti-Listeria antibody and with
-phalloidin to visualize the F-actin (Gaillot et al., 2000
). EGD
hly expressing LLO and EGD
hly alone were used as controls. We monitored quantitatively the capacity of the mutant strain to promote the disruption of the phagosomal membrane and to multiply intracellularly, by immunofluorescence microscopy. We calculated: (i) the percentage of phagosomal escape (i.e. the number of bacteria surrounded by polymerized actin/total number of bacteria in cells); (ii) the number of infected cells containing bacteria surrounded by polymerized actin; and (iii) the number of bacteria per infected cell (on a mean of 50100 infected cells), at the different times of the infections.
As shown in Table 1 and Fig. 3
, EGD
hly expressing ILO was able to efficiently disrupt the phagosomal membranes of infected macrophages. After 4 h post-infection, the percentage of bacteria surrounded by polymerized actin (reflecting the percentage of phagosomal escape) reached 87 %, a value very similar to that recorded with the strain expressing LLO. As in the case of the LLO-expressing strain, actin polymerization was visible in 100 % of the cells (Table 1
). As shown in Fig. 4
, the intramacrophagic multiplication of the ILO-expressing strain was similar to that of the LLO-expressing strain, while EGD
hly alone failed to multiply. Confirming the confocal analyses, electron microscopy analyses showed that, after 4 h infection, all the bacteria expressing ILO had multiplied and were visible inside the cytoplasm of infected BM-derived macrophages, surrounded by a mesh-work of polymerized actin (Fig. 5
).
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We next studied infection and intracellular multiplication of the ILO-expressing strain in the human hepatocellular carcinoma cell line HepG-2, by confocal microscopy. Bacterial content was monitored at 2 and 4 h after infection. Confirming the behaviour in BM-derived macrophages, the ILO-expressing strain could disrupt the phagosomal membranes of hepatocytes and multiply intracellularly as efficiently as the LLO-expressing strain. At 4 h after infection, the percentage of bacteria surrounded by polymerized actin ranged between 78 and 82 % and with both strains, actin polymerization was visible in most cells (Fig. 6a, b) and bacterial counts increased from approximately 5 bacteria per cell (at T0) to approximately 25 bacteria per cell.
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In vivo properties.
EGDhly is totally avirulent, and expression of plasmid-encoded LLO restores virulence to the strain (Dubail et al., 2000
). EGD
hly expressing ILO remained avirulent at a dose of 108 bacteria per mouse (at a 10-fold higher dose, the strain became toxic and mice died with convulsions within 2 h of infection). The ability of EGD
hly expressing ILO to multiply in the spleen and liver of infected mice was evaluated by infecting mice intravenously with the dose of 108 bacteria per mouse (at which all the mice recovered from infection). As shown in Fig. 7(a)
, the kinetics of survival in the liver showed an increase of bacterial counts up to day 2 after infection, reaching 7·7x106 bacteria per liver. The bacterial counts remained comparable at day 3 (approx. 7x106 bacteria per liver); at day 6, a significant number of bacteria still survived in the infected livers (1·8x104 bacteria per organ). By contrast, bacterial multiplication was drastically affected in the spleen. Bacterial counts dropped from 6·3x105 bacteria per spleen at day 1 after infection to only 2x103 at day 3, and bacteria were eliminated from the spleens by day 6 (<102 bacteria per spleen). Thus, at day 3 after infection, the number of bacteria expressing ILO was approximately 3500-fold higher in the liver than that in the spleen. By contrast, confirming earlier observations (Gaillard et al., 1986
; Berche, 1995
), with the LLO-expressing strain the number of bacteria in the spleen paralleled that in the liver (not shown).
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DISCUSSION |
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ILO can replace LLO for phagosomal escape and allows in vivo survival. Expression of ILO promoted efficient disruption of the phagosomal membranes of BM-derived macrophages, and intracellular multiplication of L. monocytogenes expressing ILO was essentially indistinguishable from that of the LLO-expressing strain. Moreover, expression of ILO appeared to be poorly cytotoxic. These data are strikingly different from those observed when the extracellular cytolysin PFO was expressed by L. monocytogenes in place of LLO, which revealed a high cytotoxicity of the PFO protein (Jones & Portnoy, 1994). To test whether the observed lack of virulence was mainly due to the high cytotoxicity of PFO, Portnoy and co-workers developed an elegant genetic procedure to select variants that would support normal intracytosolic growth (Jones et al., 1996
). In that study, all the non-cytotoxic PFO mutants selected, corresponding to single amino acid substitutions, were capable of mediating phagosomal escape and intracytoplasmic growth. However, they all remained avirulent in the mouse model. These data indicate that LLO possesses specific properties, and in particular a lack of cytotoxicity, which are important for the development of infection by Listeria.
In this respect, it is worth mentioning that a putative PEST sequence, suspected of controlling the intracytosolic half-life of LLO, has been identified close to the N terminus of the LLO protein (Decatur & Portnoy, 2000; Lety et al., 2001
). The deletion of this motif did not affect secretion and haemolytic activity of LLO but it did abolish bacterial virulence. We have demonstrated (Lety et al., 2002
) that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence. Furthermore, we have shown that single amino acid substitutions in the distal portion of the PEST motif are sufficient to impair phagosomal escape and to abolish the virulence of L. monocytogenes, unravelling the critical role of this region of LLO in the pathogenesis of L. monocytogenes. Although the peptide sequence of ILO is highly similar to that of LLO, this proximal region is less conserved than the rest of the protein and no PEST motif can be identified by the PEST-FIND program (http://bioweb.pasteur.fr/seqanal/interfaces/pestfind-simple.html).
Remarkably, the kinetics of survival of the ILO-expressing strain in infected mice revealed that the mutant strain could multiply in the liver up to 2 days after infection (reaching approx. 107 bacteria per organ). The persistence of a significant number of ILO-expressing bacteria in the liver 6 days after infection demonstrated a greater resistance to host defence mechanisms than an LLO-negative mutant (Gaillard et al., 1986). By contrast, bacterial multiplication was drastically affected in the spleen and the mutant strain remained avirulent. The inability of L. monocytogenes expressing ILO to persist in the spleen could be due to an increased susceptibility to proteolytic degradation of the ILO molecule in spleen cells. Other explanations are also possible. For example, an efficient infection by an ILO-expressing L. monocytogenes strain may require a higher level of expression of ILO. Alternatively, since LLO was shown to contribute to cell-to-cell spread (Dancz et al., 2002
), the avirulence of the ILO-expressing strain might be due to a defect of ILO which fails to disrupt efficiently the double vacuolar membrane entrapping the bacteria during their passage to adjacent cells. Interestingly, it has previously been shown (Conlan & North, 1994
) that neutrophils are not as important in anti-listerial defence in the spleen as in the liver during the first days of the infectious process. Neutrophils fail to substantially restrict L. monocytogenes multiplication in the spleen because the cells in which the bacterium resides in this organ (essentially monocytes and macrophages) are less permissive for its growth than are hepatocytes; even in the absence of neutrophils, L. monocytogenes is not found in large numbers inside individual spleen cells.
As mentioned earlier, in the mouse model, L. ivanovii is able to colonize the liver but not the spleen. It is thus tempting to suggest that the capacity of L. monocytogenes to multiply in the spleen might be mediated, at least in part, by specific properties of the LLO molecule that ILO does not possess. Conversely, ILO may have developed specific properties that allow L. ivanovii to develop in the livers but not in the spleens; these properties are favourable for the development of an infectious process in ruminants, their usual hosts.
Finally, the inability of L. monocytogenes expressing ILO to be virulent up to a dose of 108 bacteria per mouse (a higher dose being toxic) might be linked to the high LD50 of L. ivanovii (approx. 107 per mouse) in this animal model (Rocourt et al., 1983; Hof & Hefner, 1988
; Engelbrecht et al., 1998
). It is thus not excluded that, in other animal hosts, the recombinant strain might show some virulence.
In L. monocytogenes, the two phospholipases, PlcA (a phospholipase C specific for phosphatidylinositol) and PlcB (a broad-substrate-range phospholipase), contribute to the disruption of the phagosomal membrane in synergy with the cytolysin (Geoffroy et al., 1991; Vazquez-Boland et al., 1992
; Marquis et al., 1995
). In L. ivanovii, an additional phospholipase, the smcL-encoded sphingomyelinase, has been shown to participate in the disruption of the phagocytic vacuole and subsequent intracellular proliferation. The fact that L. ivanovii possesses an additional phospholipase suggests that the haemolysin and the PlcA/PlcB pair of L. ivanovii might be less efficient in disrupting the phagocytic vacuole and require the help of sphingomyelinase. We therefore tested whether the co-expression of SmcL and ILO by EGD
hly would restore virulence. We found that, in spite of a significant reduction in the production of ILO, the co-expression of SmcL and ILO did not alter the ability to escape from the phagosomes of BM-derived macrophages and intracellular multiplication. However, in vivo kinetics studies revealed that the strain was very rapidly eliminated from both the liver and spleen of infected mice. Production of SmcL is specific to L. ivanovii strains and is not normally required by L. monocytogenes to produce an infection. The mechanism by which SmcL impairs in vivo survival of L. monocytogenes has yet to be elucidated. At this stage, it might be due only to too low a level of ILO production.
In conclusion, the present work shows that the cytolysin produced by L. ivanovii can replace LLO for phagosomal escape, intracellular multiplication and in vivo survival of L. monocytogenes. Although the precise mechanisms of phagosomal escape are not yet understood, it has been proposed that cholesterol-dependent cytolysins may act as mediators of protein delivery to the host cytosol, like type III secretion systems of Gram-negative bacteria (Madden et al., 2001). In this respect, it has already been demonstrated that LLO is responsible for the induction of a series of signalling events (Kayal et al., 1999
; Vazquez-Boland et al., 2001
) in infected cells. Whether ILO or other related cytolysins display similar functions is unknown, but it is likely that LLO has probably gradually evolved to acquire unique features that allow optimal multiplication and survival of L. monocytogenes in the broad variety of cell types, tissues and hosts that this pathogen can infect.
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ACKNOWLEDGEMENTS |
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Received 11 September 2002;
revised 24 October 2002;
accepted 17 December 2002.
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