1 Yersinia Research Unit, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
2 General Microbiology, Faculty of Biosciences, FIN-00014 University of Helsinki, Finland
Correspondence
Elisabeth Carniel
carniel2{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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Y. pestis is a highly uniform species, which emerged about 1500 to 20 000 years ago from Yersinia pseudotuberculosis (Achtman et al., 1999), probably from a serotype O : 1b strain (Skurnik et al., 2000
). Despite their very close genetic relationship, the two species have distinct epidemiological and clinical features, and a drastically different pathogenic potential. The molecular bases for their difference in virulence are still not understood. Comparison of the genome sequences of representatives of the two species revealed an extensive genetic loss that has probably been more important than acquisition of new genes in the evolution of Y. pestis (Chain et al., 2004
). Nonetheless, 32 Y. pestis chromosomal genes and two Y. pestis-specific plasmids have been acquired by Y. pestis since its divergence from Y. pseudotuberculosis. The two Y. pestis-specific plasmids are a 101 kb plasmid called pFra (or pMT1) and a 9·6 kb plasmid called pPla (or pPst or pPCP1).
pFra encodes a phospholipase D (Hinnebusch et al., 2000; Rudolph et al., 1999
), which promotes Y. pestis survival in and colonization of the flea midgut (Hinnebusch et al., 2002
). The fact that a pFra-cured Y. pestis is still fully virulent for mice (Friedlander et al., 1995
) and African green monkeys (Davis et al., 1996
), indicates that the main property of this plasmid is not to enhance Y. pestis virulence but to support its flea-borne transmission.
The pPla plasmid encodes at least four proteins (Sodeinde & Goguen, 1988): the bacteriocin pesticin (Pst), its immunity protein (Pim), an IS100 transposase and a plasminogen activator (Pla). Activation of plasminogen by Pla damages the extracellular matrix (ECM) of cultured human epithelial cells (Lähteenmäki et al., 1998
). Plasminogen is a circulating precursor of the serine protease plasmin, whose main physiological function is to degrade fibrin clots and ECMs, thus facilitating cell migration (Plow et al., 1999
; Saksela, 1985
). Pla not only increases plasmin activity by cleaving plasminogen, but also inactivates the antiprotease
2-antiplasmin (
2AP), which is the main inhibitor of plasmin (Kukkonen et al., 2001
). Pla has been shown to play a role in Y. pestis virulence. Some pla mutants of Y. pestis were severely attenuated (one millionfold) after subcutaneous (sc) infection of mice (Sodeinde et al., 1992
). It has been proposed that Pla facilitates Y. pestis dissemination from its site of inoculation by cleaving fibrin deposits that trap the organisms, and by reducing the chemoattraction of inflammatory cells (Sodeinde et al., 1992
). However, in another study, no difference in the bacterial proliferation and local inflammatory response induced by wild-type Y. pestis or the pla mutant strain was observed at the site of injection, but a lower number of pla mutants at more distant sites, such as the draining lymph node and spleen, was noted (Welkos et al., 1997
). Furthermore, a few Y. pestis isolates lacking pPla were found to be fully virulent (Kutyrev et al., 1989
; Samoilova et al., 1996
; Welkos et al., 1997
), indicating that the role of pPla in Y. pestis pathogenesis remains to be clarified.
To determine whether acquisition of pPla may have been sufficient to confer an increased pathogenicity potential to the Y. pestis ancestor, Kutyrev et al. (1999) introduced this plasmid into Y. pseudotuberculosis to evaluate the pathogenicity of the recombinant strains for mice infected subcutaneously. Although Pla was produced and expressed on the cell surface, no significant modification of the LD50 of the pPla harbouring Y. pseudotuberculosis recombinant strains, as compared to the wild-type strain, was observed, suggesting that the presence of this plasmid is not sufficient to confer additional pathogenicity potential to the host strain. However, it was subsequently demonstrated that O-antigen (O-Ag) repeats in the lipopolysaccharide (LPS) of Y. pseudotuberculosis sterically inhibit plasminogen activation by Pla (Kukkonen et al., 2004
). The lack of Pla activity due to the presence of O-Ag repeats on the surface of Y. pseudotuberculosis could thus have accounted for the previously observed absence of increased pathogenicity of the pPla harbouring Y. pseudotuberculosis strains.
In this study, a mutation that abrogates the formation of O-Ag repeats (as in natural isolates of Y. pestis) was introduced into the chromosome of a pPla-harbouring Y. pseudotuberculosis strain. In this background, the ability of Pla to activate plasminogen and to degrade 2AP was investigated, and the impact of pPla on the virulence of the recombinant strain for mice was evaluated.
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METHODS |
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PCR.
The primers used in this study are listed in Table 2. PCRs were performed with 1 unit of Taq polymerase (Roche) or 1 unit of a 3 : 1 mixture of Taq and Pfu (Stratagene) polymerases in the supplier's buffer. PCR amplification reaction mixtures contained 10 µM each primer and 1 mM dNTPs. The PCR program involved one step at 95 °C for 5 min, followed by 30 cycles of amplification of three steps at (i) 95 °C for 30 s, (ii) 55 °C for 30 s and (iii) 72 °C for 13 min, depending on the fragment length. PCR products were maintained at 72 °C for 5 min, electrophoresed in 1 % agarose gels, and stained with ethidium bromide. Amplification of the kan cassette with long flanking homologous regions of the Yersinia target DNA was done with the PCR program described by Derbise et al. (2003)
.
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To obtain a Y. pseudotuberculosis strain that does not synthesize O side chains, primer pairs OKF/OKf1 and OKR/OKr2 were used to amplify the region encompassing the gmd and fcl genes, and primer pair 136A/136B to amplify the kan cassette (Table 2). Following the (long flanking homologous regions) LFH-PCR procedure (Derbise et al., 2003
), a linear PCR fragment composed of the kan gene with its promoter region, and flanked by
550 bp DNA homologous to the regions located on each side of gmd and fcl, was generated. This fragment was introduced by electroporation into strain IP32953(pKOBEG-sacB) and correct allelic exchange between the PCR fragment and the chromosomal gene was checked by PCR with primer pairs OKup/167, OKdown/166 and OKup/OKdown (Table 2
, Fig. 1b
). Y. pseudotuberculosis IP32953 clones with the appropriate O-Ag mutation and cured of pKOBEG-sacB were selected on LB-Sac agar plates.
The IP32953(pPla) O-Ag variant was obtained by introducing pPla-Tmp into IP32953 O-Ag by electroporation. Presence of a plasmid with the appropriate size was checked by digestion of the plasmid extract with BamHI.
Protein analysis.
To determine whether Pla is present in bacterial cell-envelope preparations, bacteria grown overnight were centrifuged, and cell suspensions were adjusted to an OD600 1·2 in PBS. Bacteria were sonicated four times for 30 s over crushed ice. Unbroken cells were removed by low speed centrifugation (1800 g), and the cell envelopes were pelleted by centrifugation at 15 000 g for 10 min at 4 °C. Total membrane proteins were separated by SDS-PAGE in 12·5 % gels, the proteins were transferred onto PVDF membranes (Hybond), and the Pla polypeptides were detected using 1 : 500 dilutions of primary anti-His6-Pla antisera (Kukkonen et al., 2001) and 1 : 10 000 dilutions of secondary anti-rabbit-IgG alkaline-phosphatase conjugate. Bound antibodies were detected with the phosphatase substrate (ECL Plus Kit, Amersham).
Extraction and analysis of LPS.
Bacteria grown overnight at 37 or 25 °C on LB plates were collected and resuspended in 2 ml PBS. The bacterial suspension was adjusted to OD420 0·4 and subjected to proteolysis for 2 h at 60 °C by the addition of 40 µg proteinase K ml1. An aliquot (1·5 ml) of this suspension was centrifuged for 3 min at 1 200 g, and the pellet was resuspended in 50 µl lysis buffer [2 % SDS, 4 % -mercaptoethanol, 10 % (v/v) glycerol, 1 M Tris-HCl pH 6·8, bromphenol blue] and heated at 100 °C for 10 min. Ten microlitres of this extract was subjected to SDS-PAGE in a 15 % polyacrylamide gel and transferred onto PVDF membranes. The LPS was detected using 1 : 1000 dilution of a primary rabbit anti-LPS antiserum raised against Y. pseudotuberculosis V2812/79 (kindly provided by Dr M. Skurnik, University of Helsinki, Helsinki, Finland), and 1 : 10 000 dilution of secondary anti-rabbit IgG alkaline-phosphatase conjugate. Bound antibodies were detected with the phosphatase substrate.
Plasminogen activation and 2AP degradation.
Kinetic measurement of plasminogen activation was performed as described by Kukkonen et al. (2001), by incubating 2x108 bacteria, 4 µg human Glu-plasminogen (American Diagnostica) and the chromogenic plasmin substrate S-2251 (Val-Leu-Lys-p-nitroaniline dihydrochloride; Chromogenix) in a total volume of 200 µl at 37 °C. Breakdown of the chromogenic substrate was measured in a microtitre-plate reader at A405. The A405 values were determined at time intervals of 15 min. The level of
2AP inactivation was measured as described by Lähteenmäki et al. (2005)
. Briefly, 5 µg
2AP ml1 was incubated overnight at 37 °C with 109 bacteria in 105 µl PBS. Gentamicin (1 µg ml1) was added to prevent bacterial growth. After the addition of 100 µl PBS, bacteria were removed by centrifugation and 168 µl supernatant was transferred into a well of a microtitre plate. Human plasmin (2·5 µg ml1; Sigma) and the plasmin substrate S-2251 (0·45 mM; Chromogenix) were added, and plasmin activity was measured at A405 as the breakdown of the substrate after 90 min incubation at 37 °C. The control for
2AP activity was produced by incubating
2AP with PBS instead of bacteria, and plasmin activity was checked in the absence of bacteria and
2AP. All measurements were carried out in duplicate and the experiments were repeated twice.
Animal infections.
Prior to infection, the presence in each Yersinia strain of known unstable genetic elements, such as the plasmid(s) and the high-pathogenicity island, was systematically verified by PCR with primer pairs located on these various elements: 18/19 (irp2), 37A/37B (caf1), 159A/159B (pla) and 160A/160B (yopM) (Table 2). Groups of five 5-week-old OF1 female mice (Iffa Credo) were inoculated subcutaneously with 0·1 ml bacterial suspension. Lethality was recorded daily for 3 weeks. The LD50 was determined according to the method of Reed & Muench (1935)
. To determine whether pPla-Tmp is stably maintained in Y. pseudotuberculosis during growth in vivo, three mice were infected subcutaneously with 108 c.f.u. of the IP32953(pPla) recombinant strain. Moribund animals were euthanized, their spleens were removed aseptically, crushed in saline and streaked onto LB plates. Individual colonies (100) were spotted onto MH and MH-Tmp agar plates. The percentage of TmpR colonies were recorded. All experiments were performed in a biosafety level 3 animal facility.
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RESULTS |
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Since the presence of O-Ag repeats was found to sterically inhibit plasminogen activation by Pla (Kukkonen et al., 2004), a Y. pseudotuberculosis derivative with a mutation in the O-Ag biosynthesis pathway was constructed. For this purpose, most of the gmd and fcl genes which encode a GDP-mannose-4,6-dehydratase and a GDP-fucose synthetase, respectively, were replaced by a kan cassette (Fig. 1b
). These two genes were chosen because they are naturally inactivated in the O-Ag gene cluster of Y. pestis (Skurnik et al., 2000
), and they act at an early step of O-side-chain assembly (in the biosynthesis of the repeat unit sugar precursors). Absence of O-Ag synthesis in the resulting IP32953 O-Ag mutant strain was evidenced by immunoblotting with an anti-LPS antibody. While the wild-type strain synthesized O side chains at 25 °C, the O-Ag mutant strain was unable to do so (Fig. 2
). pPla-Tmp was subsequently introduced into IP32953 O-Ag, yielding IP32953(pPla) O-Ag.
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DISCUSSION |
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Whether or not acquisition of pPla during Y. pestis evolution has been sufficient to confer an increased pathogenicity to this species was a key question. In an attempt to answer it, Kutyrev et al. (1999) introduced pPla into Y. pseudotuberculosis. They observed that Pla was expressed at the surface of the host strains and was processed to a smaller form designated
-Pla, as is known to be the case in Y. pestis (Kukkonen et al., 2001
; Sodeinde & Goguen, 1988
). In agreement with these results, we found in this study that introduction of pPla into Y. pseudotuberculosis led to the synthesis of Pla, to levels similar to those of Y. pestis, to its export to the bacterial membrane, and to its autoprocessing. When we looked at the plasminogen activation activity of Pla, we observed that this activity was detected in cells grown at 37 °C but not in those grown at 28 °C. In contrast to Kutyrev et al. (1999)
, we found that plasminogen activation at 37 °C was much lower than that of Y. pestis. These results may be explained by the recent demonstration that Pla requires rough LPS to activate plasminogen, but is inhibited in the presence of O-Ag repeats (Kukkonen et al., 2004
).
Y. pseudotuberculosis is known to exhibit temperature-dependent O-Ag expression (Bengoechea et al., 1998). At 28 °C, O-Ag repeats are present on the conserved core oligosaccharide and could thus prevent plasminogen activation by IP32953(pPla). At 37 °C, the LPS is rough and could allow functional Pla molecules to be present on the surface of the recombinant bacterium. The decreased plasminogen activation in IP32953(pPla), as compared to Y. pestis, might be attributed to a reduced number, but not a complete absence, of O side chains on the LPS of Y. pseudotuberculosis grown at 37 °C. This hypothesis is reinforced by our observation that in Y. pestis, which naturally harbours a completely rough LPS because of mutations in genes involved in O-side-chain assembly (Skurnik et al., 2000
), plasminogen activation activity is similar in bacteria grown at 28 and 37 °C. Further demonstration that the O-Ag repeats are responsible for the absence of plasminogen activation by IP32953(pPla) grown at 28 °C, and for a low activity in cells grown at 37 °C, was obtained by introducing a mutation that abrogates the formation of O-Ag repeats (as in natural isolates of Y. pestis) in the chromosome of IP32953(pPla). The IP32953(pPla) O-Ag mutant strain exhibited a temperature-independent plasminogen-activation activity that was much higher than that of the parental strain IP32953(pPla), and was close to that found in Y. pestis. Altogether these results demonstrate that a mutation that abrogates the synthesis of the O side chains in Y. pseudotuberculosis harbouring pPla allows a plasminogen-activation activity similar to that of Y. pestis, at both 28 and 37 °C.
In addition to increasing plasmin production by plasminogen activation, Pla also overcomes inhibition of plasmin by the serine protease inhibitor 2AP (Kukkonen et al., 2001
). In the human circulation, unbound plasmin is rapidly inactivated by plasma antiproteases, the main one being
2AP. We found that
2AP inactivation was effective in Y. pseudotuberculosis IP32953(pPla) grown at 37 °C, but was not detectable in bacteria grown at 28 °C. In contrast,
2AP degradation was similar in Y. pestis grown at 28 and 37 °C. These results again suggest a role for the LPS O side chains on the temperature-dependent capacity of Pla to inactivate
2AP in Y. pseudotuberculosis. The demonstration that an O-Ag derivative of IP32953(pPla) acquired the capacity to degrade
2AP to the same extent as Y. pestis when grown at either 28 or 37 °C confirmed this hypothesis.
Therefore, Pla-mediated increased production of plasmin, resulting from both plasminogen activation and 2AP degradation, is prevented at 28 °C in Y. pseudotuberculosis because of the presence of LPS O side chains, but is observable at 37 °C, a temperature at which O-Ag repeats are reduced or absent in vitro. However, this inhibition of O-side-chain formation at 37 °C does not appear to occur in vivo. This is demonstrated by the fact that during human infections with Y. pseudotuberculosis, high titres of antibodies directed against the O-Ag repeats of the various serotypes of this species are detected (Chung et al., 1997
; Schmidt, 1965
; Sizaret & Mollaret, 1968
; Splino et al., 1969
). The in vivo production of a smooth LPS may thus prevent the activity of Pla when a Y. pseudotuberculosis strain harbouring pPla infects its host. This could potentially explain why no difference in virulence was observed by Kutyrev et al. (1999)
between wild-type Y. pseudotuberculosis and smooth pPla-harbouring derivatives. We thus used an O-Ag mutant of IP32953(pPla), which was shown to exhibit plasminogen-activation and
2AP-degradation activities similar to those of Y. pestis, to compare its virulence to that of the wild-type strain and of the IP32953(pPla) and IP32953 O-Ag variants. First, our results confirmed the absence of a difference in mouse lethality between the pPla-harbouring and non-harbouring Y. pseudotuberculosis smooth strains. Second, they indicated that absence of O-Ag expression results in a slight decrease in Y. pseudotuberculosis pathogenicity. The role of LPS in Y. pseudotuberculosis virulence was not demonstrated until now, but was indirectly suggested by results of in vivo gene expression technologies (Darwin, 2004
). Our data further suggest that O-side-chain production is necessary for the expression of full virulence in Y. pseudotuberculosis, at least by the sc route of infection. Third, our results showed that introduction of pPla into the IP32953 O-Ag variant does not increase the pathogenicity of Y. pseudotuberculosis. On the contrary, the presence of pPla further decreased the virulence of the IP32953 O-Ag mutant strain and increased the time to death of the animals.
Altogether, our results indicate that in vitro, plasminogen-activation and 2AP-degradation activities can reach levels similar to those of Y. pestis in a Y. pseudotuberculosis variant harbouring pPla and lacking O-side-chain repeats. However, the efficient activity of Pla is not sufficient to confer a higher pathogenic potential to the Y. pseudotuberculosis host strain. The deleterious effect of a rough LPS on Y. pseudotuberculosis pathogenicity demonstrated in this study, may compensate the virulence-promoting effect of pPla. Furthermore, due to its protease activity, Pla may degrade proteins important for Y. pseudotuberculosis pathogenicity. This has been shown for YadA (Kutyrev et al., 1999
) and may be true for additional surface-exposed virulence factors. Although pPla may be necessary for the full virulence of most Y. pestis strains, this plasmid may not be sufficient by itself to explain the difference in the virulence of Y. pestis and Y. pseudotuberculosis. Additional genetic changes (mutations, deletions and/or gene acquisition) that occurred in Y. pestis upon its divergence from Y. pseudotuberculosis most likely play a critical role in the virulence of the plague bacillus. Therefore, acquisition of pPla by the Y. pestis ancestor may not have been sufficient to confer an immediate higher pathogenic potential to the host strain. Interplay between the acquired Pla protease and other bacterial components may have gradually led to subtle modifications of the Y. pestis ancestor's genetic background, and finally to the emergence of the highly pathogenic plague bacillus.
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ACKNOWLEDGEMENTS |
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Received 16 June 2005;
revised 10 August 2005;
accepted 26 August 2005.
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