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INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France
Correspondence
S. M. Roche
sylvie.roche{at}tours.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank accession numbers for the plcA, inlA and inlB nucleotide sequences were: AY367414, EF990797 and EF990802 for strain BO43; AY367410, EF990793 and EF990798 for strain CNL895807; AY367411, EF990796 and EF990801 for strain CNL895795; AY367412, EF990794 and EF990799 for strain 416; and AY367413, EF990795 and EF990800 for strain 417.
Three supplementary figures, showing immunofluorescence labelling of actin tails, nucleotide and amino acid substitutions observed in plcA, inlA and inlB genes of the Group III strains, and a schematic representation of the crystal structure of PI-PLC, and also a supplementary table, listing the PCR primers used, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dramsi et al., 1995). Both proteins are necessary and sufficient for the bacterium to invade various cell lines (Lecuit et al., 1997; Braun et al., 1999). InlA interacts with host cell E-cadherin, in a species-specific manner, to mediate the invasion of epithelial cells. InlB promotes the invasion of a wide variety of mammalian cells, such as hepatocytes and fibroblast cells, through interaction with three receptors: Met, gC1qR and glycosaminoglycans (Shen et al., 2000; Braun et al., 2000; Jonquieres et al., 2001).
After invading mammalian cells, L. monocytogenes escapes from the phagocytic vacuole by using a pore-forming bacterial toxin, listeriolysin-O (LLO), encoded by the hly gene, and two phospholipases, phosphatidyl-inositol phospholipase C (PI-PLC) and phosphatidylcholine phospholipase C (PC-PLC), encoded respectively by the plcA and plcB genes. Free in the cytosol, L. monocytogenes grows rapidly and employs ActA to induce polymerization of host actin filaments, which the bacteria use for moving intracellularly. Such movements cause protrusions which may contact neighbouring cells, resulting in the formation of double-membrane vacuoles, which are lysed by PC-PLC and LLO (Gaillard et al., 1986; Vazquez-Boland et al., 1992). This cell-to-cell spread allows plaque formation in cell monolayers, which can be used to estimate the virulence of L. monocytogenes strains (Roche et al., 2001).
L. monocytogenes is a pathogenic species, but some strains have been shown to have low virulence (Tabouret et al., 1991; Norrung & Andersen, 2000; Roche et al., 2001). In a previous study, 26 low-virulence L. monocytogenes field strains were identified using a method combining a plaque-forming assay (PFA) with subcutaneous (s.c.) injection into the left hind footpad of mice (Roche et al., 2001). The low virulence of these L. monocytogenes strains was found to be a stable trait. They also exhibited a low lethality in mice compared to virulent strains (Roche et al., 2003). These low-virulence strains, unrelated in origin and isolation date, were assigned to one of four groups based on their phenotypic characteristics, using cluster analysis (Roche et al., 2005). The cause of the low virulence of the 11 strains belonging to Group I has been identified. In fact, all these strains exhibit a mutated PrfA.
The present study was designed to identify virulence factors or cell-invasion mechanisms that were impaired in five out of six low-virulence Group III strains, which exhibited exactly the same phenotypic characteristics. These five strains are referred to as ‘Group III strains’ for the remainder of this article.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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inlA, EGDeinlB and EGDeinlAinlB originated from P. Cossart's laboratory (Institut Pasteur, Paris, France) as did the Listeria innocua BUG499 strain. The EGDeactA2 strain originated from E. Domann's laboratory (Institute of Medical Microbiology, Hospital of the Justus-Liebig University Giessen, Giessen, Germany). Escherichia coli TG1 was used for plasmid construction. The characteristics of the five low-virulence Group III strains are described in Table 1. Strains harbouring plasmid pHT1618 (Lereclus & Arantes, 1992) were grown in a culture medium containing 100 µg ampicillin ml–1 for E. coli, and 10 µg tetracycline ml–1 for Listeria. Strains harbouring plasmid pP1 (Dramsi et al., 1995) were grown with 150 µg erythromycin ml–1 for E. coli, and 8 µg erythromycin ml–1 for Listeria in liquid medium, and 1 mg erythromycin ml–1 for both E. coli and Listeria in agar medium.
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Cell culture assays.
Cell invasion assays were performed with Caco-2 or Vero cells, as described previously (Velge et al., 1997). The results were expressed as the log of the number of invading bacteria recovered after 3.5 h. Experiments were carried out in duplicate and repeated twice for each strain.
PFA were performed as described previously (Van Langendonck et al., 1998). The number of plaques was counted and the diameters of 20 plaques were measured with an Anastigmat Loupe x4 including a micrometer device (Peak).
Double fluorescence labelling of actin and bacteria.
J774-A1 macrophages were grown on eight-chamber tissue-culture glass slides (Falcon) with 5x105 cells per well. Cells were activated with Salmonella enterica serovar Typhimurium LPS (0.1 µg ml–1) the night before the assay. Cells were infected for 1 h with 1x106 c.f.u. and then incubated in culture medium containing 100 µg gentamicin ml–1 for 5 h to kill extracellular bacteria. Cells were washed and fixed overnight with cold 80 % acetone, saturated with 200 µl 10 % rabbit serum, and then incubated for 1 h with 100 µl of a 1 : 50 diluted anti-Listeria polyclonal antibody conjugated with fluorescein isothiocyanate (5688-1995, Biogenesis). Cellular F-actin was stained for 20 min in darkness at room temperature with 100 µl 1 % rhodamine-labelled phalloidin. Preparations were examined under a fluorescence microscope.
Nucleotide sequencing and cloning of virulence genes.
The inlA and inlB genes were amplified by PCR from total isolated DNA with AccuPrime Taq high-fidelity DNA polymerase (Invitrogen). An inlA–inlB fragment was amplified using primers inlA-Fwd and O24 for Group III strains and inlA-Fwd and inlB-Rev5 for the EGDe strain (see Supplementary Table S1). Nucleotide sequencing was carried out by Genome Express. Nucleotide sequences were aligned using Vector NTI (Informax).
The plcAEGDe, inlAEGDe and inlBEGDe genes were amplified with Phusion enzyme (Ozyme) using O37/O39, inlA-Fwd2/inlA-rev and inlB-Fwd/inlB-rev5 primers, respectively (Supplementary Table S1). The purified and digested plcA fragment was then introduced into digested pHT1618 vector, and the purified and digested inlA and inlB fragments were introduced into digested pP1 vector. DNA plasmids from E. coli transformants were isolated using the QIAprep plasmid kit (Qiagen). After verification by endonuclease restriction and sequencing analysis, recombinant plasmids were introduced into Group III strains by electroporation, as described elsewhere (Park & Stewart, 1990).
Complementation by allelic exchange mutagenesis.
The thermosensitive pMAD vector was used for allelic replacement of the plcA gene on the chromosome (Arnaud et al., 2004). A DNA fragment of the EGDe strain was amplified with plcAF3/plcAR3 primers using the Phusion enzyme (Supplementary Table S1). The fragment was purified and digested, and cloned into the digested pMAD vector. The recombinant vector was extracted from E. coli, and a three-step procedure was used for allele replacement in L. monocytogenes. In brief, the pMAD-plcAEGDe plasmid was introduced into the 416 strain by electroporation. The following steps were carried out as described elsewhere (Arnaud et al., 2004), except that the sensitive temperature used was 42 °C and the concentration of X-Gal was 200 µg ml–1. Several colonies were checked for erythromycin sensitivity and loss of the plasmid was analysed using PCR (Supplementary Table S1).
PI-PLC activity assays.
Detection of PI-PLC activity was checked on PI-TY agar, as described elsewhere (Notermans et al., 1991), and was analysed in the culture supernatant with tritium-labelled L-
-phosphatidyl-inositol, as previously described (Roche et al., 2005). Experiments were performed in triplicate for each strain.
Western blot analysis.
Secreted proteins were obtained as described elsewhere (Bannam & Goldfine, 1999). Protein pellets were separated in 12 % SDS–polyacrylamide gels. Western blots were probed with anti-PI-PLC antibody (provided by H. Goldfine's laboratory, University of Pennsylvania School of Medicine) (Bannam & Goldfine, 1999) and bound antibodies were detected using chemiluminescence (ECL+, GE Healthcare). Total bacterial extracts were separated in 10 % SDS–polyacrylamide gels. Western blots were probed with rabbit anti-InlB antibody (provided by P. Cossart's laboratory, Institut Pasteur, Paris, France) (Braun et al., 1999).
Mouse virulence assays.
The virulence of the strains was assessed after s.c. injection into the left hind footpad or after intravenous (i.v.) injection, as described previously (Roche et al., 2001). The results were expressed as the mean of the log number of c.f.u. per positive organ.
Statistical analysis.
The spleen colonization of i.v.-inoculated mice was analysed using a non-parametric Mann–Whitney test. Invasion assays were analysed with an analysis of variance followed by a Tukey–Kramer test. These statistical tests were performed with InStat software 2.03 (GraphPad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) showed that Group III strains (strains CNL895807, 416, 417, CNL895795 and BO43) had similar LLO and PC-PLC activities to those of the virulent EGDe strain but no PI-PLC activity. Moreover, these strains did not form plaques in HT-29 cell monolayers (Table 1
), exhibited no virulence in mouse infection assays (Table 1) and had an LD50 >1x109 c.f.u., similar to the results obtained with L. innocua. The prfA gene and the intergenic regions containing PrfA boxes were not mutated, suggesting that the transcription of virulence genes could occur. The low virulence of these strains could be related to multi-causal effects: low invasion capability, incapacity to form plaques and/or lack of PI-PLC activity.
Fluorescence labelling of ActA
As the incapacity of the Group III strains to form plaques could also be related to a non-functional ActA protein, the presence of actin tails was investigated using fluorescence microscopy with J774-A1 macrophages, which overcome the low invasion capability of bacteria. However, all Group III strains exhibited the same number of actin tails with a similar appearance to those observed with the virulent EGDe strain, unlike the EGDeactA2 strain, which did not form actin tails (see Supplementary Fig. S1). These results provide evidence that the ActA protein of Group III strains is functional and that the absence of plaques on cell monolayers is not related to ActA.
Expression of virulence proteins
To determine whether the lack of invasion and PI-PLC activity was related to an absence of InlA, InlB or PI-PLC proteins, Western-blot analysis was carried out to investigate the presence of these proteins. A protein was detected by the anti-PI-PLC serum in the precipitated culture supernatant of all the Group III strains and the EGDe strain, but not in the supernatant of the L. innocua strain. This result indicates that the lack of PI-PLC activity was not due to an absence of protein expression (Fig. 1a). Similarly, analysis of InlB expression detected a band at 63 kDa for all the Group III strains and the EGDe strain. However, the 417 strain expressed a lower quantity of InlB than the EGDe strain (Fig. 1b). Western-blot analysis carried out by the CNR (Centre National de Référence des Listeria, Institut Pasteur) detected the InlA protein for the EGDe strain, but not for any of the Group III strains (data not shown). The lack of InlA synthesis could thus explain the defective invasion of these strains into epithelial cells.
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As previously shown, all the Group III strains, none of which exhibited PI-PLC activity, showed the same 12 substitutions in the plcA gene (Supplementary Fig. S2a) that led to three mutations in PI-PLC (Supplementary Fig. S2b) (Roche et al., 2005). Analysis of the mutations showed that the isoleucine–valine substitution at position 17 (Ile17Val) was located in the signal sequence of the protein. The second substitution was located at position 119, between β-sheet II and the D helix, and led to the substitution of a phenylalanine by a tyrosine (Phe119Tyr). The last substitution was located between β-sheet VII and the G helix and changed the threonine in position 262 into an alanine (Thr262Ala). Only the Phe119Tyr and Thr262Ala mutations led to a change in the physico-chemical properties of the amino acids.
Trans complementation with a plasmid encoding plcA
In order to demonstrate that the substitutions in the plcA gene were responsible for the lack of PI-PLC activity, the pHT1618 plasmid carrying the plcAEGDe gene and its intergenic region was inserted into all Group III strains. Interestingly, trans-complementation restored the PI-PLC activity for the five strains (Fig. 2a). The trans-complemented CNL895795, 416 and BO43 strains exhibited a very high enzymic activity compared to the EGDe strain, in contrast to the trans-complemented strains CNL895807 and 417, which exhibited a lower activity. However, all trans-complemented strains exhibited PI-PLC activity on PI-TY agar, unlike their parental strains (data not shown). These results indicate that mutations in the plcA gene account for the lack of PI-PLC activity in the Group III strains.
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) (Fig. 2b). Trans complementation of the Group III strains with plasmid pP1 carrying the inlAEGDe gene allowed invasion of Caco-2 cells at the same rate as the EGDe strain. In fact, a statistically significant (P<0.01) 100-fold higher invasion rate was observed for these trans-complemented strains than for their parental strains, or their parental strains transformed with the empty plasmid (Fig. 2b). These results indicate that inlA mutation explains the inability of the Group III strains to invade epithelial cells.
Trans complementation with a plasmid encoding inlB
To analyse the role of the inlB mutations, invasion assays were performed in fibroblastic Vero cells, whose invasion is InlB-dependent (Dramsi et al., 1995). This was confirmed by the similar invasion rate of the EGDeinlA strain to that of the EGDe strain (Fig. 2c). The Group III strains invaded Vero cells 100-fold less than the EGDe strain and at the same rate as EGDeinlB and EGDeinlAinlB strains. The Group III strains trans-complemented with the pP1 plasmid carrying the inlBEGDe gene showed a statistically significant (P<0.001) 100-fold higher invasion rate in fibroblastic cells than their parental strains, or parental strains complemented with the empty plasmid (Fig. 2c). Their invasion rates were similar to those of the EGDeinlB+inlBEGDe strain and three logs superior to those of the EGDeinlB or EGDeinlB+pP1 strains. Western blots showed that trans-complemented strains had a large quantity of InlB (Fig. 1c).
Complementation with the plcA gene by allelic exchange on the chromosome
Since double or triple complementations were technically impossible due to plasmid incompatibility and defective growth, the strains were complemented with the plcAEGDe gene on the chromosome by allelic exchange, a method shown to be useful for restoring the virulence of L. monocytogenes strains (Olier et al., 2005). The plcAEGDe gene and its intergenic region were inserted into strain 416 with the pMAD vector (Arnaud et al., 2004). The integrants obtained by double crossover were checked for PI-PLC activity on PI-TY agar and confirmed using a radioactivity-based assay. Homologous recombination on the chromosome allowed different alleles of the plcA gene to be obtained. The resulting alleles represented a combination of the three different amino acids (at positions 17, 119 and 262) of the plcAEGDe and plcA416 genes. Sequencing showed that strain 59, which possesses the three amino acids of the EGDe strain in positions 17, 119 and 262 (profile 17EGDe-119EGDe-262EGDe), exhibited PI-PLC activity (Table 2). This activity was also restored in strain 114 (profile 17416-119416-262EGDe) and in strain 2 (profile 17416-119EGDe-262EGDe). In contrast, strains 112 (profile 17EGDe-119EGDe-262416), 6 (profile 17416-119EGDe-262416) and 57 (profile 17EGDe-119416-262416) did not exhibit enzymic activity. These results suggest that only the Thr262Ala mutation was responsible for the lack of PI-PLC activity in the Group III strains.
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). No plaques were found for the parental 416 strain or the strain complemented with the inlAEGDe gene (Table 3). Interestingly, when strain 114, which exhibited PI-PLC activity, was complemented with the inlAEGDe gene, its plaque-forming capacity was restored, unlike the parental strain or the strain transformed with the empty plasmid. The 114+inlAEGDe strain formed smaller but more numerous plaques than the EGDe strain (Table 3). This result is specific to the T262A substitution, as the 112+inlAEGDe strain was unable to form plaques in Caco-2 cell monolayers.
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), whereas the invasion rates of the parental 112 and 114 strains were similar to those of strains 416 and EGDeinlB. Only strain 114+inlBEGDe was able to form a similar number of plaques to the EGDe strain, but the elongated shape of the plaques made it impossible to measure their diameter (Table 3). As observed with plcAEGDe-inlAEGDe complementation, double complementation with plcAEGDe and inlBEGDe genes was necessary to restore plaque formation.
In vivo assay
Since plaque-forming capacity was restored for strains complemented with either plcA/inlA or plcA/inlB genes, the virulence level of these strains was investigated in a mouse model. As murine E-cadherin is not recognized by InlA (Lecuit et al., 1999), only the strains complemented with both inlB and plcA genes were tested. None of the complemented strains showed spleen colonization after s.c. inoculation, indicating that the double complementation did not restore virulence by this route of infection. After i.v. inoculation, the 114+inlBEGDe strain showed a statistically significant (P<0.005) sixfold higher spleen colonization [median log(c.f.u. per organ)=3.72] than the 112+inlBEGDe (2.92) and 416+inlBEGDe strains (2.76). This result suggests that the virulence was restored to a lesser extent than that of the EGDe strain (5.47). The same level of colonization observed in strains 416 (3.91) and 114+inlBEGDe could be explained by the negative effect induced by the plasmid and already observed in the invasion assays (Figs 2b, c and 3a, b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For five out of six strains in group III, the same mutations in three genes, plcA, inlA and inlB, were responsible for their inability to form plaques; this ability was restored by complementation with the corresponding genes of the virulent EGDe strain. However, although complementation with plcAEGDe and inlBEGDe partly restored the virulence of these strains in mice after i.v. inoculation, it was not sufficient to restore the in vivo virulence after s.c. inoculation. As InlA is unable to interact with murine E-cadherin, the absence of in vivo virulence of the Group III strains could not be due to the InlA mutation. These results suggest that other genes are altered in Group III strains, for example the agrA and inlJ genes, which are only expressed in vivo, or genes involved in adaptation to mouse immune defences (Autret et al., 2003; Sabet et al., 2005).
Restoration of PI-PLC activity by allelic exchange highlighted that the Thr262Ala mutation was likely responsible for the absence of PI-PLC activity of the Group III strains, whereas the two other mutations were silent mutations. Threonine at position 262 is conserved in the different L. monocytogenes strains sequenced (strains EGDe, 10403S and F6845 of serovar 1/2a, and strains CLIP80459, F2365 and H7858 of serovar 4b), despite large genotypic differences between the different serovars (Doumith et al., 2004). These results demonstrate that Thr262 plays a key role in PI-PLC activity. The crystallographic structure of PI-PLC shows that myo-inositol, the polar head-group of the PI substrate, interacts with side-chains of amino acids, including Asp204, which is spatially close to Thr262 (Moser et al., 1997). Thr262, which is located on the outer rim of the active site pocket, could interact with the glycerolipid part of PI. The Thr262Ala substitution could alter the cleavage activity of the enzyme without altering binding to myo-inositol (see Supplementary Fig. S3).
The observation of actin tails for the Group III strains indicates that bacteria could escape from the single-membrane vacuole, and that this was not prevented by the lack of PI-PLC activity. Our results suggest that PI-PLC plays a role in the lysis of the double-membrane vacuole, as inlAEGDe-complemented Group III strains did not form plaques, whereas inlAEGDe-plcAEGDe-complemented strains did. As the former invaded cells, multiplied intracellularly and formed actin tails, plcAEGDe complementation should restore the lysis of the double-membrane vacuole. This is in line with a recent report showing that PI-PLC acts synergistically with PC-PLC to dissolve the inner membrane of the double-membrane vacuole (Alberti-Segui et al., 2007).
Inactivation of InlB appears to be related to the Ala117Thr substitution which changed the hydrophobic nature of the amino acid, unlike the Val132Ile substitution. This hypothesis could be confirmed by site-directed mutagenesis. The Ala117Thr substitution is located in the LRR domain, which is both necessary and sufficient for invasion by interaction with Met, the InlB receptor (Shen et al., 2000).
Our results also demonstrate that the absence of InlA synthesis, caused by the insertion of a nonsense codon, was responsible for the low invasion rate of Group III strains into epithelial cells. Insertion of a nonsense codon leading to a truncated form of the InlA protein seems common, and previous studies have described 11 other different nonsense mutations (Jonquières et al., 1998; Nightingale et al., 2005; Felicio et al., 2007; Handa-Miya et al., 2007). Our PFA test on HT-29 cells combined with inoculation of mice allowed 8–20 % of low-virulence strains to be detected. However, strains exhibiting only an inactive InlA were not detected by our test, although 35 % of L. monocytogenes strains could have a truncated form of InlA and could thus be considered to be low-virulence strains (Olier et al., 2003; Jacquet et al., 2004). Moreover, numerous reports have demonstrated that non-haemolytic strains are avirulent, although the number of these strains is unknown, since many of them are not identified as L. monocytogenes strains when detection is based only on their haemolytic activity (Gaillard et al., 1986; Kathariou et al., 1987; Cossart et al., 1989). Overall, these results suggest that the number of low-virulence strains has been underestimated in the past and that they might easily account for 50 % of L. monocytogenes field strains. This hypothesis changes our perception of the risk of consuming contaminated food, as only a few strains, mainly belonging to serotype 4b, may exhibit a high virulence level and therefore be responsible for clinical cases, whereas numerous strains have one or more altered virulence gene and should thus be considered low-virulence strains. These results suggest that the low virulence of L. monocytogenes field strains is mainly due to point mutations in several virulence genes which cannot be detected by methods such as transcriptomic analyses, genomotyping or PFGE.
Our results also show that numerous strains have the same mutations. We have previously demonstrated that 42 % of low-virulence strains are mutated in PrfA (30 % had a PrfA Lys220Thr substitution and 12 % PrfA174-237) (Roche et al., 2005; Velge et al., 2007). In the present study, 20 % exhibited the same mutations in at least three genes (plcA, inlA and inlB). As these five strains were unrelated in origin and isolation date, we can hypothesize that this loss of virulence represents a selective advantage, probably due to better adaptation of the bacteria to the environment. However, up to now, no increase in fitness has been detected for these low-virulence strains; they are even more susceptible than virulent strains to the selective medium used to identify L. monocytogenes strains (Gracieux et al., 2003). Moreover, these results suggest that low-virulence strains have diverged from virulent strains, but each low-virulence group has followed a common evolutionary pathway. This hypothesis is currently being investigated, and understanding how they have evolved into several phylogenetic divisions may offer new opportunities for the detection, treatment and control of this widespread pathogen.
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ACKNOWLEDGEMENTS |
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Edited by: T. Msadek
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Received 29 June 2007;
revised 14 December 2007;
accepted 17 December 2007.
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