Plasmid Profile Analysis of Portuguese Borrelia Lusitaniae Strains
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Ticks and Tick-borne Diseases 1 (2010) 125–128
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Original article
Plasmid profile analysis of Portuguese Borrelia lusitaniae strains
Liliana Vitorino a , Gabriele Margos b , Líbia Zé-Zé c , Klaus Kurtenbach b,1 , Margarida Collares-Pereira d,∗
Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Lisboa, Portugal Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom c Centro de Estudos de Vectores e Doencas Infecciosas Dr. Franscisco Cambournac, Instituto Nacional de Saúde Dr. Ricardo Jorge, Águas de Moura, Portugal ¸ d Unidade de Leptospirose e Borreliose de Lyme, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, and Centro de Recursos Microbiológicos, FCT/UNL, Rua da Junqueira 96, 1349-008 Lisboa, Portugal b a
Article history: Received 30 March 2010 Received in revised form 6 July 2010 Accepted 14 July 2010 Keywords: Lyme borreliosis Borrelia lusitaniae Genome organization ospA Ixodes ricinus
Introduction Borrelia burgdorferi sensu lato (s.l.) are the causative agents of Lyme borreliosis (LB) which is the most commonly reported arthropod-borne zoonosis in North America, Europe, and the temperate zone of Asia (Steere, 2001; Masuzawa, 2004). These spirochaetes are maintained in nature in enzootic cycles involving ticks of the Ixodidae family (Kurtenbach et al., 2006; Lindgren and Jaenson, 2006). The B. burgdorferi s.l. species complex comprises 17 species (Kurtenbach et al., 2010), of which B. burgdorferi sensu stricto (s.s.), B. garinii, and B. afzelii are recognised as human pathogens (Stanek and Strle, 2009). Over the last few years, however, other Borrelia species have also been associated with human disease, e.g. B. lusitaniae. This species was isolated from 2 Portuguese patients presenting with chronic skin lesions (Collares-Pereira et al., 2004) and a vaculitis syndrome (Carvalho et al., 2008a), respectively, and its pathogenic potential was successfully established in a mouse model (Zeidner et al., 2001). B. lusitaniae is the sole species found in southern Portugal and North Africa (De Michelis et al., 2000; Younsi et al., 2005), and it has been described as being highly diverse based on phylogenies of the 5S–23S intergenic spacer region (IGS) (De Michelis et al., 2000)
and in multilocus sequence typing (MLST) analysis (Vitorino et al., 2008). The most remarkable feature of the Borrelia genome is that it consists of a linear chromosome and several circular and linear plasmids, which may constitute up to 40% of its genomic DNA (Fraser et al., 1997; Glöckner et al., 2004). Borrelia plasmids have been designated according to their shape and size: The plasmid cp32, for instance, is circular and has a molecular weight of 32 kb, whereas lp25 is a linear plasmid of 25 kb. The plasmid repertoire of different B. burgdorferi s.s. isolates can vary considerably, but some extrachromosomal elements appear to be essential, namely the circular plasmid cp26 and the linear plasmid lp54, which are present in most isolates (Casjens, 2000; Glöckner et al., 2004). The reason for this may be that they encode molecules that are critical for Borrelia survival in vitro (Byram et al., 2004; Chaconas, 2005; Stewart et al., 2005). Nevertheless, there is considerable plasmid variation in number and size among Borrelia species and strains (Xu and Johnson, 1995). This and the fact that plasmids can be easily lost during in vitro cultivation (Norris et al., 1995) potentially constitute a drawback for using plasmid fingerprinting for species characterization in Borrelia. Furthermore, the presence of multiple plasmids of similar sizes (e.g. lp28 and cp32) may complicate the accurate determination of the plasmid content (Stevenson et al., 2000). In addition, only linear plasmids are well separated by pulsed-field gel electrophoresis (PFGE) (Xu and Johnson, 1995), and their size and low-copy number can be below PFGE sensitivity, hence the number of plasmids detected by this technique is usually an underestimation. In spite
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of these limitations, plasmid fingerprinting is a useful method for a first approach to compare plasmid profiles between and within Borrelia species. Extensive plasmid content analyses have been reported for several North American B. burgdorferi s.s. strains (Casjens et al., 2000; Palmer et al., 2000) and some European B. garinii and B. afzelii isolates (Glöckner et al., 2004, 2006). The present study was undertaken to elucidate the plasmid profile for 2 Portuguese B. lusitaniae strains, one isolated from a human patient and the other one from a questing tick. Materials and methods Strain cultivation B. lusitaniae strains PoTiBL37 and PoHL1 were grown in liquid Barbour–Stoenner–Kelly-H (BSK-H) medium (Sigma® ) supplemented with gelatin pH 7.6, at 32 ◦ C. Cultures were examined weekly for motile spirochaetes by dark-field microscopy, until the density reached about 1 × 106 bacteria per ml (2–3 weeks). Low passages (≤20) have been used for both strains. For comparative purposes, the Portuguese B. garinii strains (PoTiBGr4, PoTiBGr6, and PoTiBGr20) were also used in this study. DNA extraction DNA from some passages of B. lusitaniae and B. garinii strains was firstly extracted in agarose plots according to Chu et al. (1986). Since this approach displayed low sensitivity to detect Borrelia plasmids, the following methodology was used. Around 100 ml of high density culture was spun down at 8000 rpm for 20 min, resuspended in TES (50 mM Tris–HCl, pH 8.0; 50 mM EDTA, pH 8.0; 15% sucrose) with 200 l of lysozyme (50 mg per ml), and incubated 15 min on ice. 5% sodium deoxycholate and 70 l of DEPC (diethyl pyrocarbonate) were added, and the mixture was left at room temperature for 10 min. After the addition of ammonium acetate (7.5%), the cells were incubated on ice for 15 min and centrifuged for 10 min at 10,000 rpm. The supernatant was recovered, and the nucleic acids were precipitated by adding 0.6 volumes of isopropanol and centrifugation for 6 min at the same speed. The pellet was resuspended in TE buffer and treated with RNAse (10 mg per ml) for 1 h at 37 ◦ C. To remove RNAse, extraction with chloroform was performed twice. The DNA was precipitated by adding 1/10 volume of 2.5 M NaCl and 2.5 volumes of absolute ethanol, left at 20 ◦ C for 1 h, and afterwards centrifuged at 10,000 rpm for 6 min. Finally, DNA was washed in 70% ethanol, dried at 65 ◦ C for 10 min, and dissolved in 150 l of TE buffer. PFGE Counter-clamped homogeneous electric field electrophoresis (CHEF-PFGE) was performed using the Gene Navigator System (Pharmacia). Agarose gels (1%) (Seakem GTG, FMC) in 0.5× TBE Low EDTA (45 mM Tris, 45 mM Boric Acid, 1 mM EDTA, pH 8.0) were used and 10 l of DNA was applied to each well. The electrophoresis was performed at a constant voltage (220 V) at 8 ◦ C using the following parameters: 0.3 s, 1 h; 0.5 s, 1 h; 0.7 s, 1 h; 2.0 s, 5 h; 4.0 s, 5 h. /HindIII ladder marker (125–23,130 bp; New England Biolabs) and Low Range PFG marker (2.03–194 kb; New England Biolabs) were used as molecular size markers (Zé-Zé et al., 1998). Different pulse-field parameters (0.5 s, 4 h; 1.0 s, 4 h; 2.0 s, 4 h; 4.0 s, 4 h; 8.0 s, 5 h; 10.0 s, 3 h) were also used to confirm the obtained plasmid profiles. The gels were stained with ethidium bromide and photographed under UV light (KODAK DC 290). The relative sizes of the identified plasmid fragments were
estimated using the KODAK 1D 2.0 (Kodak) software (Zé-Zé et al., 2008). Plasmid-specific PCR PCR primers to amplify the vls locus (Zhang et al., 1997) were used on B. lusitaniae strains. Two microliters ( l) of boiled culture were used as DNA template in a 50- l PCR mixture containing 1 pmol of each primer, 5 mM (each) dATP, dGTP, dCTP, and dTTP (Invitrogen), 1.75 U of Taq polymerase (Invitrogen), 3 mM MgCl2 , 0.5× BSA, and 1× Taq buffer. The PCR parameters used were as follows: an initial denaturation step at 94 ◦ C for 5 min, 35 cycles at 94 ◦ C for 1 min, 60 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by a final extension step at 72 ◦ C for 5 min. Southern hybridization To detect the linear plasmid that contains the gene encoding the OspA in B. lusitaniae PoTiBL37 and PoHuL1 strains, specific primers, 5 -GAC ACT GCC TCT GGT GAT AGC-3 (forward), 5 -CTT TCC CTT TTC CTT CTT TTG-3 (reverse), were designed against conserved regions of ospA from B. lusitaniae sequences available from GenBank. The amplification was carried out using Digoxigenin (DIG)-high prime DNA labelling kit (Roche) to probe the PCR product. The membrane was prehybridized in hybridization solution [5× standard sodium citrate (SSC), 0.1% sarcosil, 0.02% sodium dodecyl sulphate (SDS), 1× blocking solution, 50% formamide] for 1 h at 37 ◦ C. The denatured DIG-labelled DNA probe (10 min at 100 ◦ C) was added to the hybridization solution, and the membrane was incubated overnight at the same temperature. The membrane was washed twice in 2× SSC, 0.1% SDS at room temperature, twice in 0.5× SSC, 0.1% SDS at 65 ◦ C, rinsed in washing buffer (0.1 M maleic acid; 0.15 M NaCl, pH 7.5; 0.3% Tween 20), incubated for 30 min in blocking solution (provided by the manufacturer), and then in antibody solution (Anti-Digoxigenin-AP 1:10,000 in blocking solution) for 30 min. The membrane was washed twice in washing solution and equilibrated in detection buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5). The membrane was covered with chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD), incubated for 5 min at room temperature and then at 37 ◦ C for 10 min to enhance the luminescent reaction. Exposure to X-ray film lasted no longer that 45 min. Results The plasmid profiles of 2 B. lusitaniae strains, PoTiBL37 and PoHL1, isolated from a vector tick collected in Portugal and from a skin biopsy of a Portuguese patient, respectively, were determined by PFGE (Fig. 1). Several low passages of both strains were used to detect potential plasmid loss due to subsequent subculturing. Few plasmids were detected compared to the number of plasmids known for the B31 strain (12 linear and 9 circular plasmids) as follows: only 5 plasmids in PoTiBL37 (19 kb, 26 kb, 64 kb, 71 kb, and a higher one above the resolution limit of the ladder used) and 6 in PoHL1 (19 kb, 25 kb, 64 kb, 70 kb, 76 kb, and a higher one above the resolution limit of the ladder used). Plasmid profiles in PoTiBL37 and PoHL1 were almost identical, except for the largest plasmids that differ in number and size. The mean size of each fragment/plasmid was estimated from several gels by linear interpolation with 2 flanking size standards (Heath et al., 1992) using Kodak 1D 2.0 software. Profiles obtained with different running parameters displayed the same plasmid number and sizes which indicated that we detected linear plasmids (Beverly, 1988; Smith and Condemine, 1990) and that the additional plasmid found in PoHL1 was not an artifact (data not shown).
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Fig. 1. Plasmid profiles of B. burgdorferi sensu lato determined by PFGE. 1, 13: /HindIII ladder; 2, 12: low range ladder; 3–5: B. lusitaniae PoHL1, passages P6, P20, and P9, respectively; 6–8: B. lusitaniae PoTiBL37, passages P8, P16, and P17, respectively; 9: B. garinii PoTiBGr20; 10: B. garinii PoTiBGr4; 11: B. garinii PoTiBGr6. B. garinii isolates were used for comparison.
Although a plasmid of 28 kb was not detected in the plasmid profiles, primers designed to amplify the vls locus (encoded on lp28-1 and associated with infectivity of B. burgdorferi s.s.) produced a weak PCR product in PoHL1 (data not shown). In addition, we investigated the location of the ospA gene by southern hybridization. The size of the plasmid that hybridized with the ospA probe was 70 kb (Fig. 2). Discussion The number of plasmids reported here for the 2 Portuguese B. lusitaniae strains is probably lower than the total number of plasmids, mainly because (i) some of them must be in such low-copy number that they could not be detected by PFGE, (ii) the circular plasmids are difficult to detect by this approach, and (iii) we cannot rule out that some plasmids could have been lost during
Fig. 2. OspA hybridization within plasmid profiles. 1, 13: /HindIII ladder; 2, 12: low range ladder; 3–5: B. lusitaniae PoHL1, passages P6, P20, and P9, respectively; 6–8: B. lusitaniae PoTiBL37, passages P8, P16, and P17, respectively; 9: B. garinii PoTiBGr20; 10: B. garinii PoTiBGr4; 11: B. garinii PoTiBGr6. OspA probe is specific for B. lusitaniae, thus no signal was detected in B. garinii isolates.
the DNA extraction, despite using a method that enhances plasmid DNA isolation. In other Borrelia species, very small plasmids in the size range below 10 kb have been described. But even when using another DNA extraction technique (Chu et al., 1986) and completely different PFGE runs including very short pulse times to detect smaller molecules (Smith and Cantor, 1987), the number and size of the detected plasmids remained the same. The plasmid profiles of B. lusitaniae strains ranged from 19 kb to 76 kb, with a higher one above the resolution limit of the ladder used. Thus, it is not possible to accurately estimate its size (Fig. 1). The size range of the plasmids detected in B. lusitaniae greatly differed from the ones detected by PGFE in B. burgdorferi s.s., B. afzelii, and B. garinii, where the largest plasmid was 57.7 kb (Xu and Johnson, 1995). This result is in agreement to both the largest plasmid size of lp60 (GenBank accession no. NC 008277) reported in B. afzelii and other plasmids between 20 kb and 40 kb also present in the genome. In the case of B. lusitaniae, not only the number of plasmids is quite low, but it seems that this species lacks all the small plasmids described so far for the other Borrelia with medical importance in LB. In view of the fact that most of the Borrelia virulence genes are located on plasmids (e.g., genes that encode for OspC, Erps, and CRASP proteins), the low number of these genetic elements in B. lusitaniae strains could be associated with the lower infectivity reported for this species, since only 2 human isolates have been obtained so far (Collares-Pereira et al., 2004; Carvalho et al., 2008a). Furthermore, the number (0.04/100,000 inhabitants) of reported cases in Portugal (Carvalho et al., 2008b) is not as high as in other European countries (Stanek and Strle, 2009) despite a high reported infection prevalence of B. lusitaniae in ticks (De Michelis et al., 2000). Another approach to study the plasmid content is to use plasmid-specific primers and to perform PCR (Purser and Norris, 2000; Iyer et al., 2003). The lp28-1-like plasmid was not detected in both isolates. The vlsE locus and the contiguous array of vls silent cassettes are located adjacent to one another in the linear plasmid lp28-1 (Zhang et al., 1997). Given that the lack of this plasmid has been correlated with the low or intermediate infectivity of B. burgdorferi s.s. clones (Purser and Norris, 2000) and the ability to colonize tissues other than the joints (Labandeira-Rey and Skare, 2001), B. lusitaniae Portuguese isolates could have a low or an intermediate infectivity phenotype due to the absence of the vls locus. However, primers designed to amplify this gene, based on B. burgdorferi s.s. genome sequencing data, yielded a weak PCR amplicon in the PoHL1 strain, indicating that the vls locus is present in that isolate, but being located in a different genomic region than the lp28-1. There are also other plasmids that have been described as essential for Borrelia survival, such as the lp54 and the cp26, encoding the OspA and OspC proteins, respectively (Barbour, 1988; Marconi et al., 1993; Sˇ dziene et al., 1993; Stewart et al., 2005). Both genes a were detected by PCR in B. lusitaniae strains, using primers designed specifically for this genospecies. The absence of a plasmid in the size range of lp54 prompted us to investigate which plasmid would encode ospA. Surprisingly, the probe hybridized with a plasmid of 70 kb which differs considerably from the plasmid known to encode this gene in other Borrelia species (lp54). Taken together, these results underpin that B. lusitaniae is different from other Borrelia species that have been associated with pathogenicity in plasmid number and content (Palmer et al., 2000). As already reported by some authors (De Michelis et al., 2000; Younsi et al., 2005; Vitorino et al., 2008), B. lusitaniae strains display a high intraspecific genetic diversity. Hence, it will be of great interest to evaluate the plasmid profile of other Portuguese B. lusitaniae strains. This species is phylogenetically very distant from
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B. burgdorferi s.s., and its distinct plasmid content is consistent with this notion. The divergent plasmid content of B. lusitaniae may reflect its adaptation to particular hosts, such as lizards, which are now believed to be important reservoir hosts of B. lusitaniae (Dsouli et al., 2006; Richter and Matuschka, 2006). Acknowledgments The ICAT/FCUL research team involved in this work strongly acknowledges its scientific leader, Prof. Rogério Tenreiro, for the general scientific supervision and engagement in providing the needed resources and facilities. We are also very grateful to Dr. Susana Baptista for providing the tick isolate (PoTiBL37) used in this study. L. Vitorino is the recipient of FCT research grants SFRH/BD/10676/2002. References
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