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Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
Correspondence
Fang Ting Liang
fliang{at}vetmed.lsu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary material describing the determination of ID50 values, and two supplementary tables showing the influence of increasing OspA and VlsE expression on dissemination and ID50 of B. burgdorferi in SCID mice, and that constitutive ospA expression severely impairs dissemination and significantly increases ID50 in immunocompetent mice, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ohnishi et al., 2001; Schwan et al., 1995; Schwan & Piesman, 2000), consistent with a critical role for these lipoproteins in spirochaetal persistence in the vector (Neelakanta et al., 2007; Yang et al., 2004). A fresh blood meal downregulates OspA/B and upregulates OspC and others, a process that prepares B. burgdorferi for infection of mammals (Fingerle et al., 2007; Grimm et al., 2004; Pal et al., 2004; Stewart et al., 2006). The downregulation of OspA and OspB during mammalian infection is critical for the maintenance of the enzootic cycle because their expression would ultimately induce strong humoral responses to effectively block acquisition by the vector (de Silva et al., 1997; Tsao et al., 2001, 2004), regardless of whether OspA or OspB can be effectively targeted by borreliacidal antibodies in mammalian tissues (Strother et al., 2007). B. burgdorferi abundantly expresses ospC only during early infection when the antigen may be crucial (Grimm et al., 2004; Stewart et al., 2006; Tilly et al., 2006). However, its expression induces a robust humoral response that imposes tremendous pressure on the pathogen (Fung et al., 1994; Xu et al., 2006). To cause persistent infection, B. burgdorferi must downregulate ospC as the specific humoral immune response is developing (Liang et al., 2002a, b, 2004b). It is also crucial for B. burgdorferi to downregulate ospC after it is acquired by the tick, as OspC antibodies in the blood meal may kill spirochaetes that express the antigen in the vector (Gilmore & Piesman, 2000), leading to discontinuation of the enzootic cycle.
B. burgdorferi also constantly modifies its surface antigenic expression in response to tissue microenvironmental changes, including the development of humoral responses, during the course of mammalian infection (Bykowski et al., 2007; Crother et al., 2004; Gilmore et al., 2007; Liang et al., 2002a, b, 2004b). The pathogen does not actively express VlsE, the surface variable antigen identified in B. burgdorferi, during early infection (Liang et al., 2004b). After dissemination into the joint, the pathogen upregulates vlsE, while maintaining low expression levels in the skin and heart tissues in the absence of humoral immune responses (Liang et al., 2004b). As the specific humoral response develops, B. burgdorferi downregulates OspC and many other surface antigens but dramatically upregulates VlsE and BBF01 (Crother et al., 2004; Liang et al., 2002a, b, 2004b), a process that probably allows the pathogen to more effectively evade the immune system and cause persistent infection.
As an extracellular bacterium, B. burgdorferi abundantly produces outer surface lipoproteins, which are directly involved in the interplay between the pathogen and tissue microenvironments and are likely to influence its infectious behaviour. To investigate the issue, B. burgdorferi was modified to overexpress either ospA or an invariant vlsE gene, and then examined for the ID50, dissemination, tissue colonization and persistence in the murine model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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ospA were generated previously (Xu et al., 2007a, b, 2008). The constructs pBBE22-ospA' and pBBE22-vlsE' were generated in a previous study (Xu et al., 2008). The features of these clones and constructs are summarized in Table 1.
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ospA were transformed with pBBE22-ospA' or pBBE22-vlsE', as described previously (Xu et al., 2007a). Transformants were identified and plasmid content was analysed as described previously (Xu et al., 2005). Selected transformants were analysed for OspA, OspC and VlsE expression by immunoblotting probed with a mixture of FlaB, OspA and OspC mAbs, or mouse antisera raised against a recombinant VlsE, as described previously (Xu et al., 2008).
In vitro growth kinetics and serum resistance.
Spirochaetes were grown to late-exponential phase (
108 cells ml–1), diluted in Barbour–Stoenner–Kelly H (BSK-H) complete medium (Sigma) or BSK-H medium supplemented with mouse sera at the ratio of 1 : 1 (v/v) to a density of 106 cells ml–1 and cultured at 33 °C. Mouse sera were prepared from blood collected from 10 severe combined immunodeficient (SCID) mice. The bacteria grown in BSK-H medium were counted every 24 h until reaching late-exponential phase. The organisms grown in the mixture of BSK-H and sera were assessed for viability every 12 h for 3 days under a darkfield microscope.
Infectivity and pathogenicity in SCID mice.
BALB/c SCID mice (age 4 to 8 weeks; provided by the LSU Division of Laboratory Animal Medicine) were given one single intradermal/subcutaneous injection of 104 spirochaetes. Animals were examined for the development of arthritis at 2-day intervals, starting at 10 days; animals were sacrificed 1 month post-inoculation. Tibiotarsal joint, heart and skin (not from the inoculation site) specimens were used for spirochaete isolation, and DNA and RNA preparation. DNA was quantified for bacterial loads by quantitative PCR (qPCR), as previously described (Xu et al., 2005). RNA was quantified for the mRNA copy numbers of flaB, ospA, ospC and vlsE by reverse-transcription qPCR (RT-qPCR), as described previously (Liang et al., 2004a). The expression levels were presented as ospA, ospC or vlsE mRNA copy numbers per 10 000 flaB transcripts.
Infectivity in immunocompetent mice.
BALB/c mice each received one single intradermal/subcutaneous injection of 105 spirochaetes. Some animals were euthanized 1 month post-inoculation; heart, tibiotarsal joint and skin specimens were aseptically collected for spirochaete culture as previously described (Xu et al., 2005), and blood was collected for ELISAs. Other inoculated mice were euthanized 1, 2, 3 and 4 weeks post-inoculation; the inoculation site and remote skin, ear, heart and joint specimens were aseptically harvested for spirochaete isolation.
Measurement of anti-OspA and -VlsE humoral immune response.
Specific OspA and VlsE antibody end point titres were determined by ELISAs. Ninety-six-well plates (Fisher) were coated with 100 µl of 2.0 µg ml–1 recombinant OspA or VlsE per well. Sera were twofold serially diluted, starting at 1/200. Five samples drawn from naive BALB/c mice were used as a control. The ELISA was performed as previously described (Xu et al., 2006).
Chronic infectivity.
Subgroups of five BALB/c mice were given one single intradermal/subcutaneous injection of 105 spirochaetes. Mice were euthanized 4 months post-inoculation; heart, tibiotarsal joint and skin specimens were aseptically collected for spirochaete culture, as previously described (Xu et al., 2005).
Statistical analysis.
A one-way analysis of variance (ANOVA) was used, followed by a two-tailed Student's t test to calculate a P value for each pair of treatments. Fisher's exact test was used to analyse data on ear biopsy and frequency of tissue colonization. A P value 
0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), both constructs were derived from pBBE22 (Xu et al., 2008) and thus carried a copy of bbe22, and should restore the infectivity of clone 13A. Plasmid content analyses identified two clones that received each construct, namely 13A/ospA'/1, 13A/ospA'/2, 13A/vlsE'/1 and 13A/vlsE'/2. These clones had identical plasmid content: all lost cp9, lp21 and lp5, in addition to lp25 and lp56. Increased VlsE expression resulting from the introduction of pBBE22-vlsE' was easily confirmed by immunoblotting probed with mouse anti-VlsE sera (Fig. 1a). Unfortunately, due to overwhelming expression resulting from the native ospA copy, the potential contribution of pBBE22-ospA' to OspA production could not be demonstrated in clone 13A/ospA'/1 or clone 13A/ospA'/2 (Fig. 1a). The immunoblot also showed that introduction of the constructs did not influence OspC production.
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ospA. Twelve transformants were obtained; two clones, namely ospA/ospA'/1 and ospA/ospA'/2, were selectively analysed by immunoblotting. Both clones abundantly produced OspA antigen (Fig. 1b
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OspA or VlsE overproduction does not affect spirochaete growth or serum resistance in vitro
The influence of lipoprotein overproduction on spirochaete growth and serum resistance was investigated. The 13A/E22/C, 13A/ospA'/1 and 13A/vlsE'/1 spirochaetes completed a generation approximately every 7 h when grown in BSK-H, and all three genotypes survived equally well in BSK-H/sera (data not shown), indicating that overproduction of OspA or VlsE does not influence growth or serum resistance in vitro.
Constitutive ospA expression represses vlsE and ospC expression, and increased vlsE expression enhances ospA but represses ospC expression during infection of SCID mice
To confirm in vivo increased ospA and vlsE expression, subgroups of five SCID mice were challenged with 104 spirochaetes of the clones 13A/ospA'/1, 13A/ospA'/2, 13A/vlsE'/1 or 13A/vlsE'/2. As a control, an additional 10 mice were inoculated with the clones 13A/E22/C or 13A/E22/D. The two clones were previously generated by transforming the 13A spirochaetes with pBBE22 (Xu et al., 2007b). Joint swelling started to develop in all mice at approximately 10 days, and quickly developed to severe arthritis (data not shown), indicating that introduction of pBBE22-ospA' or pBBE22-vlsE' did not affect arthritis virulence.
Infected mice were euthanized 1 month post-inoculation; RNA was prepared from heart, joint and skin specimens and assessed for the relative copy numbers of ospA, ospC, vlsE and flaB mRNAs by RT-qPCR. The 13A/ospA' bacteria accumulated ospA mRNA 3189-, 1034- and 180-fold more than the genotype 13A/E22 in heart (P=6.8x10–12), joint (P=4.4x10–12) and skin tissues (P=5.1x10–15), respectively (Fig. 2a). However, they reduced ospC expression 49 and 22 % in heart (P=4.9x10–6) and skin (P=0.002), respectively, but increased expression 10.3-fold in joint tissue (P=2.9x10–9) (Fig. 2b). The genotype 13A/ospA' also reduced vlsE mRNA accumulation 4.9-, 5.8- and 2.1-fold in heart (P=5.7x10–3), joint (P=2.7x10–10) and skin tissues (P=0.03), respectively (Fig. 2c).
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). The pBBE22-vlsE' spirochaetes increased ospA expression 62-, 39- and 4.8-fold in heart (P=4.4x10–4), joint (P=6.0x10–5) and skin tissues (P=0.001), respectively (Fig. 2a), but reduced ospC expression 6.4-, 10.1- and 13.2-fold in heart (P=3.7x10–11), joint (P=1.6x10–5) and skin tissues (P=1.2x10–13), respectively (Fig. 2b).
Increasing expression of either ospA or vlsE shows a tissue-dependent influence on colonization in SCID mice
DNA was prepared from the heart, joint and skin specimens harvested from the 30 infected mice described above, and quantified for tissue spirochaete burden by qPCR as an indication of tissue colonization. As shown in Fig. 2(d), constitutive ospA expression led to a 2.2- and 1.3-fold decrease in spirochaete load in heart (P=0.03) and joint tissues (P=0.03), respectively, but a 1.7-fold increase in skin (P=1.2x10–5). Increasing vlsE expression resulted in a 3.6-, 1.6-, and 2.2-fold decrease in spirochaete load in heart (P=0.007), joint (P=0.009) and skin tissues (P=1.8x10–6), respectively. When the two genotypes with increased osp expression were compared, the 13A/ospA' spirochaetes generated bacterial loads 1.6- and 3.8-fold higher than the 13A/vlsE' bacteria in heart (P=0.005) and skin tissue (P=1.2x10–5), but registered similar loads to those of the latter in joint tissue (P=0.16).
Increasing expression of either ospA or vlsE affects dissemination but does not alter ID50 in SCID mice
The influence of increasing ospA or vlsE expression on the ID50 value and dissemination was first investigated in immunodeficient mice. Groups of three animals each received one single inoculation of 101–103 spirochaetes of the clones 13A/E22/C, 13A/E22/D, 13A/ospA'/1, 13A/ospA'/2, 13A/vlsE'/1 and 13A/vlsE'/2. Ear biopsies were taken for bacterial culture at 2 and 3 weeks post-inoculation. Animals were euthanized 1 month post-inoculation; heart, joint and skin samples were cultured for ID50 determination. No difference in dissemination was observed between the genotypes 13A/E22 and 13A/ospA' when the doses were 102 or 103 organisms, as all inoculated mice in these groups produced a positive ear biopsy at 2 weeks (Supplementary Table S1). However, all five mice that had received 10 organisms of the clones 13A/ospA'/1 and 13A/ospA'/2 and were found to be infected at the end of the experiments registered a positive biopsy at 2 weeks; in contrast, none of the four infected mice in the same dose group of the clones 13A/E22/C and 13A/E22/D produced a positive biopsy within the time frame, indicating that constitutive ospA expression facilitates dissemination (P=0.008).
When the dose was 103 organisms, the 13A/vlsE' spirochaetes disseminated to and colonized the ear tissue as quickly as genotype 13A/E22, since all inoculated mice in these groups produced a positive ear biopsy at 2 weeks (Supplementary Table S1). When the 102 dose groups were compared, the genotype 13A/vlsE' disseminated at a slower pace than the 13A/E22 spirochaetes, as all the 12 mice that had been inoculated with the clones 13A/E22/C or 13A/E22/D in the dose group produced an ear biopsy at 2 weeks, but only two of the 12 mice in the same dose group of the clones 13A/vlsE'/1 and 13A/vlsE'/2 gave a positive biopsy within this time frame, indicating that increasing vlsE expression impairs dissemination (P=6.7x10–5).
The ID50 values for the six clones were measured from six to 18 organisms in repeated experiments (Supplementary Table S1). These results allowed us to conclude that constitutive expression of either ospA or vlsE does not affect the ID50 value in immunodeficient mice (P>0.05).
Increasing the expression of vlsE but not ospA leads to clearance of spirochaetes during early infection of immunocompetent mice
Subgroups of five BALB/c mice each received one single intradermal/subcutaneous injection of 105 spirochaetes of the clones 13A/ospA'/1, 13A/ospA'/2, 13A/vlsE'/1, 13A/vlsE'/2, 13A/E22/C and 13A/E22/D. Animals were euthanized 1 month post-inoculation; heart, joint and skin samples were harvested for bacterial culture and blood samples collected for ELISAs. B. burgdorferi was grown from each specimen of the 10 mice that had received the clones 13A/E22/C and 13A/E22/D (Table 2). Similarly, the 13A/ospA'/1 and 13A/ospA'/2 spirochaetes were consistently recovered from all the skin samples as well as most of the heart and joint specimens of the 10 inoculated mice. However, no spirochaetes were recovered from the 10 mice that had been inoculated with the clones 13A/vlsE'/1 and 13A/vlsE'/2, suggesting that increasing vlsE expression abrogates the ability of B. burgdorferi to infect immunocompetent mice.
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, infection with the genotype 13A/ospA' elicited an anti-OspA response 817-fold stronger than the 13A/E22 spirochaetes (P=0.003). The anti-OspA titre resulting from inoculation with the 13A/vlsE' spirochaetes was also 7.7-fold higher than that measured in the mice infected with the genotype 13A/E22 (P=0.02). The 13A/E22 spirochaetes elicited an anti-VlsE response 4.5- and 7.8-fold higher than that of the genotypes 13A/ospA' (P=1.9x10–4) and 13A/vlsE' (P=2.2x10–5), respectively.
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). The 13A/ospA'/1 and 13A/ospA'/2 spirochaetes disseminated slowly, and all ear tissue cultures did not become positive until 4 weeks post-inoculation, although all joint samples were positive within the first week. The 13A/vlsE'/1 and 13A/vlsE'/2 bacteria were recovered from at least one tissue of each inoculated mouse within the first 3 weeks, but none of the tissues was positive at 4 weeks post-inoculation, indicating that B. burgdorferi with increased vlsE expression can initiate infection but is subsequently eliminated.
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The ID50 values of the clones 13A/E22/C and 13A/E22/D were 18 and 32 organisms, compared to 178 and 56 for the clones 13A/ospA'/1 and 13A/ospA'/2 in experiment I (Supplementary Table S2). The values were 18 and 32 for the clones 13A/E22/C and 13A/E22/D, and 178 and 178 for the clones 13A/ospA'/1 and 13A/ospA'/2, respectively, in experiment II. Taken together, the study indicated that increasing ospA expression results in a 24-fold increase in ID50 in immunocompetent mice (P=0.007).
Another defect resulting from constitutive ospA expression was a reduced frequency in colonizing heart and joint tissues. The 13A/ospA'/1 and 13A/ospA'/2 spirochaetes were grown from only 10 heart and 15 joint specimens of the 23 infected mice; in contrast, the clones 13A/E22/C and 13A/E22/D were recovered from each specimen from all of the 26 infected mice (Supplementary Table S2). Overall, constitutive ospA expression led to a 67 and 35 % decrease in frequency of colonizing heart (P=4.4x10–6) and joint tissues (P=1.1x10–3), respectively.
B. burgdorferi with constitutive ospA expression is able to persist in the skin tissue of chronically infected immunocompetent mice
Subgroups of five BALB/c mice each received one single intradermal/subcutaneous injection of 105 spirochaetes of the clones 13A/E22/C, 13A/E22/D, 13A/ospA'/1 and 13A/ospA'/2. Animals were euthanized 4 months post-inoculation. B. burgdorferi was grown from all skin and heart specimens, and nine joint samples of 10 mice that had been challenged with the clones 13A/E22/C and 13A/E22/D (Table 4). In contrast, the 13A/ospA'/1 and 13A/ospA'/2 spirochaetes were recovered from all skin specimens but from none of the heart or joint samples of 10 infected mice, indicating that constitutive ospA expression diminishes the ability of B. burgdorferi to persist in the heart or joint tissue during chronic infection of immunocompetent mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Both ospA and vlsE use 
70-dependent promoters, while the ospC promoter is RpoS-dependent (Hubner et al., 2001). Within mammals, however, ospA expression is essentially repressed, while vlsE expression is upregulated, especially during chronic infection of immunocompetent hosts (Liang et al., 2004b). Nothing is known about how this could occur. Nevertheless, our RT-qPCR results showed that constitutive ospA expression repressed vlsE and ospC expression, and that increased vlsE expression upregulated ospA but downregulated ospC in infected SCID mice. The same scenario also appeared to occur in infected immunocompetent mice, as B. burgdorferi with constitutive ospA expression elicited a weaker anti-VlsE response, but bacteria with increased vlsE expression stimulated a stronger anti-OspA response than the control. It would be interesting to address how an increase in lipoprotein gene expression can influence the expression of other lipoproteins.
To initiate an infection, B. burgdorferi must be able to evade innate immunity and colonize local tissues of the inoculation site; this ability was measured by ID50 values in immunodeficient mice. Apparently, increasing ospA or vlsE expression did not affect this ability, as increasing the expression of either did not change the value in SCID mice. However, increasing vlsE expression slightly impaired dissemination, and constitutive ospA expression apparently facilitated the process in the absence of adaptive immunity, underscoring the importance of surface antigen expression in spirochaetal dissemination. Our previous study showed that increasing expression of DbpA severely impairs dissemination (Xu et al., 2007b). Even more dramatically, overproducing DbpA restrains OspC-deficient B. burgdorferi to tissues at the inoculation site for 4 weeks without dissemination to distal tissues, further highlighting the influence of surface lipoprotein expression on dissemination (Xu et al., 2008). OspC may function as a dissemination-facilitating factor (Xu et al., 2008), and has been found to bind plasminogen (Lagal et al., 2006). Interestingly, OspA is a primary plasmin(ogen) receptor (Fuchs et al., 1994), and in vitro evidence shows that OspA helps B. burgdorferi to acquire plasmin(ogen) (Coleman et al., 1995; Hu et al., 1995; Klempner et al., 1996). Plasmin, a trypsin-like serine proteinase, is generated by limited proteolysis from its serum-derived zymogen precursor plasminogen by the urokinase-type plasminogen activator or tissue-type plasminogen activator. An in vitro study has shown that B. burgdorferi coated with plasmin gains a proteolytic activity to effectively degrade components of the mammalian extracellular matrix (Coleman et al., 1999), and an animal study has indicated that plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochaetaemia in mice (Coleman et al., 1997). It remains to be addressed whether facilitated dissemination due to constitutive ospA expression results from gaining host plasminogen.
As an extracellular pathogen, B. burgdorferi resides primarily in the extracellular matrix and connective tissues as well as between host cells during mammalian infection. The influence of surface antigen expression on this aspect was reflected by tissue bacterial load. Increasing expression of either ospA or vlsE negatively affected the ability of B. burgdorferi to colonize most tissues. This adverse effect of high vlsE expression underscores the biological significance of low expression during early infection. B. burgdorferi does not actively express vlsE but increases expression after dissemination into joint tissues in the absence of adaptive immune responses (Crother et al., 2003; Liang et al., 2004b). Low vlsE expression may also allow bacteria to more efficiently disseminate to distal tissues, as the current study showed that increasing vlsE expression led to slow dissemination in SCID mice. After dissemination, humoral responses greatly upregulate vlsE in all tissues (Crother et al., 2004; Liang et al., 2004b), an event consistent with the critical role of VlsE in immune evasion during chronic infection of immunocompetent hosts (Bankhead & Chaconas, 2007; Zhang et al., 1997).
Naive and immune statuses constitute two distinct environments to microbial pathogens. The current study used immunodeficient and immunocompetent mice to provide these environments in order to study the influence of increased surface lipoprotein expression on infectious behaviour. Increasing the expression of ospA or vlsE did not affect the ID50 value in SCID mice. In sharp contrast, increased vlsE expression diminished the ability to infect immunocompetent mice. Although a less significant effect attributed to increased ospA expression was observed in immunocompetent mice, B. burgdorferi with constitutive ospA expression registered a substantial ID50 increase and disseminated at a much slower pace. Even more dramatically, constitutive ospA expression diminished the ability to persist in both heart and joint tissues during chronic infection of immunocompetent mice.
An earlier study has shown that simultaneous constitutive expression of OspA and OspB abrogates infectivity in immunocompetent mice (Strother et al., 2007). In the current study, although constitutive ospA expression significantly increased the ID50 value and severely impaired dissemination, recombinant spirochaetes were able to persist in the skin tissue of chronically infected immunocompetent mice. Co-expressing OspA and OspB gives the immune system two targets, so that recombinant spirochaetes may be quickly cleared by the humoral responses after inoculation into immunocompetent mice. Alternatively, OspB is more effectively targeted than OspA by protective immunity, so that the anti-OspB response alone is sufficient to clear infection.
Increasing the expression of a lipoprotein appears to be an efficient way to test whether the specific antigen is effectively targeted by protective immunity. To date, four surface lipoproteins, OspC, DbpA, OspA and VlsE, have been individually examined in this way (Xu et al., 2006, 2007b). B. burgdorferi modified with increased expression of dbpA or ospA can persist in the skin tissue of chronically infected immunocompetent mice (Xu et al., 2007b). Although constitutive ospC expression diminishes the ability to cause persistent infection, recombinant spirochaetes are consistently recovered from immunocompetent mice for at least a month after infection (Xu et al., 2006). In contrast, B. burgdorferi with constitutive invariant vlsE expression is cleared within a month after inoculation into immunocompetent mice, although the production of invariant VlsE antigen under the control of its own promoter allows spirochaetes to cause persistent infection (Lawrenz et al., 2004). Taken together, these studies indicate that VlsE, when no antigenic variation occurs, is the most effective target of protective immunity.
B. burgdorferi actively modifies its surface antigen expression in order to better adapt to the tissue microenvironment during the course of mammalian infection. The current study shows that artificially increasing surface antigen expression alters infectious behaviour in a negative way, highlighting the importance of the coordination of surface antigen expression in the pathogenesis of B. burgdorferi.
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ACKNOWLEDGEMENTS |
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Edited by: G. E. Duhamel
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Received 17 April 2008;
revised 10 July 2008;
accepted 2 September 2008.
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