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Infection and Immunity, March 2008, p. 1239-1246, Vol. 76, No. 3
0019-9567/08/$08.00+0     doi:10.1128/IAI.00897-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Both Decorin-Binding Proteins A and B Are Critical for the Overall Virulence of Borrelia burgdorferi{triangledown}

Yanlin Shi, Qilong Xu, Kristy McShan, and Fang Ting Liang*

Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

Received 2 July 2007/ Returned for modification 27 September 2007/ Accepted 31 December 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 104-fold increase in the 50% infectious dose (ID50). Complementation of the mutant with either dbpA or dbpB reduced the ID50 from over 104 to roughly 103 organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Borrelia burgdorferi, the Lyme disease spirochete that is transmitted by Ixodes ticks, causes the most common vector-borne illness in North America and Europe (5, 34). Lyme disease is a multisystem disorder that can result in arthritis, neurological abnormalities, carditis, and cutaneous lesions such as erythema migrans and acrodermatitis chronica atrophicans (34). B. burgdorferi is a slow-growing extracellular bacterium but has a very low 50% infectious dose (ID50) in the murine host (1); it can also cause persistent infection despite the development of strong immune responses (31), making it one of the most invasive microbial pathogens in humans and animals.

One feature of B. burgdorferi is expression of a broad array of surface adhesive molecules (8), including two decorin-binding proteins, DbpA and DbpB (3, 13-15), a fibronectin-binding protein, BBK32 (26, 27), a glycosaminoglycan-binding protein, Bgp (24, 25), and a mammalian cell surface receptor-binding protein, P66 (7, 9). The outer membrane protein Bgp binds glycosaminoglycans (24); the outer surface lipoproteins DbpA/DbpB and BBK32 bind decorin and fibronectin, respectively (3, 13, 26, 27), in addition to binding of glycosaminoglycans (11, 12). Another outer membrane protein, P66, binds cell surface receptor integrin {alpha}IIbβ3 (7, 9). In addition, there are unidentified borrelial adhesins that interact with another cell surface receptor, called integrin {alpha}3β1 (2). Both decorin and glycosaminoglycans are important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on host cell surfaces, while fibronectin exists mainly in the ECM. As an extracellular bacterium, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host components mediated by spirochetal surface adhesins may impact various aspects of infection and thus potentially affect overall infectivity and pathogenicity. Accumulating evidence supports this notion (6). Mice deficient for decorin, a ligand of DbpA and DbpB, become less susceptible to murine Lyme disease and harbor fewer spirochetes during chronic infection (4, 22). Increasing DbpA expression significantly reduces the ID50 value but severely impairs dissemination and abolishes arthritis virulence in a murine model (39).

None of the borrelial surface adhesins has been found to play an essential role in mammalian infection. Parveen et al. reported that Bgp is not required for infection of immunodeficient mice (23), although it remains to be addressed whether the adhesin significantly contributes to infectivity in immunodeficient hosts and whether it is essential for infection of immunocompetent mice. Seshu et al. showed that a BBK32 deficiency increases the ID50 value by approximately 10-fold in immunocompetent mice (32), virtually consistent with a subsequent study by Li et al. indicating that the BBK32 gene is not essential for the life cycle of B. burgdorferi either in the tick vector or the murine host (21). A recent study by Jewett et al. also suggests that BBK32 plays a minor role in murine infection (16). Our previous study showed that the dbpBA locus is not required for murine infection (33). Although deletion of the locus was associated with an apparent defect in colonization of certain tissues during infection of immunocompetent mice, due to lack of complementation, the study concluded only that the dbpBA locus is not essential for the infection of mice (33). In the current study, to thoroughly examine the contribution of these two adhesins to the overall virulence of B. burgdorferi, the dbpBA locus was first deleted, followed by complementation with the gene dbpA or dbpB or both. This allowed us to investigate the contribution of these genes, either in combination or individually, to the infectivity, in terms of ID50 value, and tissue colonization, as reflected by bacterial load, of B. burgdorferi in the murine host.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of pNCO1T::dbpAB::aacC1. A 2,047-bp fragment covering a partial sequence of the open reading frame (ORF) BBA20, the entire ORFs for BBA21, BBA22, and BBA23, and a small portion of dbpA (BBA24) was amplified with primers P1F and P1R (Fig. 1A; Table 1) by PCR. An 1,899-bp fragment, including the entire ORFs of BBA26, BBA27, BBA28, and BBA29 and a partial BBA31 sequence, was amplified using primers P2F and P2R. The two PCR products were pooled, purified using the QIAquick PCR purification kit (Qiagen Inc., Valencia, CA), digested with NheI, repurified, and ligated. The resultant product was used as a template and amplified by nested PCR using primers P3F and P3R. The PCR product was purified, digested with Acc65I, and cloned into the TA vector pNCO1T as described previously (33) to generate pNCO1T::dbpAB, which cannot replicate in the borrelial system. A gentamicin cassette (aacC1) which confers gentamicin resistance both in Escherichia coli and B. burgdorferi (10) was amplified from the vector pBSV2G (a gift from P. Rosa and P. Stewart) using primers P5F and P5R (Fig. 1B; Table 1). The amplicon was purified, digested with XbaI, and cloned into pNCO1T::dbpAB to complete construction of the disruption plasmid pNCO1T::dbpAB::aacC1. The insert within the plasmid was sequenced to ensure it was arranged as designed.


Figure 1
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  		FIG. 1. Generation of dbpAB mutant. (A) Diagram of the dbpBA locus and adjacent ORFs. The two-gene operon is located within lp54, which carries 76 ORFs, BBA01 to BBA76. The two open arrows represent dbpA and dbpB; the filled arrows denote BBA20 to BBA23 and BBA26 to BBA31. The binding sites of eight primers, i.e., P1F to P4F and P1R to P4R, are also indicated. (B) Diagram of the disrupted dbpBA locus, showing a major portion of dbpA and the entire dbpB gene replaced with a gentamicin resistance cassette (aacC1; gray arrow). The small arrow points the dbpA residual portion (dbpA'). The binding sites of primers P5F and P5R are also indicated. (C) PCR analysis of the dbpAB mutant. The 13A spirochetes, the disruption plasmid pNCO1T::dbpAB::aacC1, and {Delta}dbpAB were used as DNA sources and subjected to PCR amplification using primers P4F and P4R (top) or primers P5F and P5R (bottom). (D) Immunoblot analysis of the dbpAB mutant. The parental clone 13A and the {Delta}dbpAB mutant were analyzed via an immunoblot probed with a mixture of FlaB MAb and mouse antisera raised against recombinant DbpA (left) or DbpB (right).

 

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  		TABLE 1. Primers used in the study

 
Deletion of the dbpBA locus. B. burgdorferi was grown in Barbour-Stoenner-Kelly H (BSK-H) complete medium (Sigma Chemical Co., St. Louis, MO) at 33°C. To delete the dbpBA locus, 10 µg of pNCO1T::dbpAB::aacC1 DNA was electroporated into the 13A spirochetes harvested from a 40-ml late-logarithmic-phase culture; the resulting gentamicin-resistant clones were screened as described previously (33). The clone 13A was derived from a highly transformable clone, B. burgdorferi B31 5A13, which harbors 20 plasmids but lacks lp25 (29). Further loss of lp56 makes the clone 13A more transformable (36, 38). The deletion of the dbpBA locus due to an insertion of the aacC1 cassette was confirmed by PCR using primers P4F and P4R, unique for the dbpBA operon, and P5F and P5R, specific for the cassette (Fig. 1B; Table 1). The lack of DbpA and DbpB expression was verified using an immunoblot probed with a mixture of a FlaB monoclonal antibody (MAb) and anti-DbpA or -DbpB sera as described previously (33). The plasmid content of dbpAB mutants was analyzed as described previously (37).

trans-complementation of a dbpAB mutant. The recombinant plasmid pBBE22 (a gift from S. Norris) was first modified to make the lacZ site available for insertion of the dbpBA operon as diagramed in Fig. 2A because this site provides the easiest way for screening a construct with an insert. pBBE22 was digested with Acc65I, purified, and circularized to generate pBSV2 via ligation. A 1,368-bp fragment including the entire BBE22 gene and potential upstream regulatory sequence was PCR amplified with primers P6F and P6R and borrelial DNA as a template and cloned into at the AatII restriction site of pBSV2 to create pME22. To generate pME22-dbpBA, a 1,665-bp fragment covering the entire dbpBA operon was PCR amplified with primers P7F and P7R and borrelial DNA as a template and cloned into pME22 after the vector was digested with restriction enzymes Acc65I and BamHI. Two large amplicons were generated by use of the plasmid pME22-dbpBA as a template and the primer pair P8F and P8R or P9F and P9R, digested with NheI, purified, and circularized via ligation to produce two complementation plasmids, namely, pME22-dbpA and pME22-dbpB.


Figure 2
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  		FIG. 2. trans-complementation of the dbpAB mutant. (A) Construction of complementation plasmids. The construction of pME22, pME22-dbpBA, pME22-dbpA, and pME22-dbpB was described in Ma- terials and Methods. All restriction enzyme sites and primer binding sites used for plasmid construction are labeled. (B) Confirmation of restored DbpA/DbpB expression by immunoblotting. The parental clone 13A, the {Delta}dbpAB mutant, and clones {Delta}dbpAB/E22/1, {Delta}dbpAB/E22/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, {Delta}dbpAB/dbpB/2, {Delta}dbpAB/dbpAB/1, and {Delta}dbpAB/dbpAB/2 were analyzed via an immunoblot probed with a mixture of FlaB MAb and mouse anti-DbpA (top) or -DbpB (bottom) sera.

 
Complementation plasmids were electroporated into a dbpAB mutant; the resulting transformants were screened as described previously (37). Identified transformants were first surveyed for the presence of lp28-1 because this plasmid is essential for persistent infection of immunocompetent hosts (18, 29). Only clones containing lp28-1 were further analyzed for plasmid content as described previously (37). Restoration of DbpA and DbpB expression was verified using immunoblots probed with a mixture of a FlaB MAb and anti-DbpA or -DbpB sera as described above.

Determination of ID50 values. The ID50 values were determined in two separate experiments performed within 7 months. In each experiment, spirochetes were grown to late log phase (108 cells per ml) at 33°C and 10-fold serially diluted with BSK-H complete medium. BALB/c mice (age, 4 to 6 weeks; provided by the Division of Laboratory Animal Medicine at Louisiana State University, Baton Rouge) each received one single intradermal/subcutaneous injection of 100 µl of spirochetal suspension. Mice were euthanized 4 weeks postinoculation; heart, tibiotarsal joint, and skin (not from the inoculation site) specimens were harvested for bacterial culture and DNA preparation as described previously (37). The ID50 value was calculated as described by Reed and Muench (30). DNA was extracted for analysis of tissue bacterial loads as described below.

Quantification of tissue spirochetal load. DNA was extracted for heart, joint, and skin specimens and quantified for the copy numbers of flaB and murine actin genes by quantitative PCR (qPCR) as previously described (37). The tissue spirochete burden was expressed as flaB DNA copies per 106 host cells (2 x 106 actin DNA copies).

Statistical analysis. A one-way analysis of variance was used to analyze data, followed by a two-tailed Student t test to calculate a P value for each two groups. A P value ≤0.05 was considered to be significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of dbpAB mutant. The dbpBA locus is located within the plasmid lp54, which carries 76 ORFs (Fig. 1A). The clone 13A, which was derived from B. burgdorferi B31 5A13, had been used in our previous studies because of its high transformability (36, 38, 39). This clone harbors 19 of the 21 plasmids; its improved transformability is due to lack of lp25 and lp56, the two plasmids that may carry restriction enzyme sites (17, 20). The disruption plasmid pNCO1T::dbpAB::aacC1 was electroporated into the 13A spirochetes; two dbpAB mutants were identified, one of which had lost lp28-1, the plasmid essential for persistent infection of immunocompetent hosts (18, 19, 29), and thus was discarded. Further plasmid content analyses revealed that the remaining clone, designated {Delta}dbpAB, had lost cp9, lp5, lp21, and lp28-4, in addition to lp25 and lp56. The insertion of the aacC1 cassette within the dbpBA locus is diagrammed in Fig. 1B and was confirmed by PCR (Fig. 1C); the lack of DbpA and DbpB expression was demonstrated by immunoblotting (Fig. 1D).

trans-complementation of the dbpAB mutant. Four trans-complementation plasmids were constructed from the recombinant plasmid pBBE22, as diagrammed in Fig. 2A, and designated pME22, pME22-dbpBA, pME22-dbpA, and pME22-dbpB. Because {Delta}dbpAB had lost lp25, the plasmid that carries the BBE22 gene, coding for a nicotinamidase essential for survival of B. burgdorferi in the mammalian environment (28), all four constructs contained a copy of the BBE22 gene. The same complementation strategy had been used by us and other investigators (20, 28, 32, 36, 39). Between 13 and 19 transformants were obtained from transformation with each construct. Plasmid analyses identified two clones receiving each construct; these eight clones were designated {Delta}dbpAB/E22/1, {Delta}dbpAB/E22/2, {Delta}dbpAB/dbpAB/1, {Delta}dbpAB/dbpAB/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, and {Delta}dbpAB/dbpB/2. All of the eight clones had the same plasmid content as {Delta}dbpAB, which lost cp9, lp5, lp21, lp28-4, lp25, and lp56. Restoration of DbpA and/or DbpB expression in these clones was confirmed by immunoblot analysis (Fig. 2B).

Both DbpA and DbpB are critical for full infectivity, and DbpA is more critical than DbpB. Groups of two or three BALB/c mice each received one inoculation of 101 to 106 spirochetes of the clone {Delta}dbpAB/dbpAB/1, {Delta}dbpAB/dbpAB/2, {Delta}dbpAB/E22/1, {Delta}dbpAB/E22/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, or {Delta}dbpAB/dbpB/2. Animals were euthanized 4 weeks later; heart, joint, and skin specimens were cultured for spirochetes for ID50 determination. In experiment I, the ID50 values for the clones {Delta}dbpAB/dbpAB/1, {Delta}dbpAB/dbpAB/2, {Delta}dbpAB/E22/1, {Delta}dbpAB/E22/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, and {Delta}dbpAB/dbpB/2 were determined at 3, 6, 3.16 x 104, 3.16 x 104, 1.78 x 103, 1.78 x 104, 3.16 x 103, and 3.16 x 103 organisms, respectively (Table 2). In experiment II, the ID50 values for these clones were 18, 18, 1.0 x 105, 1.0 x 105, 560, 1.78 x 103, 3.16 x 103, and 3.16 x 103 spirochetes, respectively, essentially reproducing the results of experiment I. By combining the two sets of data, the average ID50 value for the {Delta}dbpAB/dbpAB genotype clones was found to be 11 organisms, indicating that the {Delta}dbpAB mutant was fully competent. In sharp contrast, the genotype {Delta}dbpAB/E22 mutant produced an average ID50 value of 6.58 x 104 organisms. Overall, disruption of the dbpBA locus led to a nearly 6,000-fold increase in ID50 (P = 0.02), demonstrating that the two-gene operon is critical for full infectivity, although neither DbpA nor DbpB is essential for infection.


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  		TABLE 2. Quantitative analysis of the contribution of DbpA and DbpB to infectivity of B. burgdorferia

 
The mean ID50 values for the genotype {Delta}dbpAB/dbpA and {Delta}dbpAB/dbpB mutants were 1,475 and 3,160 organisms, respectively, which are 134-fold (P = 0.003) and 287-fold (P = 2.6 x 10–16) higher than that for the genotype {Delta}dbpAB/dbpAB mutant but 45-fold (P = 0.02) and 21-fold (P = 0.02) lower than that for the genotype {Delta}dbpAB/E22 mutant, indicating that both DbpA and DbpB are critical for infectivity. Moreover, the genotype {Delta}dbpAB/dbpB spirochetes registered a twofold-higher ID50 than the {Delta}dbpAB/dbpA spirochetes (P = 0.001), indicating that DbpA is more important than DbpB in contributing to the infectivity of B. burgdorferi.

Both DbpA and DbpB are critical for tissue colonization in heart. The ID50 value reflected only in part the overall virulence of B. burgdorferi. The {Delta}dbpAB/dbpAB spirochetes were consistently recovered from each tissue of all the 35 infected mice (Table 2). In contrast, the {Delta}dbpAB/E22 bacteria were not grown from any heart tissues of the 14 infected mice, demonstrating a critical role of the dbpBA locus in colonization of this specific tissue. Although the {Delta}dbpAB/dbpA spirochetes were recovered from 20 joint and 20 skin specimens of the 21 infected mice, only 9 of the 21 heart specimens produced a positive culture, reflecting a 57% decrease in frequency of heart tissue colonization in comparison with that for genotype {Delta}dbpAB/dbpAB spirochetes (P = 0.02). Even more dramatically, the {Delta}dbpAB/dbpB bacteria could not be recovered from any heart tissue although all the 32 joint and skin samples from the 16 infected mice produced a positive culture. Taken together, these data indicated that both DbpA and DbpB are critical for the ability of B. burgdorferi to colonize heart tissues. Moreover, there was a trend showing that DbpA might be more important than DbpB in heart colonization, as the {Delta}dbpAB/dbpA spirochetes were recovered from nine heart specimens of the 21 infected mice but the {Delta}dbpAB/dbpB bacteria were not grown from any heart tissues of the 16 infected mice. However, our statistical analysis precluded such a claim because the calculated P value (P = 0.07) was slightly larger than the cutoff value, ≤0.05.

Deficiency in either DbpA or DbpB severely impairs tissue colonization in joints but only deficiency in both significantly does in skin. The spirochetal burden was analyzed to further assess how much the DbpA/B deficiency affected tissue colonization. All heart, joint, and skin specimens that gave positive culture results, as listed in Table 2, were processed for DNA. qPCR analyses were not able to generate meaningful results from DNA samples extracted from either joint or skin specimens of the 14 mice that were infected with the clone {Delta}dbpAB/E22/1 or {Delta}dbpAB/E22/2 (data not shown), indicating very low bacterial loads in these samples and thus revealing a critical role of the dbpBA locus in colonization of both joint and skin tissues. Similarly, the {Delta}dbpAB/dbpA spirochetal DNA could not be consistently detected by qPCR in heart specimens that produced culture-positive results (data not shown), underscoring a critical role of DbpB in the colonization of this specific tissue.

qPCR analyses revealed no trend that the inoculation dose affected spirochetal loads for any of the four genotypes. Nevertheless, to make comparisons generally acceptable, qPCR data were obtained from joint and skin samples of all the 24 mice that were infected through a single inoculation with 104 spirochetes of the clone {Delta}dbpAB/dbpAB/1, {Delta}dbpAB/dbpAB/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, or {Delta}dbpAB/dbpB/2, and presented in Fig. 3. In the joint, the {Delta}dbpAB/dbpAB spirochete load was 5.6- and 8.8-fold higher than those of the genotype {Delta}dbpAB/dbpA (P = 0.03) and {Delta}dbpAB/dbpB (P = 0.02) spirochetes, respectively, indicting that both DbpA and DbpB are critical for colonization of this tissue. There was no significant difference in bacterial load registered for the genotype {Delta}dbpAB/dbpA or {Delta}dbpAB/dbpB spirochetes (P = 0.36), suggesting that the two adhesins contribute similarly to the ability of B. burgdorferi to colonize joint tissues. In skin, spirochetes of all three genotypes generated similar bacterial loads (P > 0.05), indicating that lack of either adhesin does not significantly reduce the ability of B. burgdorferi to colonize this tissue, although lack of both severely impaired this ability. Or in other words, although a deficiency in both DbpA and DbpB significantly reduces the ability of B. burgdorferi to colonize skin, either adhesin can compensate for loss of the other in colonization of the skin tissue.


Figure 3
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  		FIG. 3. Deficiency in either DbpA or DbpB severely reduces the ability of B. burgdorferi to colonize joint but not skin tissue. Joint and skin specimens were collected from all the 24 mice that were infected via a single inoculation with 104 spirochetes of the clone {Delta}dbpAB/dbpAB/1, {Delta}dbpAB/dbpAB/2, {Delta}dbpAB/dbpA/1, {Delta}dbpAB/dbpA/2, {Delta}dbpAB/dbpB/1, or {Delta}dbpAB/dbpB/2 in two separate experiments, listed in Table 2. DNA samples were prepared and analyzed for spirochetal flaB and murine actin DNA copies by qPCR. The data are expressed as spirochete numbers per 106 host cells. The three groups are labeled "dbpAB," "dbpA," and "dbpB."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The dbpAB mutant generated in the current study registered an ID50 value of 11 organisms, on average, after complementation with a dbpAB copy carried on the vector pME22, demonstrating that the mutant is fully competent in spite of having gained two antibiotic selection markers and lost six plasmids, including lp28-4, which may harbor genes important for the invasiveness of B. burgdorferi (35). This allowed us to thoroughly investigate the contributions of DbpA/DbpB, either in combination or individually, to the overall virulence of B. burgdorferi. Inactivation of the dbpBA locus resulted in nearly a 104-fold increase in ID50, indicating that the two-gene operon is critical for mammalian infection. Deficiency in either DbpA or DbpB increased the ID50 value by more than 100-fold, highlighting the importance of both lipoproteins in mammalian infection. Comparison of the two adhesins showed that DbpA was more important than DbpB for infectivity. Disruption of the dbpBA locus affected colonization in all tissues, but the defect was tissue dependent. The most significant defect was noted in heart; lack of dbpA alone abrogated the ability of B. burgdorferi to colonize this tissue, and the deficiency for DbpB led to a 57% decrease in frequency of tissue colonization. In joint, lack of DbpA or DbpB alone did not significantly reduce the frequency of tissue colonization in infected mice, but the deficiency in either DbpA or DbpB severely reduced the bacterial load. Lack of either adhesin did not affect the frequency of tissue colonization or the bacterial load in skin, but deficiency in both significantly reduced the bacterial load, indicating that either adhesin can compensate for loss of the other in the colonization of this specific tissue.

Consistent with our previous study (33), the current study showed that the dbpBA locus is not essential for murine infection. In the previous study, a dose of 105 organisms initiated an infection in almost all inoculated mice and the ability of the dbpAB mutant to colonize the heart tissue was found more severely affected. In two separate experiments of the previous study, one of the three examined mutant clones could not be recovered from any heart tissue of the five infected immunocompetent mice and a second clone was grown from only one heart specimen from the same number of infected mice (33). Such a disparity observed among different clones with similar genetic compositions was also noted in the current study. For instance, the {Delta}dbpAB/E22/1 spirochetes were recovered from all four joint samples but from none of the skin specimens of the four infected mice, while the {Delta}dbpAB/E22/2 bacteria were grown from one joint specimen and three skin specimens from the same number of infected animals in experiment I. In experiment II, however, the clone {Delta}dbpAB/E22/1 was not grown from any joint specimen but from three of the four skin samples, while the clone {Delta}dbpAB/E22/2 was recovered from one-half of the four joint and four skin specimens. This type of inconsistency was probably due to very low infectivity of the inocula and extremely low tissue bacterial loads. This explanation is supported by our analysis of bacterial loads, which revealed very low {Delta}dbpAB/E22 DNA contents in joint and skin tissues.

DbpA and DbpB apparently do not contribute equally to the infectivity of B. burgdorferi, in terms of ID50. The DbpA deficiency led to a 287-fold increase in ID50, but the lack of DbpB alone caused only a 134-fold increase. When a more precise comparison was made between the two deficiencies, DbpA showed more importance than DbpB in contribution to infectivity. However, both adhesins appear to contribute similarly to tissue colonization, especially in joint and skin. Deficiency in either DbpA or DbpB resulted in similar decreases in bacterial load in joint and did not produce any defect in skin although the lack of both significantly did. Unfortunately, the current study was unable to conclusively show whether the two adhesins contributed differently to colonization in the heart tissue. DbpA and DbpB are translated from the same bicistronic mRNA and have similar sizes (14), and both bind decorin and glycosaminoglycans, albeit exhibiting different affinities and specificities (12, 13). It would be interesting to address how DbpA can more effectively contribute to the infectivity of B. burgdorferi.

Both DbpA and DbpB more significantly contribute to the overall virulence of B. burgdorferi than another lipoprotein adhesin, BBK32. Inactivation of the BBK32 gene increases the ID50 value by only 10-fold (32) and does not significantly reduce the bacterial load in the murine host (21). In contrast, deficiency in either DbpA or DbpB resulted in a more-than-100-fold ID50 increase and also severely reduced the ability of B. burgdorferi to colonize heart and joint tissues. All three adhesins can bind glycosaminoglycans, albeit exhibiting different specificities (11, 12); in addition, DbpA/DbpB and BBK32 recognize decorin and fibronectin, respectively (13, 26). These differences may in part cause unequal contributions to the overall virulence of B. burgdorferi. The remaining factors that potentially make the difference remain to be addressed, including densities of the adhesins expressed on the spirochete surface.

The current study clearly showed that the influence of DbpA/DbpB deficiency is tissue dependent. The most notable defect was observed in heart, followed by joint and skin tissues. In heart, the lack of either DbpA or DbpB severely reduced the frequency of tissue colonization in infected mice, while a deficiency in either or even both did not affect the frequency of colonization in joints but significantly reduced the bacterial load. The least defect was observed in skin, where the deficiency in either adhesin did not affect frequency or bacterial load although the lack of both significantly did. As a primary ligand of both DbpA and DbpB (13), decorin is most abundantly expressed in skin, followed by joint and heart tissues (22). The abundant presence of decorin in skin may provide excess interactions so that the loss of either adhesin does not significantly compromise the ability of B. burgdorferi to colonize this tissue. In contrast, the heart tissue expresses much less decorin; the presence of both adhesins may be required for sufficient interactions. Alternatively, the skin tissue may express other ligands to interact with other known or unknown borrelial adhesins so that the reduced interactions of DbpA/DbpB with decorin can be compensated, or it may provide a better protective microenvironment for the pathogen, in which the contribution from the interactions of DbpA/DbpB and decorin is less important to the overall virulence of B. burgdorferi.

As an extracellular bacterium, B. burgdorferi resides primarily in the ECM and connective tissues as well as between host cells during mammalian infection, where host ligands that interact with well-characterized borrelial adhesins such as DbpA and DbpB are abundant. The interactions of B. burgdorferi with these ligands mediated by its surface adhesins potentially affect various aspects of infection and, as a result, influence its overall infectivity and pathogenicity. The current study showed that a deficiency in either DbpA or DbpB alone dramatically reduces the overall virulence by severely reducing infectivity, as measured by ID50, and tissue colonization, as measured by frequency of tissue colonization, and bacterial load. Thus, the study highlights the importance of the potential interaction of B. burgdorferi with host ligands in the pathogenesis of B. burgdorferi.


   ACKNOWLEDGMENTS
 
We thank S. Norris for providing the recombinant plasmid pBBE22 and P. Stewart and P. Rosa for providing the shuttle vector pBSV2G.

This work was in part supported by a career development award and a grant from NIH/NIAMS, an Arthritis Foundation Investigators award, and P20RR020159 (awarded to LSU) from NIH/NCRR.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathobiological Sciences, Louisiana State University, Skip Bertman Drive at River Road, Baton Rouge, LA 70803. Phone: (225) 578-9699. Fax: (225) 578-9701. E-mail: fliang{at}vetmed.lsu.edu Back

{triangledown} Published ahead of print on 14 January 2008. Back

Editor: V. J. DiRita


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Infection and Immunity, March 2008, p. 1239-1246, Vol. 76, No. 3
0019-9567/08/$08.00+0     doi:10.1128/IAI.00897-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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