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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Lyme borreliosis is a chronic, systemic illness caused by infection with Borrelia burgdorferi and the closely related spirochaetes Borrelia garinii and Borrelia afzelii. In 2000, 17 730 cases of Lyme disease were reported in the United States (Centers for Disease Control and Prevention, 2002). Transmission occurs with the bite of an infected Ixodes tick, resulting in a localized lesion called erythema migrans. B. burgdorferi then disseminate to distant sites, including the nervous system, heart and joints, where they contribute to a constellation of manifestations including neuroborreliosis, cardiac block and arthritis (Steere, 2001). B. burgdorferi is an obligate parasite of its mammalian and arthropod hosts and is not naturally transmitted by other means. Therefore, continued existence of the bacterium is reliant upon both long-term survival within, and successful transmission between, these radically different host environments.

The genetic mechanisms involved in the infection process are poorly understood. The B. burgdorferi B31 genome was sequenced recently (Fraser et al., 1997; Casjens et al., 2000) and consists of a 911 kb linear chromosome and 21 linear and circular plasmids ranging in size from 9 kb to 56 kb. The consistent presence of this large group of plasmids in Lyme disease Borrelia suggests that they play important roles in the survival and pathogenesis of these organisms (Casjens et al., 2000; Palmer et al., 2000). Sequence analysis of the plasmids revealed the presence of 175 multigene families (most of which are unique to Borrelia species) and relatively few genes with predicted functions (Fraser et al., 1997; Casjens et al., 2000). Many of the plasmid-encoded genes undergo dramatic changes in expression levels during the transition between mammal and tick environments. For example, the lipoproteins outer surface protein A (OspA) and OspB are expressed at high levels in the tick midgut but not in the tick salivary glands or within the mammalian host, whereas OspC is dramatically upregulated during tick feeding and transmission of organisms to mice (Indest et al., 2001a; Schwan et al., 1995; Schwan et al., 2000; Seshu and Skare, 2001). Temperature, pH and as yet unidentified host factors contribute to this transition (Schwan et al., 1995; Tilly et al., 1997; 2001; Akins et al., 1998; Cassatt et al., 1998; Carroll et al., 1999; Yang et al., 1999; 2000; Schwan and Piesman, 2000; 2001; Gilmore et al., 2001; Hefty et al., 2001; 2002; Indest et al., 2001b; Revel et al., 2002). The sigma factors RpoS and RpoN have been shown to play a central role in the regulation of OspC and decorin-binding protein A (DbpA) (Hübner et al., 2001). One of the linear plasmids, lp28-1, contains the VMP-like sequence (vls) locus involved in antigenic variation of the surface-exposed lipoprotein, VlsE (Zhang et al., 1997; Eicken et al., 2002). The cp26 genes guaA and guaB encode proteins homologous to purine biosynthesis enzymes, and B. burgdorferi guaA was capable of complementing an Escherichia coli GMP synthetase mutant (Margolis et al., 1994), providing evidence that plasmid-encoded genes can also provide physiological functions.

Reduced infectivity of B. burgdorferi has long been associated with repeated in vitro passage and the resulting loss of plasmids (Johnson et al., 1984; Barbour, 1988; Simpson et al., 1990; Norris et al., 1995). Recent studies indicated that absence of lp25 is associated with a loss of infectivity in C3H mice and that absence of lp28-1 results in reduced infectivity (Xu et al., 1996; Purser and Norris, 2000; Labandeira-Rey et al., 2001). The consistent hybridization of probes specific for lp25 (14/15 strains) and vls sequences from lp28-1 (15/15 strains) with a panel of North American B. burgdorferi isolates further suggests that these plasmids are needed for the survival of Lyme disease Borrelia (Palmer et al., 2000). However, the requirement for specific B. burgdorferi plasmids or genes at any stage of the infectious cycle has not been demonstrated conclusively, in large part because of the difficulties encountered with transformation and gene disruption in low-passage, infectious Borrelia (Hübner et al., 2001; Eggers et al., 2002; Elias et al., 2002; Lawrenz et al., 2002).

In this study, we determined that the lp25 gene BBE22 encodes a protein with nicotinamidase activity and is capable of restoring infectivity to a B. burgdorferi clone lacking lp25. These findings demonstrate that the plasmids of Lyme disease Borrelia fulfil important physiological as well as pathogenic functions, and represent the first fulfilment of the so-called molecular Koch's postulates (Salyers and Whitt, 2001) for a Borrelia gene.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Lp25 provides a required physiological function

In a previous study, well-characterized B. burgdorferi B31 low-passage clones were examined for infectivity, and clones lacking lp25 were shown to be non-infectious in immunocompetent C3H/HeN mice at a dosage of 105 organisms (Purser and Norris, 2000). To determine the importance of the immune response in this phenotype, the infectivities of B. burgdorferi B31 clones containing all plasmids (5A4) or lacking lp25 (5A13) or lp28-1 (5A8) were compared in immunocompetent C3H/HeNHsd and C3H/Smn.ClcrHsd-scid mice, which have a severe combined immunodeficiency (SCID) phenotype. The lp25-deficient clone 5A13 could not be cultured from either SCID or wild-type mice 2 weeks after inoculation (Table 1). B. burgdorferi 5A13 were not recovered from infected mice of either strain even if the inoculum was increased to 106 or 107 organisms (data not shown). In contrast, a B. burgdorferi B31 clone lacking lp28-1 (5A8) has decreased infectivity in immunocompetent mice (Purser and Norris, 2000), but was cultured from all tissues examined in SCID mice (Table 1). The growth of B. burgdorferi clones 5A4, 5A8 and 5A13 was also examined in the dialysis membrane chamber (DMC) model, in which borrelia are cultivated in chambers of dialysis tubing implanted within the peritoneal cavities of rats for 12–14 days (Akins et al., 1998). These ‘host-adapted’ organisms are exposed to a tissue-like environment, but are protected from antibody and cellular responses by the 8000 Da dialysis membrane barrier. In DMCs, B. burgdorferi clones 5A4 (containing all plasmids) and 5A8 (lp28-1) increased from 103 ml−1 to ≈ 107 ml−1; however, clone 5A13 (lp25) was neither visible by darkfield microscopy nor could it be rescued by culturing DMC fluid in BSK medium after explantation. In some experiments, 5A13 was implanted at a higher concentration (106 organisms ml−1) to permit visualization by darkfield microscopy; these studies revealed that organisms were still present in similar numbers after explantation, but were non-motile reflecting loss of viability. Taken together, these results suggested that gene(s) of lp25 fulfil a physiological role rather than providing an immunologically related function, as is apparently the case with lp28-1.

Table 1. . Infectivity of B. burgdorferi B31 clones lacking plasmids lp28-1 or lp25 in immunocompetent or severe combined immunodeficiency (SCID) mice. a
Mouse strain B. burgdorferi B31 cloneNo. of cultures positive/totalNo. of mice positive/total
BladderHeartJointEarAll sites
  • a . Groups of six immunocompetent C3H/HeN.Hsd or C3H/Smn.ClcrHsd (SCID) mice were inoculated intradermally with 10 5 of the indicated B. burgdorferi B31 clone. Two weeks after inoculation, the indicated tissues were cultured in BSK-II medium and examined for the growth of B. burgdorferi.

  • b

    . One culture was contaminated.

C3H/HeNHsd5A4 (all plasmids)5/5b6/66/66/623/236/6
C3H/HeNHsd5A13 (lp25)0/60/60/60/6 0/240/6
C3H/HeNHsd5A8 (lp28-1)0/60/63/61/6 4/244/6
C3H/Smn.ClcrHsd-scid5A4 (all plasmids)6/66/66/66/624/246/6
C3H/Smn.ClcrHsd-scid5A13 (lp25)0/60/60/60/6 0/240/6
C3H/Smn.ClcrHsd-scid5A8 (lp28-1)6/66/66/66/624/246/6

BBE22 confers infectivity to non-infectious, low-passage B. burgdorferi

A survey of the sequence of lp25 (Fraser et al., 1997) revealed the presence of one open reading frame (ORF), BBE22, with a predicted metabolic function. The putative product of BBE22 has 33.7% identity and 45.5% similarity to PncA, a well-characterized nicotinamidase of Salmonella typhimurium that catalyses the deamination of nicotinamide to nicotinic acid. BBE22 also exhibited significant homology to functionally defined or putative nicotinamidases/pyrazinamidases in a wide variety of eubacteria, including E. coli and Yersinia, Mycobacterium, Brucella, Ralstonia, Aquifex and Pyrococcus species. BBE22 is also a member of pfam00857, a large family of hydrolase enzymes including several nicotinamidase/pyrazinamidases (Conserved Domain Database, http:www.ncbi.nlm.nih.gov). BBE22 was therefore selected for further analysis.

A segment of DNA from lp25 containing BBE22 was amplified and inserted into the shuttle vector pBSV2 (Stewart et al., 2001), resulting in the recombinant plasmid pBBE22 (Fig. 1). The insert sequence matched that of lp25 co-ordinates 14 571–16 628 and thus included the 537 bp BBE22 ORF plus ≈ 1 kb of sequence 5′ to the gene to include potential transcriptional and regulatory regions, as well as ≈ 0.5 kb of downstream sequence. BBE22 was the only large ORF contained within the insert; however, the 183 bp gene BBE23, encoding a putative hypothetical protein, was also included to ensure that adequate upstream sequence was present for expression of BBE22. The resulting construct, pBBE22, was used to transform lp25-deficient B. burgdorferi 5A13. Infectivity of 5A13/pBBE22 and the 5A13/pBSV2 vector control was examined in C3H/HeN mice. When 105 5A13/pBBE22 were used as the inoculum, viable B. burgdorferi were recovered from all mice in each of the four tissues examined (Experiments 1 and 2 in Table 2), demonstrating that infectivity had been restored at this dosage; identical results were obtained with the 5A4 positive control, which contains lp25 and hence BBE22. In contrast, the 5A13/pBSV2 vector-only control could not be recovered from the 11 mice in these two experiments. [An additional mouse from this group in Experiment 1 was culture positive at all sites, but it was determined by polymerase chain reaction (PCR) analysis of all cultures that this mouse had been inadvertently inoculated with 5A13/pBBE22.] To examine the infectivity of 5A13/pBBE22 relative to the wild-type 5A4 further, a median infectious dose (ID50) determination was performed (Experiment 3 in Table 2). In this experiment, the ID50 determined for 5A13/pBBE22 was 178 organisms, whereas the ID50 for the wild-type clone 5A4 was 18 organisms. No cultures were positive from any of the mice infected with up to 107 5A13/pBSV2 (Experiment 3 and additional experiments), so the ID50 was > 107 for these organisms.

image

Figure 1. Construction of pBBE22. BBE22, and ≈ 1 kb and 0.5 kb of sequence 5′ and 3′ of the gene, respectively, were amplified by PCR with primers containing KpnI sites. The resulting PCR product was then digested with KpnI and ligated at the corresponding restriction site in the shuttle plasmid pBSV2, which contains the replication locus of the B. burgdorferi circular plasmid cp9 (Stewart et al., 2001). BBE23, a predicted ORF encoding a 61-amino-acid hypothetical protein, was included in the construct to ensure that the regulatory region of BBE22 was not truncated. pBBE22M is identical to pBBE22 except for a Cys[RIGHTWARDS ARROW]Ala substitution at codon 120 of BBE22. The drawing of pBSV2 is from Stewart et al. (2001); used with permission.

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Table 2. . Complementation of infectivity of B. burgdorferi 5A13 with plasmid pBBE22 in C3H/HeN mice. a
B. burgdorferi B31 clone and inoculumbID50c (no. of organisms)No. of cultures positive/total No. of mice positive/total
BladderHeartJointEarAll sites
  • a . Experimental procedure as described in Table 1.

  • b . Inoculum = 10 5 in Experiments 1 and 2.

  • c . Calculated by the method of Reed and Muench (1938).

  • d

    . One culture had bacterial contamination.

  • e . 5A13/pBSV2 was also non-infectious at doses of 10 6 or 107 organisms, as determined in a separate experiment.

Experiment 1
5A4 (all plasmids) 6/66/66/66/624/246/6
5A13 (lp25) 0/60/60/60/60/240/6
5A13/pBBE22 (BBE22+) 6/66/66/66/624/246/6
5A13/pBSV2 (BBE22–) 0/50/50/50/50/200/5
Experiment 2
5A4 (all plasmids) 6/66/66/66/624/246/6
5A13 (lp25) 0/60/60/60/60/240/6
5A13/pBBE22 (BBE22+) 6/66/66/66/624/246/6
5A13/pBSV2 (BBE22–) 0/60/60/60/60/240/6
Experiment 3 (ID50 determination)
5A418      
105 3/33/33/33/312/123/3
104 3/33/32/2d3/311/113/3
103 3/33/33/33/312/123/3
102 3/33/32/2d3/311/113/3
101 1/31/31/31/34/121/3
5A13/pBSV2 (–BBE22)>107e      
105 0/30/30/30/30/120/3
104 0/30/30/30/30/120/3
103 0/30/30/30/30/120/3
102 0/30/30/30/30/120/3
101 0/30/30/30/30/120/3
5A13/pBBE22 (+BBE22)178      
105 3/33/33/33/312/123/3
104 3/33/33/33/312/123/3
103 3/33/33/33/312/123/3
102 1/31/31/30/33/121/3
101 0/30/30/30/30/120/3

Histological examination of the tibiotarsal joints of animals infected with 105 5A13/pBBE22 for 2 weeks demonstrated that pathologic changes (tendonitis and periostitis) were present in four out of six mice, compared with six out of six mice infected with clone 5A4 (Fig. 2). The pathology scores were also lower overall (0, 0, 4, 7, 12 and 15 out of 25) than those of the 5A4-inoculated mice (10, 13, 13, 16, 17 and 18 out of 25), and were significantly different by the Student's two-tailed t-test (P = 0.022). No histopathological abnormalities were observed in mice inoculated with 5A13 or 5A13/pBSV2 controls. Thus, transformation with a BBE22-containing shuttle vector restored infectivity of 5A13 to nearly wild-type levels, although the transformant had a somewhat higher ID50 and resulted in decreased joint histopathology relative to the wild-type 5A4 control.

image

Figure 2. Occurrence of joint inflammation in mice infected with B. burgdorferi clone 5A13 complemented with pBBE22. C3H/HeN mice were injected intradermally with 105B. burgdorferi 5A4 (wild type), 5A13 (lp25) or 5A13/pBBE22 (5A13 complemented in trans with BBE22). Two weeks after infection, tibiotarsal joints were isolated, decalcified, sectioned, stained with haematoxylin and eosin and analysed for histopathology. Tissues from mice inoculated with (A) 5A4, (B) 5A13 and (C) 5A13/pBBE22. Pathological changes, including mixed inflammatory infiltrates in the tendon (T), tendon sheath (TS) and tendon sheath space (Sp), were noted in 6/6 5A4-infected mice, 0/6 5A13-infected mice and 4/6 5A13/pBBE22-infected mice (see text).

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Borrelia burgdorferi strain HP-J is a high-passage, non-infectious clone of B. burgdorferi that is missing 14 plasmids (Lawrenz et al., 2002), but can still be cultivated in vitro. HP-J was transformed with pBBE22 and tested for infectivity in SCID mice and for growth in DMCs. No organisms were recovered from tissues of the inoculated mice, nor from the DMCs, indicating that complementation with pBBE22 was not sufficient to restore infectivity to the HP-J strain (data not shown).

Mutation of BBE22 putative active site abolishes the ability to restore infectivity

The protein sequence of BBE22 was compared with those of other related nicotinamidases. Protein sequence alignments reveal highly conserved domains among all other members of the hydrolase family (pfam00857), and PncA proteins from E. coli, S. typhimurium and Mycobacterium tuberculosis have confirmed nicotinamidase/pyrazinamidase function (Frothingham et al., 1996). Based on mutational analysis of members of this family, the proposed mechanism of hydrolase activity and a high degree of conservation, the cysteine located at position 120 in the BBE22 product was postulated to be a required amino acid within the active site of BBE22. Therefore, PCR mutagenesis was used to create construct pBBE22M, which was identical to pBBE22 except for a Cys-120Ala amino acid change. B. burgdorferi 5A13 was transformed with pBBE22M and assayed for infectivity phenotype in C3H/HeN mice. No organisms were recovered from mice infected with 5A13/pBBE22M, whereas mice inoculated with controls 5A4 or 5A13/pBBE22 were consistently infected (Table 3). Therefore, complementation with the mutated form of BBE22 failed to restore infectivity.

Table 3. . Mutation abrogates the restoration of infectivity by BBE22 in C3H/HeN mice. a
Bb B31 cloneNo. of cultures positive/totalNo. of mice positive/total
BladderHeartJointEarAll sites
  • a . Experimental procedure as described in Table 1.

  • b

    . pBBE22M is identical to pBBE22 except for a Cys

  • [RIGHTWARDS ARROW]

    [RIGHTWARDS ARROW]Ala conversion at amino acid 120 of the BBE22 ORF.

5A4 (all plasmids)6/66/66/66/624/246/6
5A13/pBSV2 (–BBE22)0/60/60/60/60/240/6
5A13/pBBE22 (+BBE22)6/66/66/66/624/246/6
5A13/pBBE22M (mutated BBE22)b0/60/60/60/60/240/6

In vivo expression of BBE22 restores growth of lp25 B. burgdorferi in DMCs

Borrelia burgdorferi 5A4, 5A13/pBBE22, 5A13/pBBE22M and 5A13/pBSV2 were implanted into rat peritoneal cavities within DMCs as described previously (Akins et al., 1998). Complemented B. burgdorferi 5A13/pBBE22 not only grew to the same density as wild-type 5A4 organisms within the chambers, but also exhibited the same host-adapted protein expression pattern when examined by SDS-PAGE and silver staining (data not shown). In contrast, neither B. burgdorferi control strain 5A13/pBSV2 nor 5A13/pBBE22M (with a mutated form of BBE22) survived in DMCs.

Complementation of a Salmonella pncA nadB mutant with Borrelia BBE22

Borrelia burgdorferi construct pBBE22 was transformed into an S. typhimurium pncAnadB double mutant to determine whether the B. burgdorferi gene product expression would complement the pncA mutational deficiency and allow growth of the mutant on minimal media using nicotinamide as a sole substrate for NAD production. The S. typhimurium pncAnadB mutant used in this study is deficient in both pathways of NAD synthesis (see Discussion): one using exogenously acquired nicotinamide as a precursor and one involving de novo synthesis from aspartate (Zhu et al., 1991). This mutant will not grow in M9 minimal medium unless it is supplemented with nicotinic acid, the end-product of PncA activity. To determine whether BBE22 could complement the pncA mutation, the S. typhimurium pncAnadB mutant was transformed with pBBE22, pBBE22M or pBSV2; wild-type S. typhimurium was included as a positive control (Fig. 3). pBBE22 conferred on S. typhimurium pncAnadB the ability to grow in minimal medium + nicotinamide (Fig. 3E), whereas transformation with pBBE22M or pBSV2 did not permit growth under these conditions. To determine whether a promoter known to be expressed efficiently in E. coli would improve the complementation of the S. typhimurium mutation, BBE22 was cloned into the E. coli expression vector pQE30 to produce pQE30:BBE22. Expression of BBE22 from the T5 promoter/lac operator of pQE30 further increased the growth rate of the S. typhimurium mutant so that it closely resembled that of the wild-type strain (Fig. 3B). All strains were able to grow in the presence of nicotinic acid (Fig. 3C and F), indicating that the conversion of nicotinamide to nicotinic acid is the critical activity provided by BBE22.

image

Figure 3. Complementation of a Salmonella typhimurium pncAnadB mutant with BBE22 expressed from the T5/lac promoter of plasmid pQE30 or the native B . burgdorferi promoter. BBE22 in pQE30 (pQE30:BBE22; A–C) or the shuttle vector pBSV2 (pBBE22; D–F) was used to transform the Salmonella pncAnadB mutant. pQE30 and pBSV2 were used as vector-only controls, and pBBE22M, containing BBE22 with a Cys[RIGHTWARDS ARROW]Ala mutation at amino acid 120, was used to determine the requirement for Cys-120 for complementing activity. Wild-type (WT) S. typhimurium LT2 and the transformed pncAnadB strains were tested for growth in M9 minimal medium, M9 + nicotinamide or M9 + nicotinic acid.

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The BBE22 product has nicotinamidase activity

The Salmonella pncAnadB mutant transformed with pQE30:BBE22 was assayed for nicotinamidase activity by quantifying the release of ammonia from the conversion of nicotinamide to nicotinic acid. In the presence of nicotinamide, only Salmonella complemented with pQE30:BBE22 liberated amounts of ammonia> 1.0 µg ml−1 (data not shown). All other strains tested, including wild-type Salmonella, produced relatively small quantities of ammonia. The presence of BBE22 on a high-copy-number plasmid may explain why only the Salmonella strains harbouring the pQE30:BBE22 plasmid generated high quantities of ammonia. These results provide evidence that the BBE22 product is defined properly as a PncA homologue with nicotinamidase activity.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Lp25 provides a physiological function

Previous reports identified lp25 as an infectivity-associated plasmid (Xu et al., 1996; Purser and Norris, 2000; Labandeira-Rey et al., 2001). In the present study, B. burgdorferi lacking lp25 were assayed for infectivity in normal mice and in immunocompromised mice. The absence of an adaptive immune response had no effect on lp25-mediated infectivity: lp25-deficient B. burgdorferi were not infectious in immunologically normal nor in SCID mice, even at doses of 107 organisms (Table 1). In addition, the lp25-deficient clone did not grow in DMCs implanted in rats, an environment in which growth would not be impeded by antibodies or by inflammatory cells. Clone 5A8, which contains lp25 but lacks lp28-1 and hence the vls antigenic variation system, can only occasionally be cultured from tissues after infection of immunocompetent mice, indicating a low level of infection (Purser and Norris, 2000). However, this clone was able to grow consistently in both SCID mice (Table 1) and DMCs. We believe that the vls antigenic variation system is not required in these ‘immunologically privileged’ environments, permitting 5A8 to thrive under these conditions. In contrast, the lp25-specific factor contributing to infectivity in mammals appeared to be a gene product important in cellular metabolism, rather than one affected by the immune response. This observation led to the characterization of BBE22, the presence and functional activity of which was required for the restoration of infectivity and growth in DMCs (as summarized in Table 4).

Table 4. . Summary of results obtained with mouse infection and dialysis membrane chamber growth of B. burgdorferi strains exhibiting varied plasmid content and BBE22 expression.
B. burgdorferi strainBBE22presentInfectivity inC3H/HeN miceInfectivity inSCID miceGrowth in dialysis membrane chambers
  1. a . Intermediate infectivity phenotype; see Table 1.

5A4 (all plasmids)++++
5A8 (lp28-1)+±a++
5A13 (lp25)
5A13/pBSV2
5A13/pBBE22++++
5A13/pBBE22M+(mutated)

BBE22 has homology with PncA

Thirty-two ORFs have been identified in lp25, with most having no known homologues in other organisms (Fraser et al., 1997). BBE22 shares strong homology with many nicotinamidases/pyrazinamidases. Nicotinamidase, or PncA, is an enzyme involved in the production of NAD (Fig. 4). S. typhimurium and E. coli use PncA in the preferred exogenous pathway for NAD synthesis (Hughes et al., 1983). In the less preferred endogenous pathway, NadB, NadA and NadC catalyse the conversion of aspartate to nicotinate D-ribonucleotide. The genome of B. burgdorferi contains PncA (BBE22), PncB (BB0635), NadD (BB0782) and NadE (BB0522) orthologues, but not NadB, NadA or NadC orthologues (data not shown). Therefore, B. burgdorferi may rely upon a single pathway for NAD synthesis. B. burgdorferi lacking pncA may be able to survive and grow in vitro because BSK-II medium contains CMRL medium, an enriched tissue culture medium that contains nicotinamide, nicotinic acid, NAD and NADP. One or more of these compounds may permit the growth of lp25 clones at the same rate as clones with the full complement of plasmids. It is also possible that PncA participates in other pathways required for survival and growth in vivo.

image

Figure 4. NAD synthesis in S. typhimurium and E. coli. NAD may be synthesized via the endogenous pathway, using aspartate, or via the preferred exogenous pathway, using exogenously acquired nicotinamide as the initial substrate. The homologous genes in B. burgdorferi are indicated in parentheses; the NadBAC pathway appears to be absent, based on the lack of homologous sequences.

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The 5′ end of BBE22 predicted by Fraser et al. (1997) lacks an obvious ribosome binding site (RBS) or a nearby consensus promoter sequence. Sequencing of this region in our laboratory confirmed that the reported sequence is free of sequence errors. Interestingly, a 48 nucleotide region upstream of the predicted translational start site encodes 16 amino acids that are highly conserved in PncA homologues of other bacteria; an out-of-frame GTG codon with potential RBS and promoter sequences is located at the 5′ end of this sequence. The possibility that this gene is expressed from this potential start site through a slipped strand synthesis mechanism is under investigation.

Complementation with BBE22 restores infectivity

To determine whether the addition of BBE22 is sufficient to restore infectivity of lp25B. burgdorferi in mice, pBBE22 was constructed. pBBE22 is a recombinant shuttle plasmid containing BBE22, flanked by ≈ 1 kb of DNA upstream of the putative start codon and 0.5 kb of DNA downstream of the stop codon. pBBE22 alone restored infectivity in the lp25B. burgdorferi strain. Mutational analysis further demonstrated that BBE22 is required for the restoration of infectivity, as discussed subsequently. The small ORF BBE23 was also included in pBBE22 to ensure that the transcriptional regulatory region of BBE22 was intact in the construct; the contribution of this ORF to the restoration of infectivity (if any) has not been determined as yet.

The higher ID50 for clone 5A13/pBBE22 relative to clone 5A4 (178 Borrelia compared with 18 organisms), as well as the decreased histopathology observed, may result from the involvement of other lp25 genes in the mammalian infection process. Alternatively, these differences may be caused by plasmid copy number, toxicity or instability, incomplete trans-complementation by the recombinant vector resulting in decreased expression or other factors. As only a single time point (2 weeks) was examined, it is possible that the pBBE22-complemented lp25 strain would cause greater histopathology at later times after infection. A time course of infection, including a quantitative analysis of the number of B. burgdorferi present in affected tissues, is planned. Another important consideration for future studies is the transmissibility of the complemented strain by ticks.

Mutation of BBE22 at the predicted catalytic site abrogates complementation of infectivity

Amino acid sequence alignments of BBE22 with other characterized nicotinamidases demonstrated a high degree of conservation of the putative active site. E. coli YcaC, the family member with closest homology to BBE22, is an octameric hydrolase of unknown specificity. Colovos et al. (1998) identified Cys-118 as the putative active site because of its location in a conspicuous cleft between two ycaCgp subunits and conservation among family members. Another family member, Pyrococcus horikoshii PH999, was confirmed by Du et al. (2001) to have pyrazinamidase activity. Analysis of the crystal structure suggested that the overall fold of the PH999 protein is similar to YcaC of E. coli, and C133 was identified as part of the active site. PncA in mycobacteria is responsible for converting the antimicrobial compound pyrazinamide to its active form pyrazinoic acid and is altered in naturally occurring pyrazinamide-resistant strains. In studies by Scorpio and Zhang (1996), M. tuberculosis pyrazinamide-resistant mutants from clinical isolates were examined for common aberrant single amino acid substitutions. Possible structural effects resulting from these substitutions were assessed via the analysis of a three-dimensional model of PncA constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp. The data indicated that, in the mutants, five of the aberrant residues were located within a 6 A sphere around Cys-138, the putative active site of M. tuberculosis PncA (Scorpio and Zhang, 1996).

Based on the above information, the predicted active site for BBE22, Cys-120, was targeted for mutational analysis. pBBE22M, containing a Cys-120Ala single amino acid substitution, was unable to restore infectivity of the lp25 strain 5A13 when injected into mice (Table 3). B. burgdorferi 5A13/pBBE22M also did not grow within DMCs in rats. These results provided definitive evidence that BBE22, and not other small ORFs present in the insert of pBBE22, is the locus in lp25 that is principally required for infectivity.

Complementation of an S. typhimurium pncAnadB mutant by BBE22

To characterize further the biochemical activity of BBE22, two different constructs were electroporated into a Salmonella pncAnadB double mutant: one containing the native B. burgdorferi promoter and a second using the T5 promoter/lac operator of plasmid pQE30. Transformation with either construct permitted growth of the pncAnadB mutant in minimal medium containing nicotinamide, indicating complementation of nicotinamidase activity required for NAD biosynthesis. The somewhat reduced rates of growth of all pBSV2-containing strains in medium containing nicotinic acid (Fig. 3F) may result from deleterious effects of the shuttle vector on the growth of S. typhimurium under these conditions.

In addition, the nicotinamidase activities of cell lysates from the pQE30:BBE22-transformed mutant were also compared with those of wild-type cells and vector-only controls. Release of ammonia from the nicotinamide substrate confirmed that BBE22 conferred nicotinamidase activity. These results demonstrate that BBE22 encodes a nicotinamidase, consistent with its previous designation as pncA based on sequence homology.

BBE22 provides a physiological function in vivo

Although plasmid-encoded genes have long been suspected of being required for the lifestyle of B. burgdorferi, this report is the first definitive demonstration of an activity required for mammalian infection. The fact that BBE22/pncA fulfils a physiological function reinforces the perspective that plasmids harbour vital genes.

The simplest explanation for this observation is that the concentration of nicotinic acid in tissue is too low to maintain the growth of B. burgdorferi lacking BBE22 in infected mice or DMCs, whereas the presence of BBE22 permits the utilization of available nicotinamide as a substrate for NAD production. The normal range of nicotinic acid in human serum is 0.02–0.05 mg l−1 (≈ 0.16–0.41 µM), whereas the concentration in rat serum is reported to be 0.5–1.0 mg l−1 (≈ 4–8 µM) (Baker and Frank, 1968); we were unable to locate values for the nicotinic acid concentration in mice. These ranges are similar to the nicotinic acid concentration in the CMRL culture medium used in BSK-II Borrelia medium (0.2 µM), in which B. burgdorferi lacking BBE22 are able to grow at the same rate as wild-type cells. Thus, the results cannot be interpreted easily with available data. It is possible that BBE22B. burgdorferi are capable of assimilating and using the NAD and NADP that are also present in BSK medium, but do not have access to adequate supplies of NAD or usable precursors during mouse infection. To clarify this issue, experiments are planned to examine the growth of wild-type or BBE22B. burgdorferi in BSK medium lacking NAD or its precursors or with nicotinamide or nicotinic acid alone, as was done with S. typhimurium (Fig. 3).

The guaAB genes of cp26 encode GMP synthetase and IMP dehydrogenase and thus also appear to provide important biosynthetic activities (Margolis et al., 1994). However, most of the other plasmid-encoded proteins have no predicted physiological function and are likely to be involved in some aspect of interactions of B. burgdorferi with either the mammalian or the tick host. OspA (Schwan et al., 1995; Schwan and Piesman, 2000), OspC (Schwan and Piesman, 2000) and the OspE/F/Erp/Elp/Mlp protein families (Stevenson et al., 1995; 1998; Akins et al., 1998; 1999; Yang et al., 1999) are all differentially regulated during different stages of infection or transmission but have no obvious homologues in other bacteria. Recent studies suggest that differential binding of host complement factor H by multiple Erp proteins on the spirochaetal surface may allow B. burgdorferi to resist complement-mediated killing in a wide range of hosts (Hellwage et al., 2001; Stevenson et al., 2002). The gene encoding the VlsE antigenic variation protein undergoes extensive gene conversion during infection of mice (Zhang and Norris, 1998a,b); in addition, loss of lp28-1 containing the vls locus correlates with reduced infectivity (Purser and Norris, 2000; Labandeira-Rey et al., 2001). Coupled with the consistent presence of the same set of plasmids in freshly isolated strains and the rapid loss of many plasmids during in vitro culture, these results indicate that strong selective pressure is responsible for retention of the 21 B. burgdorferi plasmids during the infection cycle.

Future studies

The effective replacement of lp25 with a BBE22-containing plasmid may permit circumvention of the difficulties encountered in the transformation and genetic manipulation of low-passage, infectious B. burgdorferi strains. The efficiency of transformation of B. burgdorferi B31 low-passage clones with pBSV2 is decreased dramatically by the presence of lp25 or another linear plasmid, lp56 (Lawrenz et al., 2002). This effect may result from putative restriction–modification enzymes encoded by the large ORFs BBE02 (on lp25) and BBQ67 (on lp56) (Lawrenz et al., 2002). Lp56 does not appear to play a critical role in infectivity in mice (Purser and Norris, 2000). It may therefore be possible to perform gene inactivation in strains lacking lp25 and lp56, and then complement with shuttle vectors containing BBE22 (with or without the inactivated gene) to determine the roles of other B. burgdorferi genes in infectivity.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains

Borrelia burgdorferi B31 clones were cultured in BSK-II medium with 6% heat-inactivated rabbit serum, and colonies were isolated by subsurface plating, as described previously (Norris et al., 1995). Clone 5A4 contains all 19 plasmids examined, and 5A13 contains all plasmids except lp25 as determined previously by PCR and Southern blot analysis; 5A4 is highly infectious, whereas 5A13 has a low infectivity phenotype (Purser and Norris, 2000). Clone 5A8 was lacking only lp28-1 and exhibits an intermediate infectivity phenotype (Purser and Norris, 2000). B. burgdorferi was transformed with the shuttle plasmid pBSV2 or its derivatives (Stewart et al., 2001) using the conditions described by Lawrenz et al. (2002). Kanamycin (200 µg ml−1) was added to the medium for selection of transformed clones, and the presence of the transforming construct was verified by PCR (Purser and Norris, 2000).

Salmonella typhimurium wild-type strain LT2 and an isogenic pncAnadB mutant (genotype TT400 sty LT2 pncA278::Tn10d-cam nadB215::Tn10) (Zhu et al., 1991) were kindly provided by J. R. Roth and J. Grose of the University of California at Davis. The pncAnadB mutant was grown in Luria–Bertani (LB) medium (Sambrook and Russell, 2000) containing 25 µg ml−1 tetracycline and 20 µg ml−1 chloramphenicol. S. typhimurium containing the constructs pBSV2, pBBE22 or pBBE22M were grown in the presence of 50 µg ml−1 kanamycin. The expression plasmid pQE30 was obtained from Qiagen. S. typhimurium transformed with pQE30 constructs were cultured in the presence of 50 µg ml−1 carbenicillin.

Animal studies

Borrelia strains were tested for infectivity in C3H/HeNHsd (wild type) and C3H/Smn.ClcrHsd-scid mice (Harlan) by intradermal needle inoculation and culture of joint, heart, ear and bladder tissue 2 weeks after inoculation, as described previously (Norris et al., 1995). The median infectious dose (ID50) was determined by inoculation of groups of three mice with serial 10-fold dilutions of organisms and culture of tissues as indicated above (Norris et al., 1995). ID50 values were calculated using the method of Reed and Muench (1938). In some experiments, tibiotarsal joints from mice inoculated with B. burgdorferi were examined for histopathological changes. Tissues were formalin-fixed, decalcified, sectioned and stained with haematoxylin and eosin using standard methods. Specimens were randomly coded, examined in a blinded manner and evaluated for histopathology using the scoring system described by Weis et al. (1999).

For DMC studies (Akins et al., 1998), B. burgdorferi strains were cultured in BSK-II medium at 23°C to mid-log phase and inoculated into DMCs with a molecular cut-off of 8000 Da at a cell density of 103 bacteria ml−1. In some experiments, B. burgdorferi strains lacking lp25 were implanted at 106 bacteria ml−1. The chambers were implanted in the peritoneal cavities of 4- to 6-week-old Sprague–Dawley rats (Harlan Sprague–Dawley) for 12–14 days and analysed microscopically for cell numbers and by SDS-PAGE for protein expression as described previously (Akins et al., 1998).

Recombinant plasmid constructs

Standard molecular biology techniques (Sambrook and Russell, 2000) were used for the preparation of recombinant plasmid pBBE22. Briefly, B. burgdorferi B31-5A4 plasmid DNA was prepared using the Promega Wizard Plus Maxipreps DNA purification system and used as the template for PCRs. A DNA fragment corresponding to lp25 co-ordinates 14 571–16 628 (http:www.tigr.org) was amplified using primers 4735 (5′-GGGGTACCTTTTTATATTGTGAGCCG GTTT-3′) and 4734 (5′-CGGGTACCTCTATGCTATCCCCT TGTTCA-3′) (IDT). Each primer contained a 5′KpnI site (underlined). The shuttle vector pBSV2 (Stewart et al., 2001) and the BBE22 amplicon were each digested with KpnI, followed by dephosphorylation of the vector DNA using calf intestinal alkaline phosphatase (Promega) and by ligation to form the recombinant plasmid pBBE22. E. coli MC290 cells were transformed with pBBE22 by a heat shock method (Chung et al., 1989). Kanamycin-resistant colonies were selected, and PCR was used to detect the presence of the kanr and BBE22 genes. The insert of pBBE22 was sequenced and found to be identical to the previously reported sequence (Fraser et al., 1997).

BBE22 was mutagenized using the Stratagene QuikChange PCR mutagenesis kit. Mutagenic primers 4803 (5′-CGGGACTAGCATTGGATTTTGCTGTAAAAGAAACAAT ACTTGATGC-3′), and 4804 (5′-GCATCAAGTATTGTTTCTT TTACAGCAAAATCCAATGCTAGTCCCG-3′) each span co-ordinates 15196–15241 within BBE22. Underlined nucleotides (corresponding to lp25 co-ordinates 15 220 and 15 221) were changed from CA to GC, resulting in a cysteine to alanine substitution at residue 120 of BBE22. Primers 4803 and 4804 were used to amplify the entire plasmid pBBE22. E. coli TOP10F′ cells (Invitrogen) were transformed with the resulting construct pBBE22M according to the supplier's instructions, and the plasmid insert DNA was sequenced to confirm the mutation. DNA from the resulting plasmid, designated pBBE22M, was electroporated into B. burgdorferi B31 5A13 as described previously (Lawrenz et al., 2002).

pQE30:BBE22 was constructed for the expression of a His6-tagged, recombinant form of BBE22 from a T5 promoter and lac operator in E. coli and S. typhimurium. A modified form of BBE22 containing an additional 48 bp 5′ of the designated start codon (Fraser et al., 1997) was amplified using the primers 4881 (5′-GGGGATCCGCACTTATTTTAATA GATATACAAAATGATTTTTTAG-3′) and 4783 (5′-CCCTG CAGTATATTAAGCTTACTTTGGCTGTCG-3′). This construct thus contained an additional 16 in frame codons from the B. burgdorferi sequence and yielded a product with the 16 amino acids ALILIDIQNDFLESGT added between the His6 tag and the previously designated protein sequence for BBE22. The PCR product and pQE30 vector were each treated with BamHI and PstI; the vector was then dephosphorylated as described above, and the two were ligated. Expression of the product in S. typhimurium or E. coli was induced by the addition of IPTG (1.0 mM) for 2 h.

Complementation in S. typhimurium

The S. typhimurium pncAnadB double mutant was transformed by electroporation with pBSV2, pBBE22, pBBE22M, pQE30 or pQE30:BBE22. The presence of each plasmid was confirmed by PCR. S. typhimurium strains were tested for growth in 96-well plates containing 200 µl each of M9 medium with 0.2% glucose and the indicated concentrations of nicotinamide (NM) or nicotinic acid (NA) (Fluka). LB plates containing the appropriate selective antibiotics were inoculated with the strains to be examined and incubated overnight at 37°C. Approximately 20 isolated colonies per plate were suspended in 1 ml of M9 minimal medium and adjusted to an A600 of 1.0. Samples of 2 µl of each cell type were added to triplicate wells of each medium. Cultures were incubated at 37°C with shaking, and optical densities at 600 nm were measured at 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h and 48 h using a µQuant microplate spectrophotometer (Bio-Tek Instruments).

Quantitative nicotinamidase assay

Salmonella typhimurium transformants were used to examine nicotinamidase activity by a modification of the technique described by Frothingham et al. (1996). Briefly, overnight cultures of wild-type LT2 (positive control), nadBpncA mutant or strains containing pBSV2, pBBE22 or pQE30 were used to inoculate 10 ml of LB + 8 mM nicotinamide. Cultures were grown to OD600 of 0.4–0.5. Cells were centrifuged and washed three times in phosphate-buffered saline (PBS; Sambrook and Russell, 2000), resuspended in 150 µl of PBS at a density of 1 × 108 cells µl−1 and transferred to a 1.5 ml tube on ice. Each cell suspension (10 µl) was added to 90 µl of PBS with or without 8 mM nicotinamide and incubated at ambient temperature for up to 60 min. At each time point, tubes were centrifuged at 13 000 g for 1 min, and supernatants were flash frozen. These supernatants were tested for ammonia concentration using an ammonia kit (Sigma-Aldrich). The protocol was modified for use with 96-well microtitre plates. Samples of 10 µl of each supernatant were added to triplicate wells containing 100 µl of the ammonia reagent buffer. Water was used as a blank, and an ammonia-positive control was included. After taking an initial OD340 reading, 1 µl of glutamate dehydrogenase solution from the kit was added to each well. After 5 min, the OD340 was determined. The final OD340 was subtracted from the initial OD340 readings for all samples, controls and blanks. The value of the blank was then subtracted from the value of each sample and control. The resulting number was multiplied by the calibration factor 30.3, providing the concentration of ammonia in µg ml−1 present in each sample.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We are indebted to J. Grose and J. R. Roth for providing strains and advice for the S. typhimurium studies, and to P. Stewart and P. A. Rosa for providing the B. burgdorferi shuttle vector pBSV2. We thank P. J. McCormick and A. E. Johnson for assistance with cloning pBBE22, and C. Gonzalez, M. Lee, D. Wang, D. J. Botkin, M. A. McLoughlin and M. E. Mosher for technical assistance and advice. This work was supported by National Institutes of Health grants AI37277 (S.J.N.) and AI29735 (J.D.R. and M.J.C.) from the Institute for Allergy and Infectious Diseases and by the Texas Advanced Technology Program grant no. 011618-0236-1999 (S.J.N.).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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