Altered Murine Tissue Colonization by Borrelia burgdorferi followingTargeted Deletion of Linear Plasmid 17-Carried Genes
Timothy Casselli,a Yvonne Tourand,a and Troy Bankheada,b
Department of Veterinary Microbiology and Pathologya and Paul G. Allen School for Global Animal Health,b Washington State University, Pullman, Washington, USA
The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromo-some and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studyinglaboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the lin-ear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirementof these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined both in vitro andin vivo. A mutant strain lacking the genes bbd16 to bbd25 showed no deficiency in the ability to establish infection or dissemi-nate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear,heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This pheno-type was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by anadaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein C in vitro,which was correlated with the absence of the gene bbd18. To our knowledge, this is the first report involving genetic manipula-tion of lp17 in an infectious clone of B. burgdorferi and reveals for the first time the effects of lp17 gene deletion during murineinfection by the Lyme disease spirochete.
The causative agent of Lyme disease, Borrelia burgdorferi, is anobligate parasite that is maintained in nature through an en-
zootic cycle involving a tick vector and mammalian host (10, 33,55). B. burgdorferi has an unusual segmented genome comprisedof a 910-kb linear chromosome and upwards of 23 linear andcircular plasmids ranging in size from 5 to 56 kb (12, 21, 39, 56).The majority of predicted plasmid-borne open reading frames(ORFs) encode genes of unknown function, and their potentialroles in transmissibility, colonization, dissemination, and persis-tence in the mammalian host have yet to be determined.
Much of what is known about the requirement of plasmid-borne genes for infectivity in the mammalian host is derived fromstudies using clonal isolates that have spontaneously lost one ormore plasmids during in vitro cultivation and determining theability of these mutant clones to infect mice in the laboratory (29,32, 40, 48, 53, 63). Selective displacement of B. burgdorferi plas-mids through the introduction of incompatible shuttle vectors hasalso been used as a technique to assess the contribution of certainplasmids to virulence (18, 23).
The loss of several linear plasmids in particular, including lp25,lp28-1, and lp36, has been associated with a loss of infectivity of B.burgdorferi (23, 28, 31, 32, 48). Further studies have localized theseeffects to specific virulence determinant genes carried on theseplasmids, demonstrating the importance of plasmid-borne genesfor both B. burgdorferi pathogenesis and physiological functions invivo (3, 47, 57). Despite the importance of some B. burgdorferiplasmids for murine infection, clones lacking other plasmids, in-cluding cp9, lp5, lp21, lp28-4, lp38, and lp56, have been shown tobe fully infectious in mice, illustrating the variable requirement ofB. burgdorferi plasmids for infectivity in the mouse host (18, 29,32, 48).
While analysis of plasmid loss among B. burgdorferi isolates hasbeen an invaluable strategy for assessing the contribution of plas-mid-borne genes to infectivity, some plasmids are never or rarelyreported to be spontaneously lost during routine culture. There-
fore, limited information about the requirement of these con-served plasmids for infectivity is available. One such plasmid,lp17, is a 16,823-bp linear plasmid carrying 25 putative ORFs (21).While lp17 has occasionally been reported to be lost after extendedin vitro passage (24, 41, 50), this plasmid was not reported to belost in prior studies correlating plasmid loss with infectivity (18,23, 29, 32, 40, 48, 53, 63).
To date, lp17 has been primarily utilized as a tool to examinethe mechanism of linear DNA replication in B. burgdorferi (5, 13).Using targeted plasmid deletion, the majority of this plasmid hasbeen shown not to be required for viability in the noninfectiousstrain B31-A; however, a role for lp17 during infectivity or persis-tence has not been evaluated in an infectious background. Re-cently, many of the genes on lp17 have been shown to undergochanges in expression under culture conditions that mimic eitherthe tick vector or the mammalian host (1, 9, 38, 42, 58). Furtherstudies using mutant strains lacking alternative sigma factors in-volved in regulating the expression of host-specific genes showedsimilar alterations in the expression levels of lp17-borne genes (7,11, 43, 44, 49). During the course of the current study, Sarkar et al.demonstrated that expression of the lp17-borne bbd18 gene in-versely correlated with expression of outer surface protein C(ospC) in a noninfectious strain of B. burgdorferi (52). These pre-vious studies combined with the relative lack of knowledge about
Received 22 September 2011 Returned for modification 17 October 2011Accepted 14 February 2012
the involvement of this plasmid during mammalian infectionmake lp17 an interesting target for further analysis during theinfectious cycle.
In the present study, the effect of lp17 gene deletion on infec-tivity and persistence in the murine host was examined. Deletionof ORFs bbd16 to bbd25 did not affect the ability of B. burgdorferito establish initial infection in mice or to disseminate from the siteof infection to the bloodstream. However, the mutant demon-strated a delayed ability to colonize all peripheral tissues examinedand continued to exhibit reduced bladder colonization for up to56 days postinfection (p.i.). This strain displayed an increasedexpression of OspC in vitro, which correlated with the absence ofthe gene bbd18. Collectively, these results demonstrate a role forlp17 during mammalian infection and strongly support the con-tribution of this plasmid to the regulation of OspC expression.
MATERIALS AND METHODSBacterial strains, culture conditions, and DNA isolation. The B. burg-dorferi isolates B31-A and B31-5A4 (5A4) were kindly provided by GeorgeChaconas via gifts from Patti Rosa and Steven Norris, respectively. Bothare clones of the sequenced B31 type strain (21), and their respectiveinfectivities and plasmid profiles have been previously described (8, 48).These B. burgdorferi isolates were used to generate all mutant clones in thisstudy, as described below. All B. burgdorferi clones were grown at 35°C inmodified Barbour-Stoenner-Kelly medium (BSK-II) supplemented with6% rabbit serum (4). Mutant strains were grown with kanamycin (200�g/ml) or gentamicin (100 �g/ml), as indicated. Cell densities and growthphase were monitored by visualization under dark-field microscopy andcounting using a Petroff-Hausser counting chamber.
Plasmid DNA was isolated from B. burgdorferi cultures using a plas-mid midikit (Qiagen, Valencia, CA) for further analysis and/or use as thePCR template.
Plasmid construction. The deletion plasmid, pYT1014, was generatedthrough the replacement of the original deletion target region from thepreviously described construct pYT1 with DNA corresponding to coordi-nates 9188 to 10113 of lp17 (60). A 1,214-bp region of lp17 was PCRamplified from 5A4 plasmid DNA using primers P43 (5=-CAAATGACTTTTATGAAATTTGG-3=) and P44 (5=-GGAAACATCTATCTGCTTTATG-3=) and subsequently cloned into the vector pCR-Blunt II-TOPO (In-vitrogen, Carlsbad, CA). The resulting construct was digested with therestriction endonuclease EcoRI, in order to recover the 925-bp target re-gion. The discrepancy in size between the original PCR product and thepurified target region was due to the presence of an internal EcoRI site inthe cloned DNA fragment, which did not affect the desired lp17 deletion.
The left-end-deletion construct, pYT1013F, was generated in a similarmanner, using primers P41 (5=-TCACATTATTGTAATTTTTTTTTG-3=)and P42 (5=-GTGAACTTCTACTAACTTGTTTGC-3=) in order to am-plify the target region. In this instance, despite an internal EcoRI sitewithin the PCR product, full-length target was recovered through partialEcoRI digestion and subsequent screening.
After isolation and purification, lp17 target regions were ligated intoEcoRI-digested pYT1 and transformed into Escherichia coli. Plasmids iso-lated from individual E. coli clones were screened by restriction digestionto confirm the presence and correct orientation of the deletion targets.
Complementing plasmids were constructed by PCR amplification ofthe target gene, including its putative native promoter, and cloned intopBSV2G (19) at a unique SalI recognition site (introduced SalI sites areindicated in bold, as shown below). The bbd18 complement was con-structed by PCR amplification of bbd18, including 409 bp of upstreamsequence and 50 bp of downstream sequence, from 5A4 plasmid DNAusing the primers P209 (5=-GCTCGTCGACAAATATTATTTAATAATAATAAATAAAATTAAACG-3=) and P210 (5=-ATCAGTCGACCAGAATTTACTTACAATATTTAACCTTC-3=). The bbd21 complement was con-structed by PCR amplification of bbd21, including 363 bp of upstream
sequence and 50 bp of downstream sequence, using the primers P211(5=-ATCAGTCGACGCTTGTTAATCATGCTATTG-3=) and P212 (5=-GCTCGTCGACGTAGATAATTCTTTATTATTTTTTTG-3=).
Mutant generation and screening. All transformations were per-formed using electrocompetent B. burgdorferi prepared and cultivated aspreviously described (3, 51). Transformed bacteria were recovered for 24h and plated by limiting dilution with antibiotic selection to isolate clonaltransformants. Deletion mutants were initially identified by PCR screen-ing for the kanamycin resistance gene using primers P54 (5=-CATATGAGCCATATTCAACGGGAAACG-3=) and P55 (5=-AAAGCCGTTTCTGTAATGAAGGAG-3=) (8). Clones positive for the kanamycin resistancegene were further analyzed by field inversion gel electrophoresis andSouthern blot analysis to confirm the desired deletion and the absence ofthe parent plasmid. The bbd18- and bbd21-complemented strains wereconfirmed in a similar manner, utilizing primers P91 (5=-CGCAGCAGCAACGATGTTAC-3=) and P92 (5=-CTTGCACGTAGATCACATAAGC-3=) to screen for the gentamicin resistance gene by PCR (59). Prior tomouse studies, B. burgdorferi plasmid content was analyzed by PCR withprimers specific for regions unique to each plasmid, as previously de-scribed (48, 59).
In vitro growth assays. B. burgdorferi was grown to late log phase andsubcultured to a density of 105 cells/ml. Cell densities were determined at24-h intervals and expressed as the mean number of cells from threeexperiments � standard deviation. Mean values for wild-type and mutantclones were compared at each time point using Student’s t test. To com-pare overall stationary-phase cell densities, values from days 9 to 13 werepooled prior to analysis. Differences between groups were considered sta-tistically significant where P was �0.05. Data were analyzed and displayedusing the SigmaPlot (version 11) program (Systat Software, San Jose, CA).
Infection and recovery of B. burgdorferi from mice. All animal infec-tions were carried out in accordance with approved protocols from theInstitutional Animal Care and Use Committee (IACUC) of WashingtonState University (Animal Safety Approval Form 3729). Four-week-oldmale C3H/HeN (C3H; Harlan, Indianapolis, IN) and C3SnSmn.CB17-Prkdcscid/J (SCID; Jackson, Bar Harbor, ME) mice were infected by bothintraperitoneal (91% of infectious dose) and subcutaneous (9% of infec-tious dose) needle inoculation of 100 �l BSK-II containing the indicatednumber of B. burgdorferi cells. Blood, ear, heart, bladder, and joint tissueswere collected at the indicated times and cultured in BSK-II supple-mented with 20 �g/ml phosphomycin, 50 �g/ml rifampin, and 2.5 �g/mlamphotericin B. Dark-field microscopy was used to determine the pres-ence or absence of viable spirochetes for each cultured tissue sample. Asample was deemed negative if no spirochetes could be seen in 10 fields ofview after 4 weeks of culture.
Protein analysis. Total B. burgdorferi cellular proteins were resolvedusing SDS–15% polyacrylamide gel electrophoresis (PAGE). Either gelswere stained directly with Coomassie brilliant blue or proteins were trans-ferred to nitrocellulose for immunoblotting. Nitrocellulose membraneswere incubated overnight at 4°C in Tris-buffered saline (TBS) plus 0.1%Tween 20 (TBST) supplemented with 5% nonfat milk, followed by im-munoblotting (1 h, room temperature) in TBST with primary antisera, asindicated. Primary antibody included mouse anti-5A4 sera (1:1,000), rab-bit anti-FlaB (1:1,000; Rockland Immunochemicals, Gilbertsville, PA),and rabbit anti-OspC (1:1,000; Rockland Immunochemicals). After a se-ries of washes, all samples were then probed with the appropriate second-ary antibody conjugated to horseradish peroxidase (1:5,000; Jackson Im-munoResearch Laboratories, West Grove, PA), washed, and visualizedusing enhanced chemiluminescence.
For identification of unknown protein, proteins were resolved using4% to 20% precast Tris-HCl Ready Gels (Bio-Rad Laboratories, Hercules,CA) and stained directly using Coomassie brilliant blue. The band ofinterest was excised and destained, and gel slices containing the targetprotein were sent to the Mass Spectrometry and Proteomics Core Analyt-ical Facility at the University of Idaho for further processing and analysisusing liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Proteins corresponding to the identified peptides were determined bysearching the Swiss-Prot protein database using Protein Lynx GlobalServer software (Waters Corp., Milford, MA).
RESULTSThe left and right ends of lp17 display different requirementsfor viability of 5A4 in vitro. Previous work by Beaurepaire andChaconas localized the essential region of replication on lp17 to asegment of the plasmid from coordinates 7946 to 9766 containinga single intact gene, bbd14, in addition to some adjacent noncod-ing sequence (5). All of the predicted ORFs upstream or down-stream (or left and right, respectively) of this region were success-fully deleted from lp17, yielding viable mutant clones in vitro inthe noninfectious isolate B31-A. In the present study, the effects oflp17 gene deletion on both in vitro viability and in vivo infectivitywere examined in the infectious B. burgdorferi clone 5A4, whichcontains the full complement of parent plasmids reported in thesequenced B31 isolate (21, 48).
In order to generate left- and right-end lp17 mutants in 5A4, atargeted deletion strategy was utilized (Fig. 1) (3, 5, 13). Briefly,this approach involves the introduction of a replicated telomere atany position within a linear plasmid via integration of a deletionconstruct at a target site for homologous recombination. Deletionconstruct integration is followed by recognition and processing ofthe introduced internal replicated telomere by the endogenous B.
burgdorferi protein ResT (30). Telomere resolution by ResT leadsto the production of a new covalently closed hairpin end, resultingin deletion of the entire plasmid region located upstream ordownstream of the target sequence.
To generate an lp17 right-end deletion, the plasmid pYT1014was constructed (see Materials and Methods). This plasmid con-tains a kanamycin resistance gene, a replicated telomere, and aregion of target DNA homologous to lp17 corresponding to aportion of bbd14-bbd15 (Fig. 1). Strain 5A4 was transformed withthis construct, and clones were isolated under kanamycin selec-tion. As shown in Fig. 1, integration of pYT1014 into lp17 resultsin insertion of the kanamycin resistance gene and the loss of allgenes downstream of the target region, specifically, bbd16 tobbd25. Clones containing the desired mutation were initially iden-tified by PCR screening for the kanamycin resistance gene andconfirmed by visualization of total plasmid DNA separated byfield inversion gel electrophoresis and Southern blot analysis (datanot shown). The mutant clone (denoted 5A4�D16-D25) wasfound to have retained all B. burgdorferi parent plasmids, as deter-mined by PCR analysis (data not shown).
A second deletion construct, pYT1013F, was generated to tar-get the lp17 left-end genes bbd01 to bbd11 for removal (see Fig. 1for lp17 gene arrangement). Numerous attempts at deletion ofthese genes in wild-type 5A4 were unsuccessful. To confirm that
FIG 1 Targeted deletion of bbd16 to bbd25 on lp17. An E. coli plasmid, pYT1014 (light gray), containing a target region of lp17 (arrow, dark gray), a 50-bpreplicated telomere (rtel, hatched lines), and a kanamycin resistance gene (kanR, white) was generated and used to transform B. burgdorferi 5A4. Insertion of theplasmid construct occurs at the target site through a process of homologous recombination. Deletion of lp17 to the right of the target sequence, including ORFsbbd16 to bbd25, follows a telomere resolution event at the replicated telomere. The remaining truncated form of lp17 (lp17�D16-D25) is maintained due to thepresence of the replication initiator bbd14 and confers kanamycin resistance due to the presence of the integrated deletion construct. The 25 predicted ORFs oflp17 are numbered according to their gene designation and depicted as large gray arrows where size allows, with the smallest ORFs indicated by vertical lines only.The two ORFs containing authentic frameshift mutations, bbd20 and bbd23, are represented as white arrows.
the plasmid construct was functional, pYT1013F was tested in theB31-A background as previously described (5), successfully pro-ducing the desired lp17 deletion mutant (data not shown). Addi-tionally, the previously described deletion construct pYT1 (60),which targets the extreme left-end genes bbd01 to bbd04 for dele-tion, was capable of generating the desired mutation in bothB31-A and 5A4 (data not shown).
Collectively, these results demonstrate that bbd16 to bbd25 arenot required for in vitro survival of the infectious B. burgdorfericlone 5A4, which possesses the full complement of parent plas-mids. Despite the ability to generate mutations in both the left andright ends of lp17 in 5A4 (as demonstrated using pYT1 �bbd01-bbd04 and pYT1014 �bbd16-bbd25, respectively), attempts at de-leting bbd01 to bbd11 did not yield viable clones, suggesting amore stringent in vitro requirement for this region. The inabilityto delete the region containing bbd01 to bbd11 appears to be clonespecific, as mutant clones were obtained in the noninfectious iso-late B31-A. The phenotypic effects of the deletion of genes bbd16to bbd25 were further examined both in vitro and in vivo.
B. burgdorferi lacking bbd16 to bbd25 shows impairedgrowth in vitro. Both 5A4�D16-D25 and the wild-type controlstrain were grown in liquid BSK-II to determine how the loss ofbbd16 to bbd25 affects growth during in vitro cultivation.5A4�D16-D25 showed a significantly lower cell density than 5A4at all time points (P � 0.03 for all), with the exception of day 1postdilution, whereby the observed difference was not statisticallysignificant (P � 0.054) (Fig. 2). In addition to decreased cell den-sities at time points throughout the exponential phase of growth,5A4�D16-D25 showed a reduced maximum cell density duringstationary phase (5.85 � 107 � 1.41 � 107 cells/ml) compared towild-type 5A4 (1.33 � 108 � 2.51 � 107 cells/ml) (P � 0.001).
Similarly, swarming ability on semisolid BSK-II agarose plateswas reduced for 5A4�D16-D25 compared to the wild-type strain(data not shown), likely due to differences in growth between thetwo strains upon plating. These results suggest that B. burgdorferilacking bbd16 to bbd25 is somewhat impaired in its ability to rep-licate in vitro.
Deletion of bbd16 to bbd25 leads to altered tissue coloniza-
tion during infection of C3H mice. In order to examine pheno-typic differences in the ability of the mutant strain to infect mice,two groups of 15 C3H mice were needle inoculated with 1.1 � 104
spirochetes of either 5A4 or 5A4�D16-D25. Each group of 15mice was further divided into three groups of five mice (denotedgroup 1, group 2, and group 3) in order to track infection, asshown in Fig. 3.
At day 7 p.i., blood was collected from all mice and culturedfor the presence of spirochetes. Additionally, group 1 micewere sacrificed and ear, bladder, and joint tissues were isolatedand cultured. Spirochetes were successfully cultured from theblood of all mice infected with either 5A4 or 5A4�D16-D25,indicating that bbd16 to bbd25 are not required for establishinginitial infection or disseminating from the inoculation site tothe bloodstream (Table 1).
All bladder and joint tissues of group 1 mice infected withwild-type 5A4 were culture positive for spirochetes, whereas5A4�D16-D25 was not recovered from any joint tissues and wassuccessfully recovered from the bladder tissue of only one mouse(Table 1). The reduced ability to recover the mutant strain frombladder or joint tissues at day 7 p.i. suggests that bbd16 to bbd25may be required for early dissemination and/or colonization oftissues. With the exception of one 5A4-infected mouse, ear biopsyspecimens from all group 1 mice were culture negative regardlessof the infecting strain type.
Infection was tracked weekly in group 2 mice over a 4-weekperiod by culturing ear biopsy specimens (days 14, 21, and 28 p.i.),as well as heart, bladder, and joint tissues (day 28 p.i.). Ear tissuesfrom all group 2 mice infected with wild-type 5A4 were culturepositive at all time points, consistent with previous infection stud-ies using this strain (Table 2) (3). In contrast, spirochetes were notrecovered from ear tissues from any group 2 mice infected with
FIG 2 Deletion of bbd16 to bbd25 leads to decreased growth in vitro. Averagenumber of spirochetes/ml (n � 3) in liquid BSK-II of wild-type 5A4 (blackcircles) and mutant 5A4�D16-D25 (open circles) at the indicated time points.The mutant strain demonstrated a decreased cell density compared to 5A4 atall time points from day 2 to day 13 after the initial subculture. Standarddeviations from the mean are indicated by vertical bars. Means were comparedat each time point using Student’s t test, and statistically significant differencesbetween wild-type and mutant clones are denoted by asterisks (P � 0.05).
FIG 3 Sampling protocol to monitor mouse infection with B. burgdorferi.Groups of either 15 C3H or 10 SCID mice were infected by needle inoculationwith wild-type strain 5A4 (WT) or the mutant strain (�D16-D25). Infectedmice were further divided into groups of five mice each (denoted group 1,group 2, and group 3) as indicated. Blood samples were isolated from all miceat day 7 p.i. and cultured in BSK-II for the presence of spirochetes to confirminitial infection. Infection was tracked by culturing ear biopsy specimens ob-tained at the indicated times. Finally, mice were sacrificed for harvesting oftissues (heart, bladder, joint) at the indicated time points.
5A4�D16-D25 collected at day 14 p.i., and only 3/5 ear biopsyspecimens were culture positive at day 21 p.i. At day 28 p.i., all fivemice infected with 5A4�D16-D25 were culture positive for spiro-chetes from ear tissue. This timeline analysis of ear colonization ingroup 2 mice demonstrates that spirochetes lacking bbd16 tobbd25 are capable of disseminating from the bloodstream andcolonizing ear tissue; however, their ability to do so is delayed.
Interestingly, although wild-type strain 5A4 was recoveredfrom all tissue types at day 28 p.i. in group 2 mice, 5A4�D16-D25was found only in ear, heart, and joint tissues and was not recov-ered from the bladder of any of the infected group 2 mice (Table1). A similar difference in tissue colonization was observed withgroup 3 mice when infection was allowed to progress for 56 daysprior to harvesting of tissues for bacterial culture (Table 1), indi-cating that this phenotype is not restricted to early infection. Ad-ditionally, bladder tissues collected at day 28 p.i. were culture neg-ative for the mutant strain when the infectious dose was increased10-fold to 1.1 � 105 bacterial cells per mouse (data not shown).Taken together, the above-described results demonstrate that al-though B. burgdorferi lacking bbd16 to bbd25 shows delayed dis-semination/colonization of tissues, this mutant strain is capable ofcolonizing and persisting in ear, heart, and joint tissues for up to 8weeks p.i. In contrast, bbd16 to bbd25 are required for successfulcolonization of bladder tissue in C3H mice to the wild-type level,as determined by the ability to recover live spirochetes.
The minimum dose required for infection by a pathogenic or-ganism can be used as an indicator of the ability to evade the host’s
initial defense mechanisms. In order to determine whether loss ofbbd16 to bbd25 resulted in an altered efficiency of infection,groups of five mice were challenged with 10-fold serial dilutions ofeither 5A4 or 5A4�D16-D25. At 28 days p.i., mice were sacrificedand ear, heart, bladder, and joint tissues were cultured for thepresence of spirochetes to determine successful infection as well aspotential differences in tissue colonization. Spirochetes were suc-cessfully recovered from all mice inoculated with a dose of either1.1 � 104 or 1.1 � 103 cells of the wild-type or mutant strain, andthe differential ability to colonize bladder tissue between the twostrains as described above was observed at both infectious doses(Table 3). All tissue types from all mice receiving an inoculation of1.1 � 102 organisms of either strain were culture negative, with theexception of one 5A4�D16-D25-infected mouse that was success-fully infected. These results indicate that loss of bbd16 to bbd25does not significantly alter the number of spirochetes required toestablish infection in mice.
The deficiency in bladder colonization exhibited by5A4�D16-D25 is dependent on an adaptive immune response.In order to investigate the involvement of the murine adaptiveimmune response in the observed B. burgdorferi mutant pheno-types, two groups of 10 SCID mice were needle inoculated with
TABLE 1 Tissue distribution of B. burgdorferi lacking bbd16 to bbd25 in C3H mice
Tissue
No. of culture-positive samples/total no. of samples with the following challenge straina:
5A4 5A4�D16-D25
Group 1 (7)b Group 2 (28) Group 3 (56) Group 1 (7) Group 2 (28) Group 3 (56)
Totald 11/15 20/20 20/20 1/15 15/20 15/20a C3H mice were infected with 1.1 � 104 spirochetes of the indicated strain as described in Materials and Methods. See Fig. 3 for sampling protocol of mouse groups. Fifteen micewere tested with each challenge strain, and all 15 mice were infected.b Numbers in parentheses indicate the day p.i. at which mice were sacrificed and the ear, heart, bladder, and joint tissues were isolated for culture in BSK-II.c NA, heart tissue was not sampled at day 7 p.i., as culture results are confounded due to the presence of infected blood.d Total positive ear, heart, bladder, and joint cultures.
TABLE 2 Timeline analysis of infection by B. burgdorferi lacking bbd16to bbd25 in group 2 C3H mice
Tissuea
No. of culture-positive samples/total no. ofsamples with the following challengestrainb:
5A4 5A4�D16-D25
Blood (7) 5/5 5/5Ear (14) 5/5 0/5Ear (21) 5/5 3/5Ear (28) 5/5 5/5a Ear tissue was isolated for culture in BSK-II at the day p.i. indicated in parentheses.b See Fig. 3 for sampling protocol of group 2 mice. C3H mice were infected with 1.1 �104 spirochetes of the indicated strain as described in Materials and Methods.
TABLE 3 Minimum infectious dose of B. burgdorferi lacking bbd16 tobbd25 in C3H mice
Tissue
No. of culture-positive samples/total no. ofsamples with the following challenge strain atthe indicated challenge dosea:
a C3H mice were infected with indicated B. burgdorferi strain as described in Materialsand Methods. Mice were sacrificed at 28 days p.i., and ear, heart, bladder, and jointtissues were isolated for culture in BSK-II. The challenge dose is in number of cells permouse � 0.91.
1.1 � 104 spirochetes of either clone 5A4 or clone 5A4�D16-D25.Each group of 10 mice was further divided into two groups of fivemice (denoted group 1 and group 2) in order to track infection, asillustrated in Fig. 3.
Consistent with the results seen in C3H mice, spirochetes wererecovered from the blood of all SCID mice infected with either themutant or wild-type control strain (Table 4). Ear, bladder, andjoint tissues harvested at day 7 p.i. from group 1 SCID mice alsoshowed a similar pattern of tissue colonization seen with C3Hmice. Spirochetes were recovered from 9 out of the 10 5A4-in-fected bladder and joint tissues tested, with one joint tissue spec-imen being culture negative (Table 4). In contrast, only one5A4�D16-D25-infected joint tissue specimen was culture posi-tive, while all other mutant-infected tissues were culture negative.Ear tissue was not a consistent source of spirochetes from eitherstrain at day 7 p.i., with only one 5A4-infected ear tissue samplebeing culture positive. These results indicate that the deficiency inearly tissue colonization by 5A4 lacking bbd16 to bbd25 is notdependent on a functional adaptive immune system.
In contrast, the defect in bladder colonization observed at day28 p.i. for the mutant in C3H mice was not observed in SCID mice,as both wild-type and mutant strains were recovered from all tis-sue types of all group 2 SCID mice (Table 4). The detectable pres-ence of live mutant spirochetes in the bladder tissues of SCID micebut not C3H mice suggests that the reduced ability of 5A4�D16-D25 to colonize the bladder is due to the presence of a murineadaptive immune response.
Deletion of bbd16 to bbd25 leads to altered in vitro expres-sion of the known surface immunogen OspC. The dependency ofthe observed differences in bladder colonization on the presenceof an adaptive immune system suggested a possible change in theexpression of surface immunogens. The in vitro protein expres-sion profiles of both 5A4 and 5A4�D16-D25 were examined toaddress this possibility. The two strains were lysed in order toobtain proteins for analysis by SDS-PAGE. Both Coomassie-stained gels and anti-5A4 Western blots showed similar proteinexpression profiles between strains, with one notable exception.An additional band corresponding to a protein of approximately
20 kDa in size was visible in lysates from the mutant strain but wasnot detectable in wild-type 5A4 (Fig. 4A). The isolated proteinband was analyzed by mass spectrometry and identified to beOspC. Western blot analysis of cell lysates using an anti-OspCantibody confirmed increased levels of OspC in the mutant strainlacking bbd16 to bbd25 (Fig. 4B), suggesting that the deleted genesmay be involved in the negative regulation of OspC. Less strik-ingly, Coomassie-stained SDS-polyacrylamide gels also consis-tently showed a band corresponding to an unknown �66-kDaprotein with a slightly increased intensity in the mutant straincompared to wild-type 5A4; however, this band was not visualizedin the anti-5A4 Western blot.
Complementation of 5A4�D16-D25 with bbd18 leads to re-versal of the mutant OspC expression profile in vitro. A sche-matic representation of the 10 predicted ORFs deleted in5A4�D16-D25 is shown in Fig. 1. Of these ORFs, six are extremelysmall and are predicted to code for proteins of between 3 kDa and10 kDa. Of the four remaining ORFs, bbd20 and bbd23 have beenreported to contain authentic frameshift mutations, while bbd18and bbd21 are predicted to encode full-length proteins of �24 kDaand �27 kDa, respectively (21). The protein product coded for bybbd21 belongs to paralogous family 32 (PF32), a family of geneswith sequence homology to genes involved in plasmid DNA par-titioning, and previous studies have provided some evidence forthis function (5, 17). Given the information presented above,bbd18 was an attractive candidate for a single-gene complementa-tion of 5A4�D16-D25.
FIG 4 Increased expression of OspC correlates with the absence of bbd18 invitro. (A) Coomassie brilliant blue-stained gel (left) and anti-5A4 Western blot(right) of whole-cell protein preparations from wild-type strain 5A4 (WT) and5A4�D16-D25 (�D16-D25), as indicated. The overall protein expression pro-file was similar for both strains; however, one noticeable additional �20-kDaband was visible for the mutant strain, as noted by the asterisks. This band wasidentified as OspC using LC-MS/MS. Increased intensity of an �66-kDa bandwas also observed for the mutant strain in the Coomassie-stained gel, as indi-cated by the arrowhead. Numbers to the left of the gels are molecular masses(in kilodaltons). (B) Anti-FlaB (top) and anti-OspC (bottom) Western blots ofwhole-cell lysates of the wild-type, �D16-D25, and 5A4�D16-D25 clonescomplemented with either bbd18 or bbd21 in trans (�D18 and �D21, respec-tively). Although FlaB levels are comparable for all strains, OspC is detectableonly in strains lacking bbd18.
TABLE 4 Tissue distribution of B. burgdorferi lacking bbd16 to bbd25 inSCID mice
Tissue
No. of culture-positive samples/total no. of samples with thefollowing challenge straina:
5A4 5A4�D16-D25
Group 1 (7)b Group 2 (28) Group 1 (7) Group 2 (28)
Totald 10/15 20/20 1/15 20/20a SCID mice were infected with 1.1 � 104 spirochetes of the indicated strain asdescribed in Materials and Methods. See Fig. 3 for sampling protocol of mouse groups.Ten mice were tested with each challenge strain, and all 10 mice were infected.b Numbers in parentheses indicate the day p.i. at which mice were sacrificed and ear,heart, bladder, and joint tissues were isolated for culture in BSK-II.c NA, heart tissue was not sampled at day 7 pi, as culture results are confounded due tothe presence of infected blood.d Total positive ear, heart, bladder, and joint cultures.
The entire bbd18 gene along with its putative native promoterelement was cloned into the self-replicating B. burgdorferi shuttlevector, pBSV2G, which confers gentamicin resistance (19). Al-though the complemented strain would possess both kanamycinand gentamicin resistance genes (from the original deletion con-struct and complementing plasmid, respectively), attempts at re-covering transformants under both kanamycin and gentamicinselection were unsuccessful. Interestingly, transformants weresuccessfully recovered under gentamicin selection alone. Indeed,although the complemented strain was fully resistant to gentami-cin, all recovered clones remained kanamycin sensitive. PCR andSouthern blot analysis for both the kanamycin resistance gene andthe left end of lp17 showed that all bbd18-complemented cloneshad lost the truncated version of lp17 (data not shown). Unfortu-nately, all complemented clones had also lost lp25 containing thevirulence-determinant gene bbe22, rendering them unusable formouse infection studies. The remaining plasmid profile variedbetween recovered bbd18-complemented clones. An equivalentstrategy was utilized to complement the mutant strain with bbd21,including its native promoter. Similar to the bbd18-comple-mented clones, the majority of bbd21-complemented transfor-mants had lost the original lp17 deletion construct (data notshown).
As deletion of bbd16 to bbd25 resulted in the upregulation ofOspC in vitro, the mutant strain complemented with either bbd18or bbd21 was examined for levels of OspC expression. Westernblot analysis of whole-cell lysates using an anti-OspC antibodyshowed that complementation with bbd18 restored OspC expres-sion to below detectable levels (Fig. 4B). Furthermore, the bbd18-complemented strain showed a level of OspC expression belowthat of wild-type 5A4, as seen in Western blots using increasedamounts of protein sample or by overexposing the chemilumines-cence film during detection (data not shown). Conversely, com-plementation with bbd21 did not affect OspC levels compared tothose of 5A4�D16-D25, indicating that the rescued phenotypeseen for bbd18 is not an artifact of the complementing plasmidbackbone or a general effect of restoring any of the 10 deletedORFs in 5A4�D16-D25 (Fig. 4B). Although it is not knownwhether OspC expression is influenced by the remainder of thedeleted portion of lp17, these results demonstrate that bbd18 issufficient for the negative regulation of OspC in vitro.
DISCUSSIONB. burgdorferi lp17-borne genes are necessary for efficient tissuecolonization. This study reports the first genetic manipulation oflp17 in a fully infectious strain of B. burgdorferi and the resultingphenotypes both in vitro and in vivo. A mutant B. burgdorferistrain lacking ORFs bbd16 to bbd25 showed no reduction in theability to establish initial infection and disseminate to the blood-stream in immunocompetent or SCID mice, despite a detectablegrowth deficiency in vitro. Once initial infection was established,however, this mutant strain was delayed in its ability to colonizetissues regardless of the presence of a fully formed adaptive im-mune response. It is possible that the delayed ability to reach de-tectable bacterial levels in vivo might be linked to the observed invitro growth deficiencies. Alternatively, a 10-fold increase in inoc-ulum had no effect on the ability to recover mutant spirochetesfrom murine tissues at weeks 1 and 2 postinfection, perhaps sug-gesting that the observed delay is not due solely to growth defects.The mutant could be recovered from all tissues except bladder
tissue during persistent infection of immunocompetent mice, re-gardless of the infectious dose. This bladder tissue colonizationphenotype was not observed in SCID mice, suggesting that it wasdependent on the presence of an adaptive immune response. Fi-nally, the B. burgdorferi mutant lacking bbd16 to bbd25 showedincreased in vitro expression of OspC, and complementation stud-ies showed that the presence of bbd18 is sufficient for the negativeregulation of OspC protein expression in this mutant strain.
Genetic elements on the left end of lp17 are important forviability in fully infectious B. burgdorferi. The ability to deletelarge portions of lp17, including all predicted ORFs upstream ordownstream of the putative replication initiator bbd14, has beenpreviously demonstrated in the noninfectious isolate B31-A (5).Additionally, individual clones of serially passaged B. burgdorferistrain B31 that have reportedly lost lp17 altogether in conjunctionwith other plasmids have been isolated (24, 41, 50). Collectively,these studies provide evidence that lp17 is not essential for B.burgdorferi survival in vitro. Successful deletion of bbd16 to bbd25in 5A4 as reported here is consistent with the notion that lp17 isdispensable in culture.
Conversely, several attempts at deleting ORFs bbd01 to bbd11in 5A4 were unsuccessful, despite using a similar strategy as pre-viously reported (5). To ensure a proper experimental design, de-letion of bbd01 to bbd11 was attempted in B31-A, with success.The reason for the discrepancy between the ability to generate theknockout in B31-A and 5A4 is not known. One possibility is themuch higher transformation efficiency in B31-A than 5A4. Thisexplanation is unlikely, however, as the ability to consistently gen-erate other targeted deletions on lp17 would suggest that thetransformation efficiency of 5A4 is sufficient. Another possibleexplanation is that the left-end target region from coordinates7621 to 8982 is specifically not amenable to genetic manipula-tions. However, the ability to target the identical region on lp17 inclone B31-A suggests that there is nothing inherent to the left-endtarget sequence that would resist genetic manipulation. A finalpossibility is that other unknown differences between B31-A and5A4 may dictate the ability to delete genes upstream of bbd14.Interplay between genes located on lp17 and other plasmids in5A4 may not easily permit the loss of some regions of lp17 in thepresence of the full complement of 5A4 plasmids. As B31-A lacksat least 9 plasmids found in 5A4, such restrictions may not exist.As the collection of laboratory strains used to study B. burgdorferiis heterogeneous in terms of plasmid content, these results illus-trate the importance of considering strain background when in-terpreting results.
B. burgdorferi lp17-borne genes are important for bladdercolonization via adaptive immune avoidance. The urinary blad-der has been well documented to be a consistent source of B.burgdorferi in both experimentally infected and naturally exposedrodents (16, 22, 45, 54). Thus, the finding that B. burgdorferi lack-ing bbd16 to bbd25 could be detected from all peripheral tissuesexcept the bladder is surprising. The most logical explanation forthis observation is a change in the surface protein profile of themutant strain resulting in altered immunological pressures or re-ceptor-ligand interactions not conducive to bladder colonization.Of these possibilities, an altered profile of surface immunogens inthe mutant strain seems most likely, as 5A4�D16-D25 was capableof bladder colonization in SCID mice. Liang et al. previously dem-onstrated that peripheral tissues provide a protective niche froman adaptive anti-B. burgdorferi response and that the bladder and
heart tissue provided a lower level of protection than joint andskin tissue (34). As 5A4�D16-D25 demonstrated altered OspCexpression levels in vitro, aberrant regulation of this or other im-munogens may lead to a selective and more pronounced reduc-tion of mutant spirochetes in bladder tissues over tissues at othersites. Liang et al. also demonstrated that while anti-OspC antibod-ies reduce the proportion of OspC-producing spirochetes inheart, skin, and joint tissues, these antibodies effectively reducespirochete burdens only in the heart (35, 37). The same groupreported unpublished observations that a similar phenotype isobserved in bladder tissues, leading to the hypothesis that the rel-atively low spirochete burden in the bladder compared to the skinand joints of immunocompetent mice is most likely due to astrong anti-OspC immune response (34). Further experiments areneeded to determine whether the reduced ability of 5A4�D16-D25 to colonize bladder tissues is related to the increased in vitroexpression of OspC or other immunogens and to determinewhether longer-term infections will result in immune clearance inother mouse tissues.
Although a role for an immune-based mechanism for the de-fective bladder tissue colonization observed for 5A4�D16-D25 ismore compelling, altered tissue-specific adhesin/receptor-ligandinteractions cannot be ruled out. Several B. burgdorferi outermembrane proteins that bind to host factors, presumably facili-tating tissue invasiveness and colonization, in addition to immuneevasion, have been described (2, 6, 14, 15, 20, 25–27, 46). It ispossible that one of the protein products of bbd16 to bbd25 acts asa tissue-specific adhesin/receptor whose deletion results in alteredbladder colonization by the mutant strain.
An important step in characterizing the mechanisms of alteredbladder colonization described above will be identification of theindividual lp17-borne gene(s) involved. Unfortunately, attemptsat single-gene complementation in this study repeatedly resultedin the loss of the original lp17 deletion construct in conjunctionwith other plasmids, including lp25, resulting in noninfectiousclones or those lacking numerous genes that could confound in-fection results. Thus, future single-gene mutational analysis willprovide a more appropriate method for further investigation ofthis phenotype.
The lp17-resident bbd18 gene is involved in OspC regulationin vitro. The surface-exposed immunogen OspC must be down-regulated upon establishment of initial mammalian infection toavoid clearance by a robust adaptive immune response (35–37,62). However, the negative regulator(s) involved in this down-regulation of OspC has remained elusive. A previous study exam-ined the in vitro expression of OspC in high-passage-number B.burgdorferi B31 clones lacking several linear plasmids (50). In-creased expression of OspC correlated with the absence of lp17,suggesting the possible presence of a negative regulator on thisplasmid. During the course of the present study, Sarkar et al. con-firmed this hypothesis by introducing portions of lp17 into onesuch clone, B312, which lacks all B31 plasmids except cp26, lp54,and several cp32 plasmids (52). It was found that fragments con-taining bbd18 were sufficient for reducing in vitro expression ofOspC mRNA and protein to wild-type levels in this clone, leadingto the suggestion that bbd18 may be acting to suppress OspC pro-duction. Because this study involved a clone that lacked the ma-jority of B. burgdorferi B31 plasmids, the question as to whetherthe absence of bbd18 would have the same effect on OspC expres-sion in an infectious clone with the full complement of parent
plasmids remained. The data reported here strongly support this,as the in vitro expression of OspC in B31 lacking only ORFs bbd16to bbd25 correlated with the presence of bbd18 but not bbd21. Itcannot be ruled out, however, that the influence of bbd18 on theexpression of OspC may differ in the presence of the nine otherdeleted ORFs of lp17. Further studies using a bbd18 single-geneknockout are needed to distinguish these possibilities.
Given these new in vitro reports suggesting the ability of bbd18to directly or indirectly suppress OspC production, it is temptingto speculate that this is the mechanism of OspC downregulationduring persistent mammalian infection. If this is the case, the abil-ity of 5A4�D16-D25 to persist in mice for 56 days postinfection issomewhat surprising. A previous study reported that spirochetesconstitutively expressing ospC could be consistently recoveredfrom mice during the first 8 weeks of infection and were clearedonly by 11 weeks postinfection (62). A second study using a dif-ferent genetic model system reported clearance of constitutivelyospC-expressing spirochetes by 3 weeks postinfection (61). It isnot currently known how the loss of bbd18 affects ospC expressionduring persistent infection or how the infection results reportedhere for 5A4�D16-D25 relate to previous studies of B. burgdorferistrains unable to downregulate ospC.
A recent study by Xu et al. demonstrated that transcription ofospC is regulated during persistent infection via the presence of alarge cis-acting operator region consisting primarily of two in-verted repeat DNA sequences located immediately upstream ofthe ospC promoter (61). Such cis-acting elements can act as sitesfor the binding of specific transcriptional repressor proteins. In-terestingly, it was found that although deletion of the ospC oper-ator region abolished the ability of B. burgdorferi to downregulateospC during persistent infection, this palindromic sequence is notinvolved in regulation of ospC expression in vitro (61). Thus, de-letion of bbd18 appears to have a different in vitro effect on ospCexpression than deletion of the ospC operator region. It is possiblethat the bbd18-dependent downregulation of ospC in vitro re-ported here and by Sarkar et al. (52) occurs through a yet uniden-tified mechanism different from that reported by Xu et al. duringmammalian infection (61). Further studies investigating the ex-pression pattern and mode of action of bbd18 will likely identifythe stage at which bbd18 regulates ospC during the B. burgdorferiinfectious cycle.
Despite numerous previous studies investigating the involve-ment of plasmid-borne genes during the infectious cycle of B.burgdorferi, the role of lp17-borne genes during mammalian in-fection has remained unknown. This study has demonstrated forthe first time the effects of lp17 gene deletion on murine infectionby B. burgdorferi. Although the relevance of the observed pheno-types to B. burgdorferi pathogenesis is not yet known, future stud-ies investigating the mechanism of impaired bladder colonizationin the mutant strain will allow a better understanding of the mech-anisms of B. burgdorferi persistence during mammalian infection.Furthermore, the targeted deletion strategy described herein isbeing used in conjunction with more traditional gene deletionmethods to further define the role of other lp17 genes duringmammalian infection. This highly conserved yet relatively un-characterized plasmid has now been shown to encode at least oneregulator of a known mammalian virulence determinant, in addi-tion to being required for efficient colonization of murine tissues.Thus, lp17 remains an attractive target for future studies involvingthe Lyme disease spirochete.
We thank George Chaconas for the strains used in this study.This work was supported by an intramural grant from the College of
Veterinary Medicine at Washington State University.
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