Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles of Borrelia burgdorferi

Bacterial strains and growth conditions.A clonal, noninfectious isolate of B. burgdorferi strain B31 (ML23, Table 1), lacking linear plasmid 25 (lp25 negative), was used for the overexpression of CsrA_Bb (44, 45, 65). All B. burgdorferi cultures used for transformations were grown in 1% CO₂ at 32°C in Barbour-Stoenner-Kelly II (BSK-II) liquid medium (pH 7.6) supplemented with 6% normal rabbit serum (Pel-Freez Biologicals, Rogers, AR) (51, 52). In order to determine the levels of synthesis of CsrA_Bb under different host-specific conditions, we also propagated various B. burgdorferi strains (Table 1) to a density of 5 × 10⁷ spirochetes/ml in BSK-II growth medium that mimicked the tick midgut before (pH 7.6 and 23°C) and after (pH 6.8 and 37°C) a blood meal to determine if these environmental signals altered levels of CsrA. Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) and Rosetta(DE3)pLysS (Novagen, Madison, MI) strains were used for all procedures involving cloning and overexpression of recombinant proteins, respectively. The E. coli strains were cultured in Luria-Bertani (LB) broth supplemented with appropriate concentrations of antibiotics (27).

Expression of recombinant CsrA_Bb.Total genomic DNA obtained from B. burgdorferi clonal isolate MSK5 (Table 1) was used as a template to PCR amplify the full-length csrA_Bb by using the primers (csrAF and csrAR) listed in Table 3 containing appropriate engineered restriction enzyme sites. The amplicon was cloned into pCR2.1-TOPO vector (Invitrogen), transformed into E. coli TOP10 cells, and subjected to blue/white colony screening in the presence of ampicillin (100 μg/ml) and kanamycin (50 μg/ml). The insert was excised with NdeI/XhoI and ligated into pET23a expression vector. The ligated products were electrotransformed into E. coli TOP10 cells and screened by restriction enzyme digestion for the presence of the insert of the appropriate size. The junction of the plasmid containing the insert of the expected size was sequenced, and the plasmid, designated pES16 (Table 2), was used to transform the E. coli expression host (Rosetta; Novagen).

Detection of glycogen in E. coli overexpressing CsrA_Bb.E. coli (Rosetta) carrying the plasmid pES16 (Table 2) or the empty vector pET23a was propagated overnight in solid Kornberg's medium supplemented with 50 mM glucose, 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), and ampicillin (100 μg/ml) and stained for endogenous glycogen with iodine vapor as described previously (70).

Purification of rCsrA_Bb and generation of anti-CsrA_Bb serum.Recombinant CsrA (rCsrA_Bb) with a C-terminal six-histidine tag was overexpressed by inducing E. coli strains containing pES16 (pET23a-csrA_Bb) with 1 mM IPTG for 2 h (27). The bacterial pellets were disrupted using a French press in denaturing lysis buffer (8 M urea, pH 8), and the supernatants were collected, clarified by centrifugation, and subjected to affinity purification using nickel-nitrilotriacetic acid beads (Qiagen, Valencia, CA) per the manufacturer's instructions. The bound six-histidine-tagged proteins were eluted as 0.5-ml fractions with elution buffer (8 M urea, pH 4.5). The eluted proteins were analyzed on a sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel. Select fractions with the largest concentrations of eluted proteins were further purified using Amicon centrifugal filters (Millipore, Bedford, MA) to concentrate the proteins as well as to remove urea used in the purification process. An approximately 13-kDa protein was purified to homogeneity (data not shown), quantified by bicinchoninic acid assay (Pierce Thermo Fisher Scientific, Rockford, IL), and stored at −80°C until further use. The predicted molecular mass of CsrA is 9.53 kDa, but the rCsrA migrates as a 13-kDa protein, which could be attributed, in part, to the presence of the six-histidine tag. The purified rCsrA emulsified in equal volumes of Titermax (Sigma, St. Louis, MO) was used to immunize 6- to 8-week-old female BALB/c mice, and booster immunizations were given at days 14 and 21. Immunoblot analysis revealed the presence of specific antibodies to purified rCsrA_Bb in the serum obtained on day 28 postimmunization (data not shown). Infection-derived serum was obtained from C3H/HeN mice at 8 weeks postinfection following intradermal inoculation with 1 × 10³ spirochetes (MSK5, Table 1) via needle inoculation. All animal procedures were done in accordance with the approved animal use protocol from the Institutional Animal Care and Use Committee of the University of Texas at San Antonio.

Overexpression of CsrA in B. burgdorferi.The insert from pES16 (Table 2) containing the full-length CsrA_Bb with the six-histidine tag was used as a template with primers having engineered restriction enzyme sites NdeI and NotI (csrAF and csrAR2, Table 3). The amplicon was cloned into pCR2.1, digested with NdeI and NotI, and recloned into pML102 so as to facilitate expression of CsrA_Bb under the control of a constitutive borrelial promoter, P_flgB. In order to overexpress CsrA_Bb in B. burgdorferi, P_flgB-csrA_Bb-His tag was cloned into the borrelial shuttle vector pBSV2 using restriction enzymes that flank P_flgB-csrA-His tag in pCR2.1. The plasmid designated pES25 was used to transform a clonal isolate of B. burgdorferi strain B31 (ML23; lp25 negative) as described previously (51, 72). The transformants were selected on BSK-II agar overlay supplemented with kanamycin (200 μg/ml) and checked for the presence of pBSV2. The level of expression of CsrA_Bb in B. burgdorferi transformants was also verified by immunoblot assay using monospecific anti-CsrA serum.

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis.B. burgdorferi whole-cell lysates were prepared and separated on SDS-12.5% polyacrylamide gels as described previously (51, 52). The separated proteins were either visualized by Coomassie brilliant blue staining or transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Hybond-P; GE Healthcare, Buckinghamshire, United Kingdom) and subjected to immunoblot analysis. The membranes were probed with anti-OspA monoclonal antibodies, mouse anti-OspC, anti-BBK32, anti-DbpA, anti-SodA, anti-P66, and rat anti-BBA64 (15) and rabbit anti-NapA serum. The blots were developed following incubation with appropriate dilutions of horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-rat secondary antibodies using ECL Western blotting reagents (GE Healthcare).

Morphology of B. burgdorferi.The control strain (ML23/pBSV2) and overexpression strain (ES25) were grown to a density of 5 × 10⁷ spirochetes/ml and washed three times in Hanks balanced salt solution-5 mM CaCl_2-50 mM sucrose and evaluated using the Live/Dead BacLight bacterial viability kit (Molecular Probes, Invitrogen, Carlsbad, CA) in conjunction with confocal microscopy (27). The images were captured using a Zeiss LSM510 microscope and deconvolved using AutoQuantX (MediaCybernetics Inc., Bethesda, MD).

RNA extraction.RNA was extracted as previously described (73). Briefly, B. burgdorferi cultures were grown to a density of 2 × 10⁷ to 3 × 10⁷ spirochetes per ml and RNA was extracted by resuspending the bacterial pellets in RNA-Bee (Tel-Test, Inc., Friendswood, TX) at a ratio of 0.2 ml to every 10⁶ cells. Following extraction with chloroform, the RNA was precipitated with isopropanol, washed with 75% ethanol, air dried, and resuspended in RNase-free water. The RNA was treated twice at 37°C for 45 min with DNase I to remove any contaminating DNA, and the total RNA was quantified spectrophotometrically. In order to evaluate the purity of the RNA sample, real-time PCR was done using recA primers (recAFq and recARq) to detect contaminating DNA (55, 80). The RNA samples devoid of contaminating DNA were reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Real-time PCRs were set up with SYBR green PCR master mix with various oligonucleotide primers (Table 3) specific to ospC, ospA, dbpA, p66, BBA64, flaB, and BBK32 at a final concentration of 100 nM, and quantitative real-time PCR was done using the ABI Prism 7300 system (Applied Biosystems). The induction of each gene in ES25 relative to that in MSK5 or ML23/pBSV2 was normalized to the levels of recA as previously described (55, 80). The threshold cycle (C_T) values of each of the genes from three independent experiments were averaged following normalization, and the levels of induction were determined with the ΔΔC_T method, where the quantity of each transcript was determined by the equation 2^−ΔΔCT, where C_T is the cycle number of the detection threshold, as described previously (42, 73). To determine whether our real-time reverse transcription-PCR (RT-PCR) data were statistically significant, the differences between the normalized C_T values obtained for ES25 and those for the ML23/pBSV2 or MSK5 strain were subjected to a two-way analysis of variance followed by the Bonferroni post hoc test implemented in PRISM. Statistical significance was accepted when the P values were less than 0.05.

Analysis of genomic organization of csrA_Bb in the borrelial chromosome.Total RNA was extracted from MSK5 as described above, and RNA samples devoid of contaminating DNA were reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). A primer specific to the 3′ end of csrA_Bb (csrR, Table 3) was used in conjunction with primers specific to 200 bp (200bpF, Table 3) and 1,000 bp (regF, Table 3) upstream of csrA_Bb that were predicted to generate 1,157-bp and 438-bp amplicons, respectively. In addition, a primer specific to the 5′ end of csrA (csrAF, Table 3) was used with a primer (regR, Table 3) corresponding to the downstream region of csrA_Bb that would result in an amplicon of 1,163 bp. The total RNA (reverse-transcriptase-negative [−RT] control) and the cDNA generated from the total RNA from MSK5 were used as the template to determine if csrA_Bb was cotranscribed with the members of a previously characterized flgK motility operon (34). Total genomic DNA from MSK5 served as the positive control for the expected size of the amplicons that would indicate if csrA_Bb was organized within the flgK motility operon or was under the control of an independent promoter.

Overexpression of CsrA_Bb in E. coli inhibits glycogen biosynthesis/accumulation.The ORF BB0184 present in the linear chromosome of B. burgdorferi has been annotated as a homolog of CsrA/RsmA (32). In several bacterial species, CsrA/RsmA has been characterized as a small RNA binding protein capable of regulating multiple metabolic and virulence mechanisms via precise interactions with its cognate small RNA molecules CsrB and CsrC (7, 53). Amino acid sequence analysis of borrelial CsrA indicated significant sequence conservation and the presence of two regions that have been biochemically shown to contain several critical residues (Fig. 1A) that mediate the interaction of CsrA with its cognate small RNA molecule in E. coli (53). An interesting feature of the borrelial CsrA in all three sequenced species (B. burgdorferi strain B31, Borrelia afzelii PKo, and Borrelia garinii PBi) is the presence of an additional 7 amino acids at the C terminus, even though the exact contribution of these residues to the function of CsrA_Bb remains to be determined. Seminal studies of E. coli indicated that csrA negatively regulates expression of genes involved in biosynthesis of glycogen such as glgB (encoding glycogen branching enzyme), glgC (encoding ADP-glucose pyrophosphorylase), and pckA (encoding gluconeogenic enzyme phosphoenolpyruvate carboxy kinase), which results in reduced levels of glycogen synthesis and accumulation when levels of CsrA are elevated upon expression from a high-copy-number plasmid (70). Even though no typical homologs of the aforementioned genes involved in glycogen biosynthesis could be identified in B. burgdorferi based on sequence analysis, we determined if CsrA_Bb would inhibit the synthesis and accumulation of glycogen when expressed in E. coli. When the E. coli Rosetta strain carrying pES16 (pET23a/csrA_Bb) was propagated in solid Kornberg's medium supplemented with 50 mM glucose and 1 mM IPTG, there was reduced accumulation of glycogen as determined by the exposure to iodine vapor compared to that in the E. coli Rosetta strain carrying the empty vector pET23a (Fig. 1B). This “complementation” analysis (in the presence of an intact copy of csrA in the E. coli Rosetta strain) demonstrated that CsrA_Bb has several features similar to that of the E. coli CsrA to mediate the suppression of glycogen biosynthesis, even though this may not be its primary role in the pathophysiology of B. burgdorferi.

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FIG. 1.

Overexpression of CsrA_Bb results in inhibition of glycogen synthesis in E. coli. (A) Sequence analysis of CsrA_Bb revealed significant conservation of amino acid residues with the CsrA homologs present in various bacterial species. Several residues (*) within the two subdomains (indicated by boxes) that have been shown to be critical for binding of CsrA to small noncoding RNAs in other bacterial species are conserved in CsrA_Bb (53). (B) Overexpression of CsrA_Bb inhibits glycogen synthesis in E. coli. The E. coli Rosetta expression host containing either pES16 (pET23a/csrA_Bb) or pET23a alone was grown overnight on solid Kornberg's medium supplemented with 50 mM glucose and 1 mM IPTG at 37°C and exposed to iodine vapor, and the intense brown coloration of the colonies is indicative of the endogenous glycogen levels.

csrA_Bb is part of the flgK operon of B. burgdorferi.Bioinformatic analysis of the genomic location of csrA in three different borrelial strains that have been sequenced revealed that it is downstream of several ORFs associated with motility. Ge et al. (34) demonstrated that the flgK motility operon consists of flbF (BB0180; unknown function), flgK (BB0181, encoding HAP1), flgL (BB0182, encoding HAP3), and orfX (BB0183, encoding FliW). The analysis of the whole-genome sequence of B. burgdorferi facilitated the annotation of the ORF immediately downstream of what was characterized as the flgK motility operon as csrA (BB0184). In order to determine if csrA_Bb is indeed a part of the flgK operon, we performed RT-PCR using primers specific to csrA_Bb and to regions up- and downstream of csrA_Bb similar to procedures described previously (34, 62). As shown in Fig. 2A, when primer 2 (200bpF, within BB0183) and primer 4 (csrAR) were used with the total cDNA from MSK5, there was an amplification of the 431-bp fragment (Fig. 2B, lane 2) consistent with the size of the fragment generated using the total genomic DNA (Fig. 2B, lane 3). This observation indicated that csrA_Bb was part of the flgK operon and is cotranscribed with its upstream neighbor, BB0183. Similarly, primer 1 (regF, within BB0182; Table 3) and primer 4 (csrAR, Table 3) amplified an 1,154-bp fragment when the total cDNA was used (Fig. 2B, lane 6) consistent with the size of the amplicon generated using total genomic DNA (Fig. 2B, lane 7). This observation further confirmed that csrA_Bb is indeed a part of the flgK operon, as it is cotranscribed with BB0182 (flgL). There was no amplification when either of the primer sets (primers 1 and 4 or primers 2 and 4) was used with the total RNA (−RT control) as the template (Fig. 2B, lanes 1 and 5, respectively). Moreover, when primer 5 (regR, Table 3) was used with primer 3 (csrAF, Table 3), there was no amplification with the cDNA generated from total RNA of MSK5, as BB0186 is in the orientation opposite that of csrA_Bb (Fig. 2B, lane 10). However, the same primer set generated an 1,163-bp amplicon when total genomic DNA was used as the template (Fig. 2B, lane 11). When primers 3 and 4 specific to csrA_Bb were used, a 243-bp amplicon was generated with the cDNA as the template (Fig. 2B, lane 14), consistent in size with what was observed with total genomic DNA (Fig. 2B, lane 15). There was no amplification when total RNA (−RT) was used as the template with any of the primer sets used in this analysis (Fig. 2B, lanes 1, 5, 9, and 13) or with any of the primer sets used with PCR master mix without template (Fig. 2B, lanes 4, 8, 12, and 16). Taken together, these observations indicate that csrA_Bb is cotranscribed with its upstream neighbors BB0183 and BB0182 and that the last ORF in the flgK operon is indeed csrA_Bb. The csrA homolog of another related spirochete, Treponema pallidum, is also organized similarly to that of csrA_Bb (Fig. 2C). Moreover, in Bacillus subtilis, the csrA homolog is located upstream of hag, which in turn has been characterized as being involved in the motility of B. subtilis (84).

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FIG. 2.

csrA_Bb (bb0184) is cotranscribed with bb0182-bb0183 and is the terminal ORF of the flgK motility operon. (A) Schematic representation of a region of the flgK motility operon that has been previously shown to extend from bb0180 to bb0183 (34). The arrows with numbers refer to the primers (Table 3) used in the RT-PCR depicted in panel B. (B) RT-PCR products separated on a 1% agarose gel stained with ethidium bromide. Lanes 1 to 4, PCR with primer sets 2 and 4; lanes 5 to 8, PCR with primer sets 1 and 4; lanes 9 to 12, PCR with primer sets 3 and 5; lanes 13 to 16, PCR with primer sets 3 and 4. The templates used in the PCRs are from MSK5 and are as follows: for lanes 1, 5, 9, and 13, total RNA (−RT control); for lanes 2, 6, 10, and 14, cDNA (+RT); for lanes 3, 7, 11, and 15, total genomic DNA; for lanes 4, 8, 12, and 16, PCR master mix with no template (double-distilled H₂O control). Lanes M and M2, molecular size markers in kilobases (M) or base pairs (M2) as indicated on left and right sides, respectively, of the image. The image was generated using the Versadoc imaging system (Bio-Rad Laboratories, Hercules, CA). (C) Genomic organization corresponding to the location of csrA_Bb in the linear chromosome of B. burgdorferi. A similar organization of genes surrounding csrA_Bb is also observed in the sequenced species Borrelia afzelii PKo and Borrelia garinii PBi (not shown). CsrA is also located downstream of homologs of flagellar biosynthesis in Treponema pallidum and upstream of hag in B. subtilis, mediating regulation of morphology by altering levels of Hag.

CsrA_Bb synthesis is elevated under conditions that mimic the midgut of fed ticks.In order to determine if the level of CsrA_Bb expression is dependent on the environmental signals present in the tick midgut before and after a blood meal, we analyzed protein lysates from MSK5, ML23, and ML23/pBBE22 propagated at pH 7.6 and 32°C and pH 7.6 and 23°C (unfed-tick midgut conditions) and pH 6.8 and 37°C (fed-tick midgut conditions), respectively, using monospecific anti-CsrA_Bb antibodies. As shown in Fig. 3, the level of CsrA_Bb was elevated under fed-tick conditions in all three strains (Fig. 3B, lanes 3, MSK5, ML23, and ML23/pBBE22) while CsrA_Bb was absent under unfed-tick conditions (Fig. 3B, lanes 2, MSK5, ML23, and ML23/pBBE22). The level of CsrA_Bb was reduced in MSK5 at pH 7.6 and 32°C compared to the level at pH 6.8 and 37°C, whereas it was absent in both ML23 and ML23/pBBE22 when the spirochetes were propagated at pH 7.6 and 32°C. There were increased levels of OspC in all three strains of B. burgdorferi (as indicated by the arrow in Fig. 3A) consistent with the previous studies that demonstrated significant increases in the levels of this lipoprotein under environmental conditions that mimicked the conditions of the midgut of fed ticks. In addition, the Coomassie blue-stained gel also served as a control for the relative loading of various samples, indicating that the increased level of CsrA_Bb observed under fed-tick conditions was not an artifact of varied levels of protein separated on the SDS-polyacrylamide gel. We (51, 52) and others (38) have previously demonstrated that the levels of BBA64 encoded on linear plasmid 54 (lp54) increase when B. burgdorferi is propagated under fed-tick conditions (15, 36, 37, 66). Immunoblot analysis using rat anti-BBA64 serum demonstrated that the level of BBA64 is indeed elevated in B. burgdorferi under fed-tick conditions (Fig. 3C, lanes 3) and coincided with the increased levels of CsrA_Bb under conditions mimicking the midgut of fed ticks (Fig. 3B, lanes 3) in MSK5, ML23, and ML23/pBBE22. There were no detectable levels of either CsrA_Bb (Fig. 3B, lanes 2) or BBA64 (Fig. 3C, lanes 2) in any of these controls when they were propagated under conditions similar to those of the midgut of unfed ticks (pH 7.6 and 23°C). When these three control strains were propagated at pH 7.6 and 32°C, only MSK5 had detectable levels of CsrA_Bb and BBA64 (Fig. 3B and C, lanes 1), whereas neither of these proteins was detected under the above conditions in both ML23 and ML23/pBBE22 (Fig. 3B and C, lanes 1). These observations suggested that the levels of CsrA_Bb may, therefore, contribute to the alteration in the levels of determinants (such as OpsC and BBA64) of B. burgdorferi that facilitate its adaptation to the mammalian host following transmission from ticks. We decided to exploit the lack of synthesis of CsrA_Bb in ML23 when propagated at pH 7.6 and 32°C to determine if constitutive overexpression of CsrA_Bb (under the control of a heterologous promoter, P_flgB) will facilitate delineation of its role independently of the culture conditions that induced its synthesis. Moreover, we also intended to develop a strategy to isolate the homolog(s) of the cognate small RNA molecule interacting with CsrA_Bb that has been reported to regulate gene expression in other bacterial systems.

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FIG. 3.

Increased synthesis of CsrA_Bb under fed-tick conditions. B. burgdorferi strain B31 clonal isolates MSK5, ML23, and ML23/pBBE22 were propagated under various conditions mimicking the unfed- or fed-tick midgut conditions such as pH 7.6 and 32°C (lanes 1), pH 7.6 and 23°C (lanes 2), and pH 6.8 and 37°C (lanes 3). (A) Total protein samples were separated on an SDS-12.5% polyacrylamide gel and stained with Coomassie brilliant blue. The arrow corresponds to OspC, and the markers on the left indicate the molecular masses of protein standards in kilodaltons. (B and C) Immunoblot analysis using monospecific serum against CsrA_Bb (B) and BBA64 (C). The blots were developed by using the enhanced chemiluminescence system. The numbers to the left of each panel indicate the molecular masses in kilodaltons.

Overexpression of CsrA_Bb in B. burgdorferi alters its morphology.We overexpressed CsrA_Bb (ES25) under the control of a constitutive promoter, P_flgB (35), with a C-terminal six-histidine tag in an lp25-deficient, clonal isolate of B. burgdorferi (ML23) by using the borrelial shuttle vector pBSV2 (78). Surprisingly, we found that the colonies on BSK-II agar overlays that grew in the presence of kanamycin (conferred by the shuttle vector) were compact compared to those of the parental strain (data not shown). Analysis of spirochetes following Live/Dead staining with confocal microscopy revealed significant differences between ES25 and the parental strain ML23/pBSV2 (Fig. 4). The cells from the parental strain had typical “wave-form” morphology, whereas those from ES25 appeared to be more “rod shaped” with reduced motility as visualized under dark-field microscopy (56). ES25 did not exhibit the typical corkscrew motility observed with the control strains but was motile as determined by dark-field microscopy. The altered morphology upon overexpression of CsrA_Bb was observed at different culture densities ranging from 1 × 10⁶ to 5 × 10⁷ spirochetes/ml, indicating that it was independent of accumulation of various metabolites and/or changes in the conditions of the medium, and the morphology was persistent even after six in vitro passages of ES25. These observations indicated that the altered morphology is essentially due to overexpression of CsrA_Bb under the control of the P_flgB promoter. Moreover, we have not observed such persistent changes in morphology when other borrelial determinants such as sodA or a variety of antibiotic resistance markers have been expressed under the control of P_flgB (27, 52, 74, 75).

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FIG. 4.

Cell morphology of B. burgdorferi upon overexpression of CsrA. The spirochetes propagated in BSK-II medium supplemented with 6% normal rabbit serum at pH 7.6 and 32°C were washed, visualized using a Zeiss LSM510 microscope, and deconvolved using AutoQuant X following Live/Dead staining. Representative images are shown indicating the loss of “wave-form” morphology observed upon overexpression of CsrA_Bb in ES25.

Overexpression of CsrA_Bb results in reduced expression of FlaB.In order to further characterize the effect of overexpression of CsrA, we analyzed the total proteins from ML23/pBSV2 and ES25 on a 12.5% SDS-polyacrylamide gel. Coomassie blue staining of the gels revealed a significant reduction in the levels of an approximately 41-kDa protein in ES25 compared to the levels of a similar-sized protein in the control strain (ML23/pBSV2) as well as in a clonal isolate of B. burgdorferi strain B31 (MSK5) carrying the full complement of plasmids (Fig. 5A). Mass spectrophotometric analysis of this protein isolated from ML23/pBSV2 (indicated by an asterisk in Fig. 5A) identified three different tryptic peptides with sequence identity with FlaB (BB0147) as shown in Fig. 5E. The N-terminal peptide (MIINHNTSAINASR) had 100% identity with the N-terminal sequence of FlaB, the endoflagellin of B. burgdorferi. Sequence analysis of additional peptides (ASDDAAGMGVSGKINAQIR and AINFIQTTEGNLNEVEK) had one amino acid difference from the deduced amino acid sequence of FlaB of B. burgdorferi (32). Moreover, immunoblot analysis of total protein lysates with anti-FlaB monoclonal antibodies indicated that there was less FlaB in ES25 than in ML23/pBSV2 and MSK5 (Fig. 5C). The reduced levels of FlaB coincided with increased levels of CsrA_Bb observed in ES25 by immunoblot analysis using monospecific serum against CsrA_Bb (Fig. 5B). The levels of CsrA_Bb were significantly lower in both parental strain ML23/pBSV2 and MSK5 when they were propagated under conditions similar to those for ES25 at pH 7.6 and 32°C (Fig. 5B). CsrA_Bb in the borrelial isolates, as determined by immunoblot analysis, migrates as an approximately 18-kDa protein, which is almost twice the predicted size. While this difference in the migration pattern could be due to dimerization, translational readthrough, or other unknown modifications, the specificity of the anti-CsrA serum to this band (18 kDa) in the total lysates of B. burgdorferi clearly demonstrates that this protein is indeed CsrA_Bb. This conclusion is also supported by the increased detection of a similar-sized protein upon overexpression of CsrA_Bb in ES25 (Fig. 5B). All three isolates had similar levels of P66, an integrin binding porin (76), providing evidence that increased expression of CsrA_Bb has effects on only a select set of proteins. Taken together, these observations indicate that the levels of FlaB are reduced upon overexpression of CsrA_Bb in B. burgdorferi. The effect of overexpression of CsrA on levels of FlaB of B. burgdorferi provides an added significance to its genetic location within the flgK operon, as shown in Fig. 2, even though the direct role of CsrA_Bb in regulating FlaB expression is yet to be determined. It should be pointed out that ML23/pBVS2 at pH 7.6 and 32°C exhibits detectable levels of CsrA_Bb (Fig. 5B, lane 2) compared to those in ML23 and ML23/pBBE22 (Fig. 3, lanes 1, ML23 and ML23/pBBE22). The samples depicted in Fig. 2 and 5 were from two separate experiments, and the effects of changes in the composition of the growth medium and cell density and the consequent changes in pH could have contributed to the increased levels of CsrA_Bb in ML23/pBVS2, even though all three strains have essentially the same genetic background with or without the borrelial shuttle vector pBSV2. We, therefore, decided to determine if the increased level of CsrA_Bb in ES25 observed under the unfed-tick conditions (Fig. 6J, lane 1), reflecting the levels from the heterologous promoter P_flgB, is sufficient to induce increased synthesis of proteins such as OspC and BBA64 which are not normally elevated in B. burgdorferi under unfed-tick conditions (51, 52).

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FIG. 5.

Overexpression of CsrA in B. burgdorferi results in reduced expression of FlaB. A clonal isolate of B. burgdorferi strain B31 with the full complement of plasmids (MSK5), the parental strain ML23/pBSV2, and CsrA overexpression isolate ES25 were grown in BSK-II growth medium with 6% normal rabbit serum at pH 7.6 and 32°C to a density of 5 × 10⁷ spirochetes/ml. (A) Total protein lysates were separated on an SDS-12.5% polyacrylamide gel and stained with Coomassie brilliant blue. The markers on the left indicate the molecular masses of protein standards in kilodaltons. An approximately 41-kDa protein that was present in ML23/pBSV2 and reduced in expression in ES25 is indicated with an asterisk. (B to D) Immunoblot analysis using monospecific anti-CsrA_Bb serum (B), anti-FlaB monoclonal antibodies (C), or monospecific anti-P66 serum (D). (E) Sequences of peptides obtained on spectrophotometric analysis of the tryptic digests of the band indicated by an asterisk in ML23/pBSV2 are indicated in underlined boldface. The sequence of the first peptide (residues 1 to 14) had 100% identity to the deduced amino acid sequence of FlaB of B. burgdorferi. The other two peptides had one residue difference in each from the deduced sequence of FlaB from B. burgdorferi.

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FIG. 6.

Overexpression of CsrA results in increased expression of borrelial determinants associated with pathogenesis. Equivalent numbers of spirochetes from MSK5, ML23/pBSV2, and ES25 (as shown in Fig. 5A) propagated in BSK-II medium with 6% normal rabbit serum at pH 7.6 and 32°C to a density of 5 × 10⁷ spirochetes/ml were resolved by SDS-PAGE, immobilized onto PVDF membranes, and probed with monoclonal antibodies or antisera as described in Materials and Methods. (A to H) Immunoblots were developed with B. burgdorferi infection-derived mouse serum (A), anti-OspA (B), anti-OspC (C), anti-BBK32 (D), anti-DbpA (E), anti-BBA64 (F), anti-NapA (G), or anti-SodA (H). (I to L) Overexpression of CsrA_Bb results in increased synthesis of OspC and BBA64 under unfed-tick midgut conditions. The CsrA_Bb overexpression strain, ES25, was propagated under conditions mimicking either the unfed-tick midgut (pH 7.6 and 23°C) (lanes 1) or the fed-tick midgut (pH 6.8 and 37°C) (lanes 2) to a density of 5 × 10⁷ spirochetes/ml. Total protein lysates were resolved by SDS-PAGE and either stained with Coomassie blue (I) or immobilized onto PVDF membranes and probed with anti-CsrA_Bb (J), anti-OspC (K), or anti-BBA64 (L). The numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens.

Overexpression of CsrA alters expression of lipoproteins of B. burgdorferi.We further examined the levels of various lipoproteins in B. burgdorferi overexpressing CsrA_Bb. Antisera generated against low-dose infection of MSK5 in C3H/HeN mice recognized several antigens that were upregulated in ES25 compared to ML23/pBSV2 (Fig. 6A). The levels of expression of these lipoproteins were elevated in the clonal isolate MSK5, which correlates with increased expression of CsrA_Bb. We used monoclonal antibodies or monospecific sera to further identify the proteins differentially expressed in ES25. While the levels of OspA appears to be the same in ML23/pBSV2 and ES25 (Fig. 6B), there was a significant increase in the levels of OspC in ES25 over those in the control strain (Fig. 6C). This was also true with other lipoproteins such as BBK32 (Fig. 6D) and DbpA (Fig. 6E), which are well-characterized adhesins mediating adherence of B. burgdorferi to fibronectin (64, 75) and decorin (39), respectively. In addition, there was increased expression of BBA64 in ES25 while there was little or no expression of this lp54-encoded protein in both the parental control strain and MSK5 when these strains are propagated in BSK-II medium at pH 7.6 and 32°C. Previous studies have shown that increased expression of BBA64 was predominantly observed upon propagation of B. burgdorferi under in vitro growth conditions that mimic the tick midgut after the ingestion of a blood meal, namely, BSK-II growth medium at pH 6.8 and 37°C (21, 51, 52, 85). Therefore, the increased synthesis of BBA64 in ES25 can be attributed to the increased levels of CsrA_Bb. The levels of NapA and SodA (Fig. 6G and F) were found to be similar in both the parental control strain and ES25, suggesting that the overexpression of CsrA_Bb does not seem to have a significant effect on proteins that have been characterized as mediating resistance to oxidative stressors in B. burgdorferi (13, 27, 47). Except for DbpA, the levels of OspC, BBK32, and BBA64 were higher in ES25 than in MSK5. The levels of NapA and SodA were also similar between MSK5, ML23/pBSV2, and ES25, suggesting a lack of significant correlation of levels of these proteins with the expression of CsrA_Bb.

Overexpression of CsrA_Bb results in increased synthesis of OspC and BBA64 under unfed-tick midgut conditions (pH 7.6 and 23°C).In order to determine if CsrA_Bb can exert regulatory effects on proteins that have been previously shown to be regulated by RpoS, we analyzed ES25 propagated under either unfed-tick conditions (pH 7.6 and 23°C) or fed-tick conditions (pH 6.8 and 37°C). Immunoblot analysis using anti-CsrA_Bb serum demonstrated that increased levels of CsrA_Bb in ES25 (in comparison to ML23 or ML23/pBBE22 [Fig. 3B]) grown under unfed-tick conditions (Fig. 6, ES25, lanes 1) are essentially due to the constitutive overexpression of csrA_Bb under the control of a heterologous promoter, P_flgB. Moreover, there was a significant increase in the levels of CsrA_Bb (Fig. 6J) when ES25 was propagated under fed-tick conditions (pH 6.8 and 37°C), which is due to the additive effects of increased expression from both the heterologous and native promoters of csrA_Bb. Interestingly, the levels of OspC (Fig. 6K) and BBA64 (Fig. 6L) in these samples were similar under the two types of culture conditions, suggesting that the overexpression of CsrA_Bb from a constitutive promoter is sufficient to induce the levels of these lipoproteins that have been previously shown to be induced following the shift from pH 7.6 and 23°C to pH 6.8 and 37°C in an RpoS-dependent mechanism (41, 85, 86). The aforementioned observations demonstrate that CsrA_Bb contributes to the induction of a select set of lipoproteins independently of the signal-dependent regulation seen when the spirochetes are cultivated at different temperatures or pHs. It is not possible to ascertain if overexpression of CsrA_Bb induces increased expression of RpoS without alterations in temperature or pH, which in turn mediates the upregulation of OspC or BBA64 from these studies. Taken together, overexpression of CsrA_Bb appears to increase the levels of select lipoproteins, which may or may not be directly mediated by a concomitant increase in the levels of RpoS in ES25. It would be interesting to determine the levels of one or more genes, not under the control of the rpoS regulon, that could potentially be regulated only by CsrA_Bb.

Effect of overexpression of CsrA_Bb on transcriptional levels of select ORFs.In order to confirm if the changes in the levels of proteins were due to transcriptional or posttranscriptional regulation by CsrA_Bb (85), we used quantitative real-time RT-PCR of total RNA obtained from MSK5, ML23/pBSV2, and ES25. The transcript from recA was used to normalize the transcript levels of the three strains. As shown in Fig. 7, there was a statistically significant increase in the transcript levels of ospC (21.9-fold difference) in ES25 over those in the control strain ML23/pBSV2, which suggests that the increased level of this lipoprotein is due to transcriptional upregulation, even though it is not possible from this study to rule out the contribution of posttranscriptional events. There were no significant differences in the transcript levels of other genes between either ES25 and ML23/pBSV2 or ES25 and MSK5. Therefore, the increased levels of synthesis of multiple borrelial proteins observed with ES25 are probably due to posttranscriptional and translational events.

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FIG. 7.

Real-time RT-PCR analysis of select determinants upon overexpression of CsrA_Bb. RNA isolated from MSK5, ML23/pBSV2, and ES25 propagated in BSK-II growth medium at pH 7.6 and 32°C was subjected to RT-PCR as described in Materials and Methods. All samples were normalized relative to the recA value, and the ΔC_T values obtained from three independent experiments for each transcript were averaged. The ΔΔC_T for each transcript from ES25 relative to MSK5 (open bars) or from ES25 relative to ML23/pBSV2 (black bars) is shown as difference on the y axis. The asterisk indicates a sample whose C_T value was statistically significant (i.e., P value less than 0.05) in ES25 compared to ML23/pBSV2. The C_T values obtained for ES25 versus ML23/pBSV2 or MSK5 were subjected to a two-way analysis of variance followed by a Bonferroni post hoc test implemented in PRISM.