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Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Correspondence
Michael V. Norgard
michael.norgard{at}utsouthwestern.edu
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ABSTRACT |
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These authors contributed equally to this work.
The array data discussed in this paper have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSM284306–284322.
A supplementary table listing the oligonucleotide primers used in this study and two supplementary figures showing representative scanned microarray images of 1723 B. burgdorferi ORFs are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Steere et al., 1983), remains the most common arthropod-borne illness in the United States (CDC, 2007). B. burgdorferi has a complex enzootic life cycle that involves an arthropod (Ixodes tick) vector and a mammalian host (most typically small rodents) (Fikrig & Narasimhan, 2006; Hovius et al., 2007). For B. burgdorferi to maintain its presence in nature, it must transit between these two dramatically different environments, and adapt to them efficiently by altering its gene expression patterns (Fikrig & Narasimhan, 2006; Hovius et al., 2007; Rosa et al., 2005; Singh & Girschick, 2004). Many studies have now shown that certain environmental signals, such as temperature, pH, cell density, nutrient availability and other mammal-specific signals, modulate the temporal expression of a number of borrelial membrane (lipo)proteins (Caimano et al., 2007; Ojaimi et al., 2003; Revel et al., 2002; Stevenson et al., 2006; Yang et al., 2000, 2003b).
Along these lines, our laboratory and others have previously reported the existence in B. burgdorferi of an alternative sigma factor (RpoN/
54–RpoS/S) regulatory cascade that governs the expression of outer surface (lipo)protein C (OspC), decorin-binding protein A (DbpA), multicopy lipoprotein-8 (Mlp8), fibronectin-binding protein BBK32, and other potential virulence-associated proteins (Caimano et al., 2004, 2005, 2007; Fisher et al., 2005; He et al., 2007; Hubner et al., 2001; Smith et al., 2007; Yang et al., 2003a, b, 2005). In this regulatory pathway, Rrp2, a putative response regulator (enhancer-binding protein) of a two-component sensory transduction system, is likely activated via phosphorylation through a histidine kinase (Hk2) (Fraser et al., 1997; Yang et al., 2003a). Activated Rrp2 then ostensibly interacts with the RpoN holoenzyme, and allows open complex formation for RpoN-dependent transcription of rpoS (Burtnick et al., 2007; Yang et al., 2003a). RpoS is then available for the transcription of those genes under its control, such as ospC, dbpA, mlp8 and bbk32. In addition, RpoS also is activated by the small non-coding RNA DsrABb in response to changes in temperature (Lybecker & Samuels, 2007).
Although recognition of the RpoN–RpoS cascade was an important first step in elucidating regulatory networks in virulent B. burgdorferi (Hubner et al., 2001), its discovery has engendered many important questions. For example, how extensive is this pathway in B. burgdorferi; how many genes are affected? Is every RpoN-dependent gene influenced solely via RpoN control over rpoS, or does RpoN control genes independent of its interaction with rpoS? Are there indirect effects; does the pathway regulate other regulators? Are there genes also downregulated by the pathway, as has been implied by the reciprocal regulation of OspC and OspA (Caimano et al., 2005; Schwan et al., 1995; Schwan & Piesman, 2000)? Does Rrp2 activation serve only to allow RpoN-dependent transcription, or does the putative DNA-binding domain of Rrp2 serve some other function(s) in B. burgdorferi? To begin to address some of these questions, Fisher et al. (2005) employed microarray analyses and concluded that there are three patterns of distinct and overlapping regulation in B. burgdorferi, including 254 genes regulated by RpoN alone, 94 genes regulated by RpoS alone, and 51 genes regulated by both RpoN and RpoS. That study raised a number of paradoxes, however, not the least of which was the notion that a majority of the genes regulated by RpoN were independent of its activation of rpoS. More recently, Caimano et al. (2007) performed transcriptional profiling of B. burgdorferi cultivated in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats or rabbits; they observed significant upregulation of rpoS and many RpoS-dependent genes, as well as RpoS-mediated repression of genes in response to mammalian host-specific signals. In the current study, to analyse the regulatory interrelationships between Rrp2, RpoN and RpoS more closely, we took the approach of implementing gene microarrays to compare the transcriptional expression profiles of rrp2, rpoN and rpoS mutants cultivated in vitro under conditions conducive to activation of the RpoN–RpoS pathway. We found that the vast majority of genes had overlapping patterns of regulation by Rrp2, RpoN and RpoS. Moreover, the functions of many of the genes under the regulatory control of Rrp2, RpoN and RpoS have yet to be defined. The combined results substantiate the close interplay between Rrp2, RpoN and RpoS, and thus further confirm their importance in virulence expression by the Lyme disease spirochaete.
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METHODS |
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) and rrp2, rpoN and rpoS mutants were cultivated in Barbour–Stoenner–Kelly (BSK-H) Complete medium (Sigma-Aldrich) (Pollack et al., 1993) at 37 °C and 5 % CO2. After the pH of the medium had been adjusted to 6.8 using 1M HCl, BSK-H medium was inoculated with spirochaetes to a final concentration of 1x103 cells ml–1. Spirochaetes were enumerated by dark-field microscopy, and bacteria were harvested when the culture reached stationary phase (
1x108 bacteria ml–1; 9 days post-inoculation). To ensure activation of the RpoN–RpoS pathway in the cultures, RpoS and OspC expression was monitored as described below.
Generation of B. burgdorferi mutants employed in this study.
The rpoN mutant BbJSB18-B2 used in this study has been described previously (Smith et al., 2007). Prior to use of this clone in microarray comparisons, genetic complementation of the rpoN mutation was performed on BbJSB18-B2 to ensure that this mutant, which was non-infectious, contained all the plasmids necessary for mammalian infection. To achieve this, a 1.7 kb region of B. burgdorferi strain 297 DNA encoding the rpoN gene and 375 bp upstream of the ORF was PCR-amplified using the primers priAH125 and priAH144 (Hubner et al., 2001) (see Supplementary Table S1) and cloned into pGEM-T easy (Promega). Following confirmation by sequence analysis, the insert was excised by digestion with BamHI and BclI and ligated into the borrelial shuttle vector pJD55, which had been linearized with BamHI. pJD55 is a derivative of pJD44 (Revel et al., 2005) in which the aph[3']-IIIa marker is replaced with PflgB-Kan of pBSV2 (Stewart et al., 2001). BbJSB18-B2 was transformed with the resulting construct, designated pJSB208B, as described previously by Yang et al. (2005), and transformants were selected using kanamycin at a concentration of 160 µg ml–1. Clones were confirmed by plasmid recovery, as described by Blevins et al. (2007).
The rpoS mutant was constructed using an approach similar to that described by Hubner et al. (2001), except that the erythromycin-resistance marker in the mutagenesis construct pALH386 was replaced with the streptomycin-resistance marker from pKFSS1 (Frank et al., 2003). To achieve this, the PflgB-aadA marker was first PCR-amplified from pKFSS1 (Frank et al., 2003) using the primers PflgB-Bam-5' and Sp/Sm-Bam-3' (Supplementary Table S1). The PCR fragment then was cloned into pCR-XL-TOPO (Invitrogen) to generate pXY205. Following confirmation of pXY205 by DNA sequence analysis, the PflgB–aadA marker was excised using BamHI and treated with T4 DNA polymerase to form a blunt-end fragment. pALH362, a suicide vector containing a 4.6 kb region of strain 297 DNA encompassing rpoS (Hubner et al., 2001), was digested with BbsI and also treated with T4 DNA polymerase to blunt the ends. The fragments were then ligated to generate pJSB19, and the resulting clones were confirmed by restriction digestion and PCR analysis. B. burgdorferi transformed with pJSB19 were selected using streptomycin at a concentration of 150 µg ml–1, and resulting clones were confirmed by PCR analysis as described by Hubner et al. (2001). For genetic complementation of the rpoS mutant, the 4.2 kb region of B. burgdorferi strain 297 DNA in pXY240 (Smith et al., 2007), which includes 1.9 kb upstream and 1.5 kb downstream of the rpoS ORF, was cloned into the shuttle vector pJD44 (Revel et al., 2005). This was achieved by removing the 4.2 kb region from pXY240 by digestion with KpnI and XbaI and ligating it into pJD44 digested with the same enzymes. The resulting construct, designated pJSB259, was transformed into BbJSB19-A7B, as described previously (Yang et al., 2005).
The rrp2 mutant was generated by transforming B. burgdorferi strain 297 with the suicide vector pXY201A (Yang et al., 2003a), which introduced a point mutation (G239C) within the putative C4 ATP-binding motif of Rrp2; this mutation abolishes the ATPase activity that is essential for RpoN-dependent activation of RpoS. Transformants, selected using 60 ng erythromycin ml–1, were confirmed as described by Yang et al. (2003a). Complementation of the rrp2 mutant was achieved by transforming the mutant with the suicide vector pXY206B (Yang et al., 2003a); transformants were selected using 150 µg streptomycin ml–1.
Plasmid profiling was performed on all mutants and complemented strains as described previously (Hubner et al., 2001; Yang et al., 2003a). All mutants and complemented strains contained the same plasmid profile as the B. burgdorferi parental strain 297 (data not shown). The infectivity of the mutants and complemented strains was assessed using the murine needle-challenge model of Lyme borreliosis (Hagman et al., 1998). University of Texas (UT) Southwestern is accredited by the International Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC), and all animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical Center. Prior to infection, the density of bacteria in each culture was determined carefully using dark-field microscopy. Groups of 3- to 5-week-old C3H/HeJ (Jackson Laboratory) or C3H/HeN (Charles River Laboratories) mice were infected via intradermal injection. At 2 or 6 weeks post-infection, ear punch biopsies were recovered from mice and placed in BSK-II media containing borrelia antibiotic mixture (BAM; Sigma-Aldrich), and the outgrowth of spirochaetes in each of these cultures was assessed using dark-field microscopy.
SDS-PAGE and immunoblot analysis.
RpoS and OspC expression was assessed by SDS-PAGE and immunoblot analysis, as described previously (Yang et al., 1999). Briefly, spirochaetes were harvested from stationary-phase cultures and washed twice in sterile 0.9 % (w/v) saline solution. A volume of whole-cell lysate equivalent to 4x107 bacteria was loaded to each lane on a 12.5 % acrylamide gel. Resolved proteins were either stained with Coomassie Brilliant Blue or transferred to nitrocellulose membranes for immunoblot analysis. A mAb directed against RpoS, designated 6A7-101, was produced in collaboration with the Antibody Production Core Facility at UT Southwestern Medical Center. OspC was detected using anti-OspC mAb 1B2-105 (Smith et al., 2007). To confirm equal loading of bacteria in each lane, immunoblotting for the flagellar core protein (FlaB) was performed using a chicken IgY anti-FlaB antibody (a gift from Dr Kayla Hagman, UT Southwestern Medical Center). Immunoblots for RpoS were developed by chemiluminescence using the ECL Plus Western Blotting Detection System (Amersham Biosciences), whereas membranes for OspC and FlaB were developed colorimetrically using 4-chloro-1-naphthol as the substrate.
B. burgdorferi microarray construction.
Oligonucleotides representing 1723 putative ORFs of B. burgdorferi B31MI and 19 random-sequence 70-mer negative controls were synthesized as described elsewhere (Terekhova et al., 2006). The oligonucleotides were resuspended in 150 mM sodium phosphate, pH 8.5 (Microarrays Inc.), to a concentration of 40 µM and printed on CodeLink activated slides (Amersham Biosciences), using a Custom arrayer (Microarrays Inc.). Each oligonucleotide was printed in quadruplicate on each array. Arrays were blocked post-printing as per the CodeLink instructions (Amersham Biosciences) with 50 mM ethanolamine. Attachment of probe DNA was confirmed by Microarrays Inc. proprietary Veriprobe assay. Prior to hybridization, arrays were stored under desiccation.
Comparative genomic DNA hybridization.
Bacterial genomic DNA was isolated using a Wizard genomic DNA purification kit (Promega) according to the manufacturer's instructions. DNA was quantified using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies). Samples (4 µg) of genomic DNA from strains 297 (test) and B31 (reference) were labelled with Alexa Fluor 555 and 647, respectively, using the Bioprimer Plus Array CGH Indirect Genomic Labelling System (Invitrogen) according to the manufacturer's protocol. Prior to hybridization, slides were pre-hybridized in a solution of 5xSSC (1xSSC=0.15 M sodium chloride/0.015 M sodium citrate, pH 7), 0.1 % SDS, and 0.1 % (w/v) BSA for 30 min at 55 °C. Following pre-hybridization, slides were washed five times in distilled water, dipped in 2-propanol, dried by centrifugation, and used immediately in the hybridization. After purification of labelled DNA using the Qiaquick PCR purification kit (Qiagen), equal amounts of labelled 297 and B31 DNA were combined and applied to arrays. Slides were covered with lifter coverslips (Erie Scientific), placed into a humidified hybridization chamber (Ambion), and the chamber was placed into a hybridization oven set at 50 °C. After hybridization overnight, slides were washed twice with 2xSSC/0.1 % SDS for 15 min at 42 °C, twice with 0.1xSSC/0.1 % SDS for 15 min at room temperature, and twice with 0.1xSSC for 1 min at room temperature. Washed slides were then dried by centrifugation and scanned immediately. Microarray scanning and data analysis were performed as described previously (Terekhova et al., 2006).
Extraction of RNA from B. burgdorferi.
RNA was extracted from three biological replicates of wild-type parental strain B. burgdorferi 297 and the rpoS, rpoN and rrp2 mutants using Trizol reagent (Invitrogen) according to the manufacturer's protocol. An additional phenol/chloroform extraction was performed, after which the RNA was precipitated using 2-propanol. Digestion of contaminating genomic DNA in the RNA samples was performed using RNase-free DNase I (GenHunter Technology) and removal of DNA was confirmed by PCR amplification using flaB1 primers specific for the B. burgdorferi flaB gene (Supplementary Table S1). RNA quality was determined using the Agilent Bioanalyser 2100 (Agilent Technologies) in the Microarray Core Facility in the UT Southwestern Medical Center.
Synthesis and labelling of cDNA.
cDNA was synthesized from extracted RNA and labelled with Cy3 or Cy5 using the Amersham post-labelling kit according to the manufacturer's instructions (Amersham Biosciences), with minor modifications. Briefly, 10 µg total RNA was converted to cDNA using CyScript reverse transcriptase, in the presence of 1 µl random nonamers (Amersham Biosciences) and 4.5 µg random hexamers (Invitrogen). The resulting cDNA was labelled with Cy3 or Cy5. Cy3- or Cy5-labelled cDNA from RNA extracted from parental strain 297 was then combined with the corresponding Cy5- or Cy3-labelled cDNA generated from RNA derived from the mutants; e.g. Cy5-labelled 297 cDNA and Cy3-labelled mutant cDNA or Cy3-labelled 297 cDNA and Cy5-labelled mutant cDNA. Labelled probes were purified using the Qiaquick PCR purification kit (Qiagen) and used in subsequent microarray experiments.
Microarray scanning and data analysis.
For comparative transcriptional microarray analysis, slides were hybridized with labelled probes as described above. Hybridized slides were scanned on an Axon 4000B microarray scanner using GenePix Pro 6.1 (Molecular Devices). The image (two representative scans are shown, to give an indication of their quality, in Supplementary Figs S1 and S2) was analysed using the GenePix program, and data then were analysed with Acuity 4.0 microarray informatics software according to the manufacturer's instructions (Molecular Devices), using a ratio-based normalization method and a cutoff value of a twofold change. Briefly, raw data were first normalized using a ratio-based normalization method to equalize the means and medians of the features to 1. Additionally, the features that were flagged by the software as ‘bad’, ‘absent’ or ‘not found’ were also excluded from further analysis. Statistical analyses were performed using the one- and two-sample significance test (P <0.05) in the Acuity program, which is a one-sample t test. Differentially expressed genes were identified by both fold change and statistical significance.
Quantitative RT-PCR analysis.
Real-time quantitative RT-PCR (qRT-PCR) was employed to validate selected data from the microarray experiments. Specific primers (listed in Supplementary Table S1) for 17 B. burgdorferi genes were designed by using Primer Express software (Applied Biosystems) and validated using 10-fold dilutions (80–0.0008 ng) of B. burgdorferi genomic DNA in an absolute quantification test on an ABI 7500 qRT-PCR system (Applied Biosystems). Standard curves created for all primers had a slope of –3.3±0.3 (data not shown). For measuring gene expression, cDNA was generated from 1 µg of the parental and mutant B. burgdorferi RNAs used in microarray experiments using the SuperScript III Platinum Two-Step qRT-PCR kit according to the manufacturer's protocol (Invitrogen). qRT-PCR (in quadruplicate) using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) was then performed and the relative quantification method (
CT) was used to calculate the variation of gene expression between B. burgdorferi strain 297 and corresponding mutants. The borrelial flaB gene was used as an endogenous control to normalize all qRT-PCR data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). At 2 or 6 weeks post-infection, ear-punch biopsies were collected from mice and placed in BSK-II medium. As shown in Table 1, spirochaetes were recovered from samples of all mice inoculated with wild-type strain 297, or the complemented strains. In contrast, no spirochaetes were recovered from samples of mice inoculated with the rrp2, rpoN or rpoS mutants. Notably, whereas rpoN and rpoS mutants of B. burgdorferi have been reported to be non-infectious for mice (Caimano et al., 2004; Fisher et al., 2005), this is the first study, to our knowledge, to show that an rrp2 mutant also lacks the ability to infect mice. These results indicate that all three regulators are required by B. burgdorferi to infect mammals.
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1x108 bacteria ml–1) in BSK-H Complete medium at a reduced pH (pH 6.8) and 37 °C (Yang et al., 2000). Consistent with previous reports (Hubner et al., 2001; Smith et al., 2007; Yang et al., 2003a), both RpoS and OspC were expressed at high levels in wild-type strain 297, but not in the rrp2, rpoN or rpoS mutant (Fig. 1). When induction of the RpoN–RpoS pathway by strain 297 was confirmed in a set of cultures, bacteria were collected for the purpose of isolating total RNA for use in subsequent microarray experiments.
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). Although the oligonucleotides employed in this array have been used by others for transcriptional profiling of B. burgdorferi strain 297 (Caimano et al., 2007), we assessed further the suitability of this newly printed microarray for analysing gene expression in strain 297. Comparative DNA hybridization was performed using genomic DNA extracted from strains 297 and B31. Consistent with earlier reports (Caimano et al., 2007; Terekhova et al., 2006), 95 % of ORFs from B. burgdorferi 297 were detected using this B31-based microarray. These results corroborated the suitability of using a microarray designed from B31 sequence information for transcriptional profiling of strain 297. It should be noted, however, that due to sequence variation between the ospC genes of strains B31 and 297, expression of OspC was not detected in strain 297 upon transcriptional profiling (below) or via genomic hybridization.
Correlation between microarray data and qRT-PCR
To validate the results of the microarray analysis, qRT-PCR was performed on 17 genes from various categories of gene expression profiling (see below and Table 2). In an initial assessment of data correlation, the ratio of transcripts from each strain, as determined by qRT-PCR and microarray, was compared; a correlation coefficient (r) of 0.61 was observed (Fig. 2a). When the log-transformed ratios were compared, an r value of 0.89 was observed (Fig. 2b). Irrespective of these r values, when examining the absolute levels of gene expression for each given gene, similar trends were observed between these experimental conditions (Fig. 2, Table 2). These combined results indicated that the differences observed in mRNA expression levels obtained by qRT-PCR correlated well with those obtained from DNA microarray analyses.
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). Among these genes, 97 were upregulated more than twofold in wild-type 297 (compared with expression levels in the corresponding mutant). Although ospC was not identified in this group of 98 genes due to the variation of ospC sequence between B. burgdorferi strains 297 and B31 (Caimano et al., 2007), qRT-PCR analysis (using 297-specific primers for ospC) showed that ospC expression was upregulated 1848-, 500- and 1408-fold, by Rrp2, RpoN and RpoS, respectively (Table 2
). One gene, bba62, was upregulated 6-, 5- or 3-fold in the rrp2, rpoN or rpoS mutant, respectively. Earlier studies have shown that bba62, which encodes the Lp6.6 lipoprotein, is strongly downregulated in an RpoS-dependent manner when B. burgdorferi is cultivated in DMCs implanted into the peritoneal cavities of rats (i.e. under mammalian infection-like conditions) (Caimano et al., 2005, 2007). In agreement with these findings, bba62 was downregulated in B. burgdorferi by RpoS in our study, even under in vitro culture conditions. Comparing gene expression profiles of the rrp2, rpoN and rpoS mutants, 44, 34 and 25 genes, respectively, were upregulated more than 10-fold in wild-type 297. As shown in Fig. 3, most of the highly induced genes were located either on the chromosome (34/98) or on plasmids lp54 (21/98) and cp32s (16/98).
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; Hagman et al., 1998; Shi et al., 2008). In addition, eight genes encoding proteins related to chemotaxis were found to be induced by Rrp2, RpoN and RpoS. These include the chemotaxis protein methyltransferase-encoding gene bb0040 (cheR-1), two purine-binding chemotaxis protein-encoding genes, bb0565 (cheW-2) and bb0670 (cheW-3), the chemotaxis histidine kinase-encoding gene bb0567 (cheA-1), the chemotaxis response regulator-encoding gene bb0672 (cheY-3), bb0671 (cheX) and two methyl-accepting chemotaxis protein-encoding genes, bb0680 (mcp-4) and bb0681 (mcp-5). For B. burgdorferi, chemotaxis and motility are presumed to be important for colonization, dissemination into deeper tissues, and for the transition between different hosts (Fraser et al., 1997; Motaleb et al., 2005; Shi et al., 1998). Three lipoprotein-encoding genes, bba36, bba62 and bbi42, as well as two predicted outer-membrane protein-encoding genes, bbq03 and bbk53, came within the highly regulated category (Brooks et al., 2006; Setubal et al., 2006). These putative outer surface proteins may contribute to interactions between B. burgdorferi and its hosts. In addition, a few polypeptides annotated as antigens, including five putative P35-encoding genes (bba73, bba64, bba66, bbk32 and bbh32), one S1 antigen-encoding gene (bba05), and one S2 antigen-encoding gene (bba04), were also found to be induced by Rrp2, RpoN and RpoS.
The genome of B. burgdorferi contains one linear chromosome and numerous linear and circular plasmids (Fraser et al., 1997). Several plasmids, such as lp25, lp28-1 and lp36, are essential for borrelial infectivity (Jewett et al., 2007; Purser & Norris, 2000; Stewart et al., 2005). Plasmid lp54 has been implicated as being important for the survival of B. burgdorferi in both tick and mammalian hosts, largely because of the presence of the ospAB and dbpBA operons (Caimano et al., 2005; Hagman et al., 1998; Neelakanta et al., 2007; Shi et al., 2008; Yang et al., 2004). Many other genes encoded on lp54 have been found to be differentially regulated in response to temperature, pH and other mammalian-derived signals, which are presumed to be mediated, at least in part, by RpoS (Caimano et al., 2005; Clifton et al., 2006; Ojaimi et al., 2003; Revel et al., 2002). Consistent with these previous findings, our data provided further evidence that 22 lp54-encoded genes are regulated by RpoS. In addition, lp36 has been reported to be vital for B. burgdorferi infection of mammal hosts (Jewett et al., 2007). In this regard, studies have shown that B. burgdorferi infectivity in mice was attenuated by the inactivation of lp36-encoded bbk32 (encoding a fibronectin-binding protein) (Seshu et al., 2006) or bbk17 (encoding an adenine deaminase, adeC, which converts adenine to hypoxanthine) (Jewett et al., 2007), thereby underscoring the importance of lp36 in B. burgdorferi pathogenesis. A previous study also found that bbk32 and bbk17 are regulated in response to mammalian signals (Revel et al., 2002). More specifically, He et al. (2007) found that bbk32 is regulated by Rrp2, RpoN and RpoS. Consistent with all of these reports, our data showed that the lp36-encoded bbk32, bbk52.1, bbk53, bbk07 and bbk17 were all positively regulated by Rrp2, RpoN and RpoS (Table 3).
In Escherichia coli, RpoS controls many genes involved in cellular metabolism and the stress response (Dong et al., 2008; Farewell et al., 1998; Loewen et al., 1998). Similarly, many B. burgdorferi genes encoding proteins with various putative physiological functions were induced by Rrp2, RpoN and RpoS; these genes included bb0251 (leuS), bb0777 (apt), bbk17 (adeC), bb0257 (cell division), bb0842 (arcB), bb0329 (oppA-2), bba34 (oppAV), bb0313 (ftsJ), bb0797 (mutS), bb0637 (nhaC-1), bbd20 (transposase), bb0646 (hydrolase), bb0728 (nox) and bb0729 (gltP) (Fraser et al., 1997). Among these genes, bb0329 and bba34 encode two putative periplasmic oligopeptide-binding proteins, which facilitate the transport of small peptides and essential amino acids (Fraser et al., 1997; Medrano et al., 2007). In addition, bb0728 and bb0729 are presumed to form an operon involved in the uptake of the amino acid glutamate (Fraser et al., 1997).
Genes likely regulated by Rrp2, RpoN and RpoS
A second subset of 47 genes was most likely regulated by all three of Rrp2, RpoN and RpoS (Table 4). This conclusion derives from the fact that genes in this category were regulated by at least two of the three regulators active in the Rrp2–RpoN–RpoS pathway, and trends favoured regulation by all three members of the pathway. For example, 21 genes were regulated by Rrp2 and RpoN, 24 were regulated by Rrp2 and RpoS, and two (bbq37 and bb0076) were regulated by both RpoN and RpoS. Except for gene bbh01 and the pseudogene bbh09.1, the fold changes of expression for all of the other genes were less substantial (<10-fold) than those genes that were characterized above as being regulated by Rrp2, RpoN and RpoS. When the expression data for all 47 genes in this category were re-examined in an analysis in which the twofold cutoff value and the P value threshold of 0.05 constraints were relaxed, the trends in gene expression changes for all of these genes were similar in all three of the mutants (Table 4). For example, if a gene was repressed or induced by two regulators (e.g. Rrp2 and RpoN), it also was repressed or induced by the third regulator (e.g. RpoS). To further analyse this trend, qRT-PCR was employed to examine the expression of two representative genes in this category: bba74 (oms28) and bbh01. In the microarray experiments, bba74 and bbh01 were not influenced significantly by RpoN and RpoS, respectively. However, qRT-PCR showed that both bba74 and bbh01 were indeed regulated more than twofold by RpoS, RpoN and Rrp2 (Table 2), further substantiating the high probability that genes placed in this category are, in fact, likely influenced by all three of Rrp2, RpoN and RpoS. Among the genes in this category, most have unknown functions, but some genes [bb0012 (hisT), bb0076 (ftsY), bb0254 (recJ), bb0312 (cheW-1), bb0344 (uvrD), bb0386 (rpsG), bb0461 (dnaX), bb0588 (pfs-2), bba74 (oms28), and the putative plasmid partition proteins bbo32 and bbu05] likely encode physiological functions (Fraser et al., 1997). bba16 (ospB), which belongs to the ospAB operon that is essential for B. burgdorferi colonization and survival within tick midguts (Neelakanta et al., 2007; Yang et al., 2004), was downregulated 3.6-, 2.1 and 1.8-fold, respectively, by Rrp2, RpoN and RpoS. In our test, we also found that bba15 (ospA) was downregulated 1.8-, 1.3- and 1.3-fold, respectively, by Rrp2, RpoN and RpoS. Although the fold change of ospA was just below the detection threshold (twofold) in our microarray data analysis, both ospA and ospB exhibited a similar trend of change for all three mutants.
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). These findings are inconsistent with the hypothesis that RpoN modulates borrelial gene expression via rpoS. However, qRT-PCR analysis of the expression of three of these RpoN-regulated genes (bb0403, bb0673 and bb0834) showed that all three genes were only moderately affected by the mutation of RpoN (less than twofold) (Table 2). Furthermore, additional analysis of the microarray results revealed marked similarity in the overall patterns of regulatory effect. For example, in all three mutants, the relative trends and fold changes in expression were approximately equivalent for each of these 12 genes potentially regulated by RpoN alone (Table 5). An additional three genes were identified during the transcriptional comparison that were predicted to be regulated by RpoS alone: bbk33, bbm41 and bb0702 (kdtB) were upregulated 3.4-, 3.1- and 2.4-fold, respectively. However, validation of the microarray via qRT-PCR for one of these genes, bb0702, showed that expression of bb0702 was similarly upregulated by RpoS (2.1-fold), RpoN (1.6-fold) and Rrp2 (2.0-fold) (Table 2). Taken together, these data trends suggest that these genes are not uniquely influenced by RpoN or RpoS.
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; Yang et al., 2003a) that, upon activation, ostensibly activates the alternative sigma factor RpoN; activated RpoN then promotes the transcription of rpoS (Hubner et al., 2001; Yang et al., 2003a). Experimental testing of this cascade, however, has been hampered in part by the fact that we and others have been unable to inactivate rrp2 by insertional or other mutagenesis approaches (Burtnick et al., 2007; Yang et al., 2003a). As a way of partially circumventing this obstacle, a point mutation (G239C) was introduced in the putative ATP-binding domain of Rrp2, thereby abolishing RpoN-dependent rpoS expression (Yang et al., 2003a). Comparative microarray analyses then were performed to determine the gene expression profiles of B. burgdorferi strain 297 and the rrp2 mutant. Our data showed that 37 and 69 genes were either upregulated (2.2–5.8- fold) or downregulated (2.1–5.9-fold), respectively, in the wild-type B. burgdorferi strain 297 (Table 6). Several of these genes are particularly noteworthy. First, three genes encoding putative plasmid partition proteins, bbs35 (5.9-fold) on cp32-3, bbr33 (2.6-fold) on cp32-4, and bbk21 (2.6-fold) on lp36, appeared to be repressed by Rrp2. These plasmid partition proteins may be essential for maintaining the sensitive balance among the numerous endogenous B. burgdorferi plasmids. Furthermore, the virulence-associated gene bbe22 (pncA), encoding a nicotinamidase (Purser et al., 2003), was also repressed by Rrp2, as were the putative glycerol uptake and metabolism genes glpA, glpF and glpK (Fraser et al., 1997). Finally, our data imply that Rrp2 may contribute to a general stress-like response in B. burgdorferi, inasmuch as the heat-shock protein-encoding genes bb0518 (dnaK-2) and bb0264 (dnaK-1) were regulated by Rrp2.
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), qRT-PCR data revealed that bb0160 and bb0835 were regulated 0.83- and 1.38-fold, respectively, by Rrp2 (Table 2). The qRT-PCR data also indicated that, with the exception of bb0519, these genes were regulated at a similar level by RpoS, RpoN and Rrp2 (Table 2). These data suggest that it is difficult to draw definitive conclusions about regulatory events based on microarray data that exhibit only slight fold changes in individual gene expression patterns. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54)–RpoS (S) alternative sigma factor regulatory cascade (Hubner et al., 2001; Yang et al., 2003a) was an important advance in understanding borrelial gene regulation, but represented only a first step towards deciphering key adaptive responses needed by B. burgdorferi to sustain itself in nature. It is now clear that this regulatory pathway is vital for modulating virulence expression by B. burgdorferi (Caimano et al., 2004, 2005, 2007; Fisher et al., 2005; He et al., 2007; Neelakanta et al., 2007; Yang et al., 2004), but its recognition has prompted many new questions and has spawned a number of controversies. For example, the results of Fisher et al. (2005) have called into question the linearity of the RpoN–RpoS pathway. From comparative microarray studies with rpoN and rpoS mutants generated in strain B31, those authors concluded that three patterns of regulation exist in B. burgdorferi: 254 genes were regulated by RpoN alone (but not by RpoS), 94 genes were regulated by RpoS alone (but not by RpoN), and 51 genes were regulated by both RpoN and RpoS. The most surprising of these observations was the very high number of genes seemingly regulated by RpoN or RpoS alone (254 and 94 genes, respectively). There were a number of potential caveats, however, in these findings. First, quantitative RT-PCR analysis revealed that rpoS expression decreased in the rpoN mutant by only 2.63-fold (Fisher et al., 2005), a level inordinately low given the well-documented dependence of rpoS expression on RpoN (Hubner et al., 2001). Second, globally, only slight changes in gene expression were observed by Fisher et al. (2005) in their comparative microarray experiments; thus, changes that were considered significant were, at best, modest. For example, for the genes regulated by RpoN or RpoS alone, changes in gene expression had ranges from only 0.49- to 2.28-fold and 0.55- to 2.64-fold, respectively (Fisher et al., 2005). Even for those genes categorized as being regulated by both RpoN and RpoS, the relative changes in gene expression were slight. Many genes that were shown to be significantly induced by RpoS, including bba25 (dbpB), bba66 and bb0844 (Caimano et al., 2007; Clifton et al., 2006; Hubner et al., 2001), were regulated only moderately (less than fourfold) by RpoS in their microarray experiments (Fisher et al., 2005). In addition, bba24 (dbpA) was found to be induced 1.6-fold by RpoS alone, but not by RpoN, which is inconsistent with previous reports (Hubner et al., 2001; Yang et al., 2003a). We contend that it is unwise to draw conclusions about gene regulation from microarray data displaying such relatively small changes. This is particularly true for B. burgdorferi, which is sensitive to many as yet unknown culture variables (Callister et al., 1990; Nelson et al., 1991; Picken et al., 1997; Wang et al., 2004; Yang et al., 2001).
As noted earlier, the minimal change in the expression of rpoS mRNA (2.63-fold) by the rpoN mutant used in the study by Fisher et al. (2005) was in marked contrast to our observed change of 19-fold in an analogous rpoN mutant, suggesting that the culture conditions employed by Fisher et al. (2005) were not optimal for induction of the Rrp2–RpoN–RpoS pathway. For the experiments described in the current study, we optimized our in vitro cultivation conditions to maximize induction of the Rrp2–RpoN–RpoS pathway and minimize variability. First, BSK-H complete medium was used in the present study, whereas Fisher et al. (2005) used BSK-II. This is relevant because it has been reported that BSK-H medium is superior to BSK-II for use in gene expression studies (Yang et al., 2001). Second, in every experiment, bacteria were cultivated using the same lot of BSK-H medium that was prescreened for the capacity to promote borrelial gene regulation (Smith et al., 2007). Third, the medium used in our study was adjusted to pH 6.8, an important condition for maximizing induction of the Rrp2–RpoN–RpoS pathway (Yang et al., 2000). Fourth, care was taken to ensure that all cultures were grown to similar high spirochaete densities (1x108 bacteria ml–1). Fifth, SDS-PAGE and immunoblot analysis was used to confirm induction of RpoS and OspC by wild-type 297 in each set of cultures. Only after it was determined that the Rrp2–RpoN–RpoS pathway was induced significantly was the RNA isolated from these bacteria and then used in microarray experiments. Efforts such as these to ensure the robust induction of the Rrp2–RpoN–RpoS pathway were not implemented in the study of Fisher et al. (2005).
The intention of our study was twofold. First, we wished to investigate further the interrelationships among Rrp2, RpoN and RpoS, with emphasis on their functional interplay. As such, this is the first study, to our knowledge, to exploit rrp2, rpoN and rpoS mutants of B. burgdorferi in gene microarray experiments to study the transcriptional influences of all three major components of the pathway. Although microarray data generated from single observations (single time points) can be subject to indirect effects, the slow growth of B. burgdorferi in vitro would serve to minimize such effects. In our comparative transcriptional profiling experiments with the rrp2, rpoN and rpoS mutants, it was clear that 98 genes were under the combined regulation of Rrp2, RpoN and RpoS. Caimano et al. (2007) examined the RpoS regulon in B. burgdorferi cultivated in either DMCs or under more typical in vitro growth conditions. When comparing our data with the microarray data of Caimano et al. (2007), an excellent correlation was observed: 68 (67 induced genes and one repressed gene, bba62) of the 98 genes in our group of targets that were definitively regulated by Rrp2–RpoN–RpoS were also present in the microarray data of those authors. We believe that this level of concordance serves to validate our data, as well as those of Caimano et al. (2007), particularly given the tendency for microarray data to vary between laboratories.
In addition to the 98 genes under the control of Rrp2, RpoN and RpoS, an additional group of 47 genes were identified whose expression is also most likely modulated by these three regulators. This conclusion is warranted based on the similarity in the changes in the expression of each given gene among all three mutants. In other words, if one particular gene was regulated by RpoS, it was also found to be regulated by RpoN, although at a less than twofold change or with a P value of >0.05. Finally, microarray data identified a third group of three and 12 genes, which were regulated by RpoS or RpoN alone, respectively. However, the relative changes in expression of these genes were modest (less than fourfold). Considering that there was substantial overlap in the genes regulated by both RpoN and RpoS, and the fact that there were only a few genes regulated by RpoN or RpoS individually (all of which exhibited only slight differences in expression levels), our data provide strong evidence that RpoN regulates the RpoS-mediated adaptive response in B. burgdorferi.
In B. burgdorferi, the putative response regulator Rrp2 has been shown to be required for the RpoN-dependent transcription of rpoS (Burtnick et al., 2007; Yang et al., 2003a). To further confirm the linearity of the pathway, we compared herein the transcriptomes of the wild-type strain 297 and the rrp2 mutant. In addition to the 98 genes known to be regulated by Rrp2–RpoN–RpoS, our data showed that 106 genes were likely regulated by Rrp2 alone in B. burgdorferi. However, for each gene in the group regulated by Rrp2 alone, the changes in gene expression were modest (i.e. less than sixfold). Unfortunately, further investigation into this Rrp2-mediated branch of gene regulation, which ostensibly is independent of the RpoN–RpoS sigma factor cascade, represents a formidable challenge, because all efforts to date to completely disrupt rrp2 (via insertion or deletion) in B. burgdorferi have been unsuccessful (Burtnick et al., 2007; Yang et al., 2003a). Given the fact that mutations in rpoN and rpoS are not lethal for in vitro growth of B. burgdorferi (Caimano et al., 2004; Fisher et al., 2005; Hubner et al., 2001), the inability to totally inactivate rrp2 has led to the supposition that rrp2 likely serves some other essential role that is independent of its activation of RpoN-dependent transcription. Thus, it is hypothesized that, in addition to its presumed enhancer-binding activity (for RpoN), Rrp2 downregulates the expression of other genes whose products are toxic when they are aberrantly expressed in B. burgdorferi. The rrp2 mutant used in this study was theoretically deficient only in its capacity to hydrolyse ATP and thus unable to activate RpoN-dependent transcription, but its putative DNA-binding capability should have been left intact. Therefore, it is reasonable that we would not have been able to identify the toxic targets repressed by Rrp2 by employing the ATP-binding site mutant utilized in the current study.
The second major focus of this study was to identify B. burgdorferi ORFs differentially regulated by the RpoN–RpoS pathway that potentially contribute to B. burgdorferi virulence and pathogenesis. To date, only relatively few virulence factors in B. burgdorferi, such as OspAB, OspC, DbpBA and BBK32, have been determined definitively to be under the regulatory control of this pathway (Grimm et al., 2004; Hagman et al., 1998; Neelakanta et al., 2007; Pal et al., 2004; Seshu et al., 2006; Shi et al., 2008; Yang et al., 2004). As such, continued efforts are warranted to elucidate other potential B. burgdorferi virulence factors. In this regard, our data confirmed that the RpoN–RpoS pathway is central to B. burgdorferi, in that it controls the differential expression of many predicted outer surface proteins, cell envelope constituents and putative metabolic genes. It is likely that some of these gene products facilitate the successful colonization and survival of B. burgdorferi within a mammalian host, and ultimately progression to disease. Further research will focus on the identification of new potential virulence factors in the infective life cycle of B. burgdorferi, with the goal of providing further insights into the molecular pathogenesis of this important arthropod-borne bacterial pathogen.
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ACKNOWLEDGEMENTS |
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Edited by: G. E. Duhamel
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Received 25 April 2008;
revised 3 June 2008;
accepted 6 June 2008.
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 J. S. Blevins, H. Xu, M. He, M. V. Norgard, L. Reitzer, and X. F. Yang Rrp2, a {sigma}54-Dependent Transcriptional Activator of Borrelia burgdorferi, Activates rpoS in an Enhancer-Independent Manner J. Bacteriol., April 15, 2009; 191(8): 2902 - 2905. [Abstract] [Full Text] [PDF]
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V. B. Mulay, M. J. Caimano, R. Iyer, S. Dunham-Ems, D. Liveris, M. M. Petzke, I. Schwartz, and J. D. Radolf Borrelia burgdorferi bba74 Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals J. Bacteriol., April 15, 2009; 191(8): 2783 - 2794. [Abstract] [Full Text] [PDF]
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 Z. Ouyang, M. He, T. Oman, X. F. Yang, and M. V. Norgard A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi PNAS, March 3, 2009; 106(9): 3449 - 3454. [Abstract] [Full Text] [PDF]
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