RNA-Seq-based analysis of changes in Borrelia burgdorferi gene expression linked to pathogenicity
© Wu et al.; licensee BioMed Central. 2015
Received: 27 September 2014
Accepted: 26 December 2014
Published: 13 March 2015
Lyme disease is a global public health problem caused by the spirochaete Borrelia burgdorferi. Our previous studies found differences in disease severity between B. burgdorferi B31- and B. garinii SZ-infected mice. We hypothesized that genes that are differentially expressed between Borrelia isolates encode bacterial factors that contribute to disease diversity.
The present study used high-throughput sequencing technology to characterize and compare the transcriptional profiles of B. burgdorferi B31 and B. garinii SZ cultured in vitro. Real-time quantitative RT-PCR was used to validate selected data from RNA-seq experiments.
A total of 731 genes were differentially expressed between B. burgdorferi B31 and B. garinii SZ isolates, including those encoding lipoproteins and purine transport proteins. The fold difference in expression for B. garinii SZ versus B. burgdorferi B31 ranged from 22.07 to 1.01. Expression of the OspA, OspB and DbpB genes were significantly lower in B. garinii SZ compared to B. burgdorferi B31.
The results support the hypothesis that global changes in gene expression underlie differences in Borrelia pathogenicity. The findings also provide an empirical basis for studying the mechanism of action of specific genes as well as their potential usefulness for the diagnosis and management of Lyme disease.
Borrelia burgdorferi is the causative agent of Lyme disease, the most prevalent tick-borne zoonosis and an important emerging infectious disease in Europe, North America, and Far Eastern countries . The Borrelia burgdorferi complex consists of 18 proposed and confirmed genospecies . The obligate parasites are transmitted by ticks of Ixodes spp., with disease symptoms and severity varying among B. burgdorferi genospecies. B. garinii is primarily associated with neuroborreliosis , B. afzelii with crodermatitis chronic athrophicans , and B. burgdorferi s.s is the major cause of Lyme arthritis .
Despite its small genome, the spirochaetes possess complex cellular machinery for regulating gene and protein expression. B. burgdorferi expresses specific subsets of genes throughout its life cycle, both in the arthropod vector and vertebrate host [6,7]. In one study of bacterial protein expression in infected mouse tissues, VlsE, OspC, and decorin-binding protein (Dbp)A were expressed at high levels in joints and dermal tissues, while OspC and DbpA were also detected in the heart , demonstrating tissue-specific protein expression. A comparative analysis of protein expression profiles of three strains of B. burgdorferi (B31, ND40, and JD-1) demonstrated large differences in the percentage of peptide coverage of proteins . The application of genome, transcriptome, interactome, and immunoproteome analyses can reveal complexities of bacterial physiology and pathogenesis; in addition, the development of massively parallel cDNA sequencing (RNA-seq) techniques is enabling more comprehensive and accurate assessments of eukaryote  and prokaryote  transcriptomes.
Our previous studies found differences in disease severity between B. burgdorferi B31- and B. garinii SZ-infected mice, particularly affecting the brain, heart, liver, and spleen tissues . Differential gene expression facilitates spirochaetal survival and promotes disease pathogenesis. In the present study, RNA-seq was employed to compare the transcriptome profiles of B. burgdorferi B31 and B. garinii SZ isolates during in vitro culture. The differences in gene expression profiles between the two species of spirochetes provide insights into disease-specific mechanisms.
B. burgdorferi B31 and B. garinii SZ were used in this study. B31 was purchased from the American Type Culture Collection (Manassas, VA, USA) and had undergone five in vitro passages. B. garinii SZ was isolated from Dermacentor ticks collected in Shangzhi county of Heilongjiang province in China . The strains were cultured in BSK-H medium in a 33°C incubator and observed under a dark-field microscope every other day. Cells were harvested by centrifugation at a speed of 5,000 × g during logarithmic phase and washed twice with phosphate buffered saline (PBS).
RNA was extracted using TRIzol reagent (Invitrogen). RNA concentration and quality were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA (10 μg) was pooled from three individual cells of each strain and used to construct two cDNA libraries following the mRNA sequencing sample preparation guide (Illumina, San Diego, CA, USA). Paired-end DNA sequencing was carried out in two lanes (one per library) on an Illumina HiSeq 2000 following the manufacturer’s protocol. The 16S and 23S rRNA was removed from total RNA using the MICROBExpresst Bacterial mRNA Purification Kit (Ambion, Foster City, CA, USA) according to the manufacturer’s protocol.
Sequence assembly and annotation
The 100-bp paired-end Illumina reads from the B. burgdorferi B31 (82,056,756 reads) and B. garinii SZ (145,680,918 reads) libraries were combined for de novo assembly. Reads that were of low quality (≥80% with Phred score < 20) or complexity (>80% with single, di-, or trinucleotide repeats) or were < 20 bp were removed. The processed reads were then assembled using the CLC Genomics Workbench v.5.5 [14,15] with wordsize = 45 and minimum contig length ≥ 200. The resulting assembled sequences and singletons were combined and processed to remove duplicates using a custom Perl Script; contigs were then assembled using CAP3 EST to obtain the final unigenes.
Functional annotation of unigenes was achieved by searching for analogous sequences in EMBL and Swiss-Prot databases using an E-value ≤ 1e − 5. Hierarchical functional categorization for gene ontology (GO) terms was accomplished using BLAST2GO, which was also used to identify genes represented among the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
Real-time quantitative reverse transcription (qRT)-PCR
Real-time qRT-PCR was used to validate data from RNA-seq experiments. Gene-specific primers (Additional file 1: Table S7) were designed using Primer Express software (Applied Biosystems, Carlsbad, CA, USA). The relative quantitation (ΔΔCt) method was used to evaluate differences between the two genospecies for each gene examined. The flaB amplicon was used as an internal control to normalize all data. Removal of genomic DNA and reverse transcription (Takara Bio Inc., Otsu, Japan) were performed for each sample and standard without reverse transcriptase to confirm the absence of genomic DNA.
Results and discussion
Whole-transcriptome profiling of bacteria has been widely used to evaluate global changes in gene expression . RNA-seq-based transcriptome analyses of pathogens during infection yields a robust, sensitive, and accessible dataset that enables the assessment of the regulatory interactions driving pathogenesis . Our previous studies revealed differences in disease severity between B. burgdorferi B31- and B. garinii SZ-infected mice ; the present study used RNA-seq to determine the transcriptional profiles of B. burgdorferi B31 and B. garinii SZ isolates during in vitro infection. This is the first comprehensive analysis of gene expression in this organism; the findings are discussed in the context of pathogenesis, diagnosis, and management of Lyme disease.
Sequence assembly and annotation
Genes with the highest transcript levels in B. garinii SZ
Log 2 (fold change)
Erf superfamily protein
Mlp lipofamily protein
Outer surface protein E
Outer surface protein D
Basic membrane protein D
Borrelia membrane P13 family protein
Outer membrane protein Omp121
Membrane-associated protein P66
Genes with the highest transcript levels in B. burgdorferi B31
Log 2 (fold change)
Decorin binding protein B DbpA/B
Outer surface protein A
Outer surface protein B (OspB)
Basic membrane protein A
Complement regulator-acquiring surface protein 3 precursor protein ErpP
Surface lipoprotein P27
Membrane protein insertase YidC
Flagellar M-ring protein FliF
Putative uncharacterized protein
Purine transport proteins
The uptake of preformed purines by spirochete represents the first step in the purine salvage pathway, which is critical for the infection of mammalian hosts by B. burgdorferi. The genes bbb22 and bbb23, which are present on circular plasmid 26, encode key purine transport proteins that are essential for hypoxanthine, adenine, and guanine transport , while inosine-5′-monophosphate dehydrogenase (encoded by GuaB) and guanosine monophoshpate synthase (encoded by GuaA) are two key enzymes in the purine salvage pathway . GuaA and B were significantly upregulated in B. burgdorferi B31 as compared to B. garinii SZ (Additional file 1: Tables S1 and S2). Genes encoding bifunctional purine biosynthesis protein (PurH) and non-canonical purine nucleoside triphosphate (NTP) pyrophosphatase were also identified. These findings suggest that this transport system is a potential target for antimicrobial agents in the treatment of Lyme disease.
Confirmation of RNA-seq data by qRT-PCR
Given the increasing incidence of and medical concerns related to Lyme disease, vaccination, drug treatment, and pathogenic mechanisms have received considerable attention. Some insight is gained from the study of other faculative pathogens such as those responsible for cholera and malaria using high-throughput cDNA sequencing techniques on organisms grown in laboratory medium or isolated from infected hosts [11,32]. Thus, RNA-seq-based transcriptome analyses of pathogens during infection offer robust, sensitive, and accessible datasets for evaluating regulatory mechanisms driving pathogenesis .
The availability of fully sequenced genomes offers new opportunities to identify genotype–phenotype relationships and undertake global genomic, proteomic, and transcriptomic analyses to investigate the biological significance of paralogous gene families and other unique features of genomes. The present study is the first to characterize the transcriptome of B. burgdorferi, the causative agent of Lyme disease. Some novel genes, including a bifunctional PurH and non-canonical purine NTP pyrophosphatase, were also identified that could potentially be targeted by antimicrobial agents for disease treatment. Moreover, the differential expression of specific factors observed between Borrelia genospecies could explain the variation in disease pathogenicity. These findings provide a framework for future studies examining the molecular mechanisms underlying the pathogenicity of Lyme disease.
This study was financially supported by the “973” Program (2010CB530206),NSFC (31272556, 30972182, 31072130, 31001061), “948” (2010-S04), Key Project of Gansu Province (1002NKDA035), NBCITS. MOA (CARS-38), Specific Fund for Sino-Europe Cooperation, MOST, China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019). The research was also facilitated by EPIZONE (FOOD-CT-2006-016236), ASFRISK (211691), ARBOZOONET (211757) and PIROVAC (KBBE-3-245145) of the European Commission, Brussels, Belgium.
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