Date Published: June 20, 2014
Publisher: Public Library of Science
Through a series of follow up experiments, the authors have come to understand that the genotype and therefore the in vivo phenotype of the Δbbk46/vector B. burgdorferi clone (1470), the data for which are shown in Table 4, were incorrect. In the publication, the mutant clone was reported to contain all of the B. burgdorferi plasmids of the wild type parent clone; however, this finding was interpreted in error and the authors now understand that in addition to lacking gene bbk46, the mutant clone also lacks plasmid lp28-1. It has been reported previously that B. burgdorferi clones lacking lp28-1 are unable to evade the host immune response resulting in the inability of the spirochetes to persist in mice [1–5]. Because the Δbbk46/vector B. burgdorferi clone (1470) lacks lp28-1, this suggests that the persistence phenotype (lack of spirochete reisolates in the tissues of infected mice 3 weeks post inoculation) shown in Table 4 is a result of the missing lp28-1 plasmid rather than the bbk46 gene. The authors have just completed careful re-derivation of the Δbbk46/vector B. burgdorferi clone (1607), genotype analysis and analysis of the phenotype of the new clone in mouse infectivity. The authors now find that the new Δbbk46/vector B. burgdorferi clone (1607), which contains all of the B. burgdorferi plasmids of the wild-type parent clone, demonstrates wild type serology and tissue reisolation in the 9 out of 9 mice inoculated with 1×104 spirochetes. These data confirm that the persistence phenotype reported for the Δbbk46/vector B. burgdorferi clone (1470) in Table 4 of the publication is due to the loss of the lp28-1 plasmid and not the deletion of the bbk46 gene. The authors have confirmed that the reported genotypes of all other clones in the publication are correct.
Tisha Choudhury Ellis1, Sunny Jain1, Angelika K. Linowski1, Kelli Rike1, Aaron Bestor2, Patricia A. Rosa2, Micah Halpern1, Stephanie Kurhanewicz1 and Mollie W. Jewett1*
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. The bbk46 gene was dispensable for B. burgdorferi infection in mice. Our findings highlight the power of the IVET-based approach for identification of B. burgdorferi in vivo-expressed genes, which might not be discovered using other genome-wide gene expression methods. Further investigation of the novel in vivo-expressed candidate genes will contribute to advancing the understanding of molecular mechanisms of B. burgdorferi survival and pathogenicity in the mammalian host.
Lyme disease is caused by tick-bite transmission of the pathogenic spirochete Borrelia burgdorferi. An increased understanding of how B. burgdorferi survives throughout its infectious cycle is critical for the development of innovative diagnostic and therapeutic protocols to reduce the incidence of Lyme disease. One of the major difficulties blocking this effort has been genome-wide identification of the B. burgdorferi genes that are expressed in the mammalian host environment. Using in vivo expression technology (IVET) in B. burgdorferi for the first time, we have identified B. burgdorferi genes that are expressed during an active murine infection. We demonstrate that candidate gene bbk46, encoded on essential linear plasmid 36, is highly expressed in vivo and, unlike some other known B. burgdorferi in vivo-induced genes, is not RpoS regulated. The bbk46 gene, however, was not found to be required for B. burgdorferi infection in mice. Further studies focused on analysis of the novel B. burgdorferi in vivo-expressed genes identified using IVET will provide insight into the ability of this pathogen to survive in the mammalian host.
Lyme disease is a multi-stage inflammatory disease caused by the pathogenic spirochete Borrelia burgdorferi, which is transmitted by the bite of an infected tick . B. burgdorferi has an enzootic life cycle that requires persistence in two disparate environments, the arthropod vector and the mammalian host. B. burgdorferi is well adapted to modulate its expression profile in response to the different conditions encountered throughout its infectious cycle . Although the specific environmental signals that induce changes in spirochete gene expression are not fully defined, it has been reported that changes in temperature, pH, the presence or absence of mammalian blood, as well as changes in bacterial growth rate, can affect patterns of gene expression [2–8]. DNA microarray analysis and proteomics have been used to examine changes in the global expression profile of B. burgdorferi grown under in vitro conditions that partially mimic the tick and mouse environments [3–5]. A rat dialysis membrane chamber (DMC) implant model, together with microarray technology, has been used to help identify B. burgdorferi genes expressed in response to mammalian host-specific signals [7–10]. Although the data reported in these studies provide insight into the molecular mechanisms of gene regulation, they may not fully reflect the patterns of B. burgdorferi gene expression during an active mammalian infection. Furthermore, transcriptome analysis of B. burgdorferi during murine infection has proven difficult given that spirochete loads in the blood and tissues are too low to recover sufficient spirochete RNA for direct microarray analysis .
Mammalian host-adapted spirochetes demonstrate a 100-fold decrease in ID50 relative to in vitro grown spirochetes. It is clear that B. burgdorferi modulates its gene expression profile at different stages of the infectious cycle . The infectious dose of wild-type B. burgdorferi varies depending on the environment from which the spirochetes are derived. For example, the 50% infectious dose (ID50) of spirochetes derived from partially fed ticks has been found to be two orders of magnitude lower than that of log phase in vitro grown B. burgdorferi . In order to quantitatively assess the impact that adaptation to the mammalian environment has on B. burgdorferi infectivity the 50% infectious dose (ID50) of spirochetes derived directly from the mammalian host was determined and compared to that of log phase in vitro grown spirochetes. B. burgdorferi are only transiently present in the blood of immunocompetent mice , whereas spirochetes persist longer in the blood of immunocompromised mice . Therefore, the blood of severe combined immunodeficiency (scid) mice infected with B. burgdorferi was used as a source of spirochetes adapted to the mammalian environment. Strikingly, an inoculum containing approximately eight in vivo-derived spirochetes was able to infect five out of six mice, whereas, 5,000 in vitro grown spirochetes were required to obtain this level of infectivity (Table 1). The ID50 for in vivo-derived spirochetes was found to be less than eight organisms. In contrast, the ID50 for in vitro grown spirochetes was calculated to be 660 organisms. These data indicate that mammalian host-adapted spirochetes are 100-fold more infectious than in vitro grown spirochetes, likely due to appropriate coordinate expression of in vivo-expressed genes important for murine infectivity.
In this study we have successfully adapted and applied for the first time an IVET-based genetic screen for use in B. burgdorferi for the purpose of identifying spirochete genes that are expressed during mammalian infection. Historically, genetic manipulation of low passage, infectious B. burgdorferi has been challenged by the low transformation frequencies of these spirochetes, preventing application of classic in vivo genetic screening techniques such as in vivo expression technology (IVET) and signature-tagged mutagenesis (STM)  to identify B. burgdorferi genetic elements important for pathogenicity. However, advances in the understanding of the B. burgdorferi restriction modification systems that inhibit transformation [34,50–53] have recently allowed construction and characterization of a comprehensive STM mutant library in infectious B. burgdorferi . The foundation for our strategy for development of IVET in B. burgdorferi was based upon the spirochete’s requirement of lp25 for both restriction modification and virulence functions. Spirochetes lacking lp25 are highly transformable but non-infectious in mice [27–29,34]. Restoration of the lp25-encoded pncA gene to lp25− spirochetes restores wild-type infectivity  but maintains high transformation frequency. At the time of the development of the pBbIVET system the true start codon of the pncA gene was not defined; therefore, the promoter-less pncA gene construct in the pBbIVET plasmid used an engineered AUG start codon and was missing the first 24 nucleotides of the now defined pncA ORF . Furthermore, this construct was purposefully designed without a ribosome binding site (RBS) and was dependent upon the cloned B. burgdorferi DNA fragments to contain both a promoter and a functional RBS. Although we acknowledge that this requirement may have limited the number of clones identified in our screen, during development of the BbIVET system we found that inclusion of an RBS sequence in the promoterless pncA construct resulted in vector-driven PncA production in the absence of a promoter. Thus, in order to reduce the possibility of recovering false positive clones, the pBbIVET system was designed without an RBS. The enzyme Tsp509I was selected to generate the DNA fragments for the pBbIVET library because the AATT restriction site of this enzyme is present approximately every 58 bp in the B. burgdorferi B31 genome. However, it is possible that DNA fragments generated with this enzyme will not result in sequences that contain a 3′ RBS appropriately distanced from the start codon of the pncA ORF, thereby limiting the number of clones identified in the screen.
Ethics statement. The University of Central Florida is accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care. Protocols for all animal experiments were prepared according to the guidelines of the National Institutes of Health and were reviewed and approved by the University of Central Florida Institutional Animal Care and Use Committee (Protocol numbers 09-38 and 12–42).