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In vivo expression technology identifies a novel virulence factor critical for Borrelia burgdorferi persistence in mice.

Ellis TC, Jain S, Linowski AK, Rike K, Bestor A, Rosa PA, Halpern M, Kurhanewicz S, Jewett MW - PLoS Pathog. (2013)

Bottom Line: Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.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.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America.

ABSTRACT
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. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

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Schematic representation of the pBbIVET vector.Features of this vector include: 3XTT, the transcriptional terminator sequence for bmpB[22] repeated in triplicate; pncA, promoterless pncA gene; flgBpkan, kanamycin resistance cassette; zeo, zeocin resistance marker; ColE1, E. coli origin of replication; ORFs 1, 2, 3, B. burgdorferi cp9 replication machinery. The EcoRI restriction site was used to clone the B. burgdorferi control in vivo-expressed promoter, ospCp, as well as the B. burgdorferi (Bb) gDNA library, in front of the promoterless pncA gene. The pBbIVET vector was derived from the B. burgdorferi shuttle vector pBSV2* [80].
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ppat-1003567-g001: Schematic representation of the pBbIVET vector.Features of this vector include: 3XTT, the transcriptional terminator sequence for bmpB[22] repeated in triplicate; pncA, promoterless pncA gene; flgBpkan, kanamycin resistance cassette; zeo, zeocin resistance marker; ColE1, E. coli origin of replication; ORFs 1, 2, 3, B. burgdorferi cp9 replication machinery. The EcoRI restriction site was used to clone the B. burgdorferi control in vivo-expressed promoter, ospCp, as well as the B. burgdorferi (Bb) gDNA library, in front of the promoterless pncA gene. The pBbIVET vector was derived from the B. burgdorferi shuttle vector pBSV2* [80].

Mentions: We have developed a genome-wide genetic screening method to identify B. burgdorferi genes that are expressed during mouse infection using an in vivo expression technology (IVET) approach [12], [13]. The in vivo expression technology vector, pBbIVET, carries the B. burgdorferi bmpB Rho-independent transcription terminator sequence [22] repeated in triplicate (3XTT), to prevent any read-through promoter activity from the pBSV2* Borrelia shuttle vector backbone, followed by the promoter-less in vivo-essential pncA gene (Figure 1) [23], [24]. Spirochetes lacking linear plasmid (lp) 25 are non-infectious in mice and severely compromised in the tick vector [25]–[31]. The pncA gene, located on lp25, encodes a nicotinamidase that is sufficient to restore murine infectivity to B. burgdorferi lacking the entire lp25 plasmid [24]. Genetic transformation of low-passage, infectious B. burgdorferi occurs at low frequency and efficiency hampering introduction of a complex DNA library into an infectious background [32], [33]. Because B. burgdorferi clones lacking lp25 and lp56 demonstrate increased transformability [34], we isolated a clonal derivative of the low-passage infectious clone A3 that lacks both lp25 and lp56. This clone was designated A3 68-1 [35]. Clones A3 and A3 68-1 were transformed by electroporation with 20 µg of a Borrelia shuttle vector. The transformation frequency and efficiency of A3 68-1 was determined relative to that of the A3 parent. As expected, genetic transformation of A3 68-1 occurred at a high frequency and efficiency. We recovered approximately 2,000 transformants/ml in clone A3 68-1, whereas no transformants were recovered with a parallel transformation of clone A3 (data not shown). In order to test the function of the B. burgdorferi IVET system, the promoter for the in vivo essential ospC gene [36], was cloned in front of the promoter-less pncA gene in pBbIVET (Figure 1), creating plasmid pBbIVET-ospCp. This plasmid, along with pBbIVET alone, was transformed into the non-infectious, low-passage, highly transformable B. burgdorferi clone A3 68-1. All clones were tested for their abilities to infect groups of 6 C3H/HeN mice at an infectious dose 100 (ID100) of 1×104 spirochetes [37], indicating the presence or absence of an active promoter sufficient to drive expression of pncA thereby restoring infectivity. Spirochetes were reisolated from the ear, bladder and joint tissues of 5/6 mice infected with B. burgdorferi harboring pBbIVET-ospCp. No spirochetes were reisolated from mice (0/6) infected with B. burgdorferi carrying the promoter-less pBbIVET alone. Together these data demonstrated that our promoter trap system functioned with a known in vivo active promoter.


In vivo expression technology identifies a novel virulence factor critical for Borrelia burgdorferi persistence in mice.

Ellis TC, Jain S, Linowski AK, Rike K, Bestor A, Rosa PA, Halpern M, Kurhanewicz S, Jewett MW - PLoS Pathog. (2013)

Schematic representation of the pBbIVET vector.Features of this vector include: 3XTT, the transcriptional terminator sequence for bmpB[22] repeated in triplicate; pncA, promoterless pncA gene; flgBpkan, kanamycin resistance cassette; zeo, zeocin resistance marker; ColE1, E. coli origin of replication; ORFs 1, 2, 3, B. burgdorferi cp9 replication machinery. The EcoRI restriction site was used to clone the B. burgdorferi control in vivo-expressed promoter, ospCp, as well as the B. burgdorferi (Bb) gDNA library, in front of the promoterless pncA gene. The pBbIVET vector was derived from the B. burgdorferi shuttle vector pBSV2* [80].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757035&req=5

ppat-1003567-g001: Schematic representation of the pBbIVET vector.Features of this vector include: 3XTT, the transcriptional terminator sequence for bmpB[22] repeated in triplicate; pncA, promoterless pncA gene; flgBpkan, kanamycin resistance cassette; zeo, zeocin resistance marker; ColE1, E. coli origin of replication; ORFs 1, 2, 3, B. burgdorferi cp9 replication machinery. The EcoRI restriction site was used to clone the B. burgdorferi control in vivo-expressed promoter, ospCp, as well as the B. burgdorferi (Bb) gDNA library, in front of the promoterless pncA gene. The pBbIVET vector was derived from the B. burgdorferi shuttle vector pBSV2* [80].
Mentions: We have developed a genome-wide genetic screening method to identify B. burgdorferi genes that are expressed during mouse infection using an in vivo expression technology (IVET) approach [12], [13]. The in vivo expression technology vector, pBbIVET, carries the B. burgdorferi bmpB Rho-independent transcription terminator sequence [22] repeated in triplicate (3XTT), to prevent any read-through promoter activity from the pBSV2* Borrelia shuttle vector backbone, followed by the promoter-less in vivo-essential pncA gene (Figure 1) [23], [24]. Spirochetes lacking linear plasmid (lp) 25 are non-infectious in mice and severely compromised in the tick vector [25]–[31]. The pncA gene, located on lp25, encodes a nicotinamidase that is sufficient to restore murine infectivity to B. burgdorferi lacking the entire lp25 plasmid [24]. Genetic transformation of low-passage, infectious B. burgdorferi occurs at low frequency and efficiency hampering introduction of a complex DNA library into an infectious background [32], [33]. Because B. burgdorferi clones lacking lp25 and lp56 demonstrate increased transformability [34], we isolated a clonal derivative of the low-passage infectious clone A3 that lacks both lp25 and lp56. This clone was designated A3 68-1 [35]. Clones A3 and A3 68-1 were transformed by electroporation with 20 µg of a Borrelia shuttle vector. The transformation frequency and efficiency of A3 68-1 was determined relative to that of the A3 parent. As expected, genetic transformation of A3 68-1 occurred at a high frequency and efficiency. We recovered approximately 2,000 transformants/ml in clone A3 68-1, whereas no transformants were recovered with a parallel transformation of clone A3 (data not shown). In order to test the function of the B. burgdorferi IVET system, the promoter for the in vivo essential ospC gene [36], was cloned in front of the promoter-less pncA gene in pBbIVET (Figure 1), creating plasmid pBbIVET-ospCp. This plasmid, along with pBbIVET alone, was transformed into the non-infectious, low-passage, highly transformable B. burgdorferi clone A3 68-1. All clones were tested for their abilities to infect groups of 6 C3H/HeN mice at an infectious dose 100 (ID100) of 1×104 spirochetes [37], indicating the presence or absence of an active promoter sufficient to drive expression of pncA thereby restoring infectivity. Spirochetes were reisolated from the ear, bladder and joint tissues of 5/6 mice infected with B. burgdorferi harboring pBbIVET-ospCp. No spirochetes were reisolated from mice (0/6) infected with B. burgdorferi carrying the promoter-less pBbIVET alone. Together these data demonstrated that our promoter trap system functioned with a known in vivo active promoter.

Bottom Line: Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.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.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America.

ABSTRACT
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. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

Show MeSH
Related in: MedlinePlus