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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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Expression of the bbk46 gene is upregulated during murine infection and is RpoS-independent.Total RNA was isolated from bladder tissue collected from (A) mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation (in vivo, gray bars) and from log phase in vitro grown spirochetes (in vitro, white bars) or (B) stationary phase temperature-shifted stationary phase in vitro grown wild-type (white bars) or ΔrpoS (gray bars) B. burgdorferi. RNA was reverse transcribed to cDNA using random hexamer primers. The expression of bbk46, flaB and ospC were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and a standard curve analysis method. The mRNA levels of the bbk46, flaB and ospC gene transcripts were normalized to that of the constitutive recA gene. The data are expressed as the gene transcript/recA transcript. The data represent the average of triplicate qRT-PCR analyses of 3 biological replicates. Error bars represent the standard deviation from the mean.
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ppat-1003567-g003: Expression of the bbk46 gene is upregulated during murine infection and is RpoS-independent.Total RNA was isolated from bladder tissue collected from (A) mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation (in vivo, gray bars) and from log phase in vitro grown spirochetes (in vitro, white bars) or (B) stationary phase temperature-shifted stationary phase in vitro grown wild-type (white bars) or ΔrpoS (gray bars) B. burgdorferi. RNA was reverse transcribed to cDNA using random hexamer primers. The expression of bbk46, flaB and ospC were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and a standard curve analysis method. The mRNA levels of the bbk46, flaB and ospC gene transcripts were normalized to that of the constitutive recA gene. The data are expressed as the gene transcript/recA transcript. The data represent the average of triplicate qRT-PCR analyses of 3 biological replicates. Error bars represent the standard deviation from the mean.

Mentions: Our BbIVET screen identified gene bbk46 as a putative in vivo-expressed gene. The BbIVET screen was designed to identify B. burgdorferi DNA fragments that are expressed in vivo and did not discriminate between those promoters that are specifically induced in vivo and those promoters that are expressed both in vitro and in vivo. Therefore, quantitative reverse transcription PCR (qRT-PCR) was used to validate the expression of bbk46 in vivo and to determine whether bbk46 expression was upregulated in vivo compared to in vitro. Total RNA was isolated from bladder tissue collected from mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation as well as log phase in vitro grown spirochetes. RNA was converted to cDNA using random hexamer primers and the mRNA level of each target gene was measured relative to the constitutive recA gene using quantitative PCR. The gene expression levels of flaB and ospC were also measured as control constitutively-expressed and in vivo-induced genes, respectively. These data demonstrated that although bbk46 was expressed during in vitro growth, expression of this gene was increased more than 100-fold during mammalian infection (Figure 3A). Consistent with their known patterns of gene regulation, flaB expression was relatively unchanged in vivo compared to in vitro; whereas, ospC demonstrated a nearly 1000-fold increase in expression in vivo compared to in vitro (Figure 3A). Moreover, the relative amount of bbk46 expression during in vitro growth was found to be approximately 10-fold more than that of ospC. Whereas, the in vivo expression levels of genes bbk46, ospC and flaB were similar.


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)

Expression of the bbk46 gene is upregulated during murine infection and is RpoS-independent.Total RNA was isolated from bladder tissue collected from (A) mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation (in vivo, gray bars) and from log phase in vitro grown spirochetes (in vitro, white bars) or (B) stationary phase temperature-shifted stationary phase in vitro grown wild-type (white bars) or ΔrpoS (gray bars) B. burgdorferi. RNA was reverse transcribed to cDNA using random hexamer primers. The expression of bbk46, flaB and ospC were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and a standard curve analysis method. The mRNA levels of the bbk46, flaB and ospC gene transcripts were normalized to that of the constitutive recA gene. The data are expressed as the gene transcript/recA transcript. The data represent the average of triplicate qRT-PCR analyses of 3 biological replicates. Error bars represent the standard deviation from the mean.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757035&req=5

ppat-1003567-g003: Expression of the bbk46 gene is upregulated during murine infection and is RpoS-independent.Total RNA was isolated from bladder tissue collected from (A) mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation (in vivo, gray bars) and from log phase in vitro grown spirochetes (in vitro, white bars) or (B) stationary phase temperature-shifted stationary phase in vitro grown wild-type (white bars) or ΔrpoS (gray bars) B. burgdorferi. RNA was reverse transcribed to cDNA using random hexamer primers. The expression of bbk46, flaB and ospC were quantified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and a standard curve analysis method. The mRNA levels of the bbk46, flaB and ospC gene transcripts were normalized to that of the constitutive recA gene. The data are expressed as the gene transcript/recA transcript. The data represent the average of triplicate qRT-PCR analyses of 3 biological replicates. Error bars represent the standard deviation from the mean.
Mentions: Our BbIVET screen identified gene bbk46 as a putative in vivo-expressed gene. The BbIVET screen was designed to identify B. burgdorferi DNA fragments that are expressed in vivo and did not discriminate between those promoters that are specifically induced in vivo and those promoters that are expressed both in vitro and in vivo. Therefore, quantitative reverse transcription PCR (qRT-PCR) was used to validate the expression of bbk46 in vivo and to determine whether bbk46 expression was upregulated in vivo compared to in vitro. Total RNA was isolated from bladder tissue collected from mice infected with 1×105 wild-type B. burgdorferi three weeks post-inoculation as well as log phase in vitro grown spirochetes. RNA was converted to cDNA using random hexamer primers and the mRNA level of each target gene was measured relative to the constitutive recA gene using quantitative PCR. The gene expression levels of flaB and ospC were also measured as control constitutively-expressed and in vivo-induced genes, respectively. These data demonstrated that although bbk46 was expressed during in vitro growth, expression of this gene was increased more than 100-fold during mammalian infection (Figure 3A). Consistent with their known patterns of gene regulation, flaB expression was relatively unchanged in vivo compared to in vitro; whereas, ospC demonstrated a nearly 1000-fold increase in expression in vivo compared to in vitro (Figure 3A). Moreover, the relative amount of bbk46 expression during in vitro growth was found to be approximately 10-fold more than that of ospC. Whereas, the in vivo expression levels of genes bbk46, ospC and flaB were similar.

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