In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice

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

Spirochetes lacking <i>bbk46</i> retain seroreactivity in mice.

<p>Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), <i>bbk46</i>::<i>flaB</i>_p-<i>aadA</i>/pBSV2G (Δ<i>bbk46</i>/vector) and <i>bbk46</i>::<i>flaB</i>_p-<i>aadA</i>/pBSV2G-<i>bbk46</i> (Δ<i>bbk46</i>/<i>bbk46⁺</i>) at a dose of 1×10⁴ spirochetes per mouse. (A) Total protein lysate from <i>B. burgdorferi</i> clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.</p

List of primers used in this study.

a<p>Lowercase indicates all non-<i>B. burgdorferi</i> sequence.</p

Schematic representation of the pBbIVET vector.

<p>Features of this vector include: 3XTT, the transcriptional terminator sequence for <i>bmpB </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003567#ppat.1003567-Ramamoorthy1" target="_blank">[22]</a> repeated in triplicate; <i>pncA</i>, promoterless <i>pncA</i> gene; <i>flgB</i>_p<i>kan</i>, kanamycin resistance cassette; <i>zeo</i>, zeocin resistance marker; ColE1, <i>E. coli</i> origin of replication; ORFs 1, 2, 3, <i>B. burgdorferi</i> cp9 replication machinery. The EcoRI restriction site was used to clone the <i>B. burgdorferi</i> control <i>in vivo</i>-expressed promoter, <i>ospC</i>_p, as well as the <i>B. burgdorferi</i> (<i>Bb</i>) gDNA library, in front of the promoterless <i>pncA</i> gene. The pBbIVET vector was derived from the <i>B. burgdorferi</i> shuttle vector pBSV2* <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003567#ppat.1003567-Bestor2" target="_blank">[80]</a>.</p

Expression of the <i>bbk46</i> gene is upregulated during murine infection and is RpoS-independent.

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

The <i>bbk46</i> gene is required for persistent infection of immunocompetent mice.

a<p>Determined 3 weeks post inoculation by serological response to <i>B. burgdorferi</i> total protein lysate and recombinant OspC protein. NA, not applicable.</p>b<p>Number of mice positive for spirochete reisolation/number of mice analyzed. NA, not applicable.</p

Amino acid alignment of the putative members of the immunogenic protein P37 family encoded on lp36.

<p>Shown is an amino acid alignment of the P37 protein family members BBK45 (GenBank accession no. NP_045617.2), BBK46 (translated <i>bbk46</i>, Genbank GeneID: 1194234), BBK48 (GenBank accession no. NP_045619.1) and BBK50 (GenBank accession no. NP_045621.1). Amino acids identical to the consensus sequence are shaded. The predicted SpLip lipobox sequence <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003567#ppat.1003567-Setubal1" target="_blank">[81]</a> is indicted with five stars. Dashes represent spaces introduced for optimal sequence alignment. The positions of the two stop codons in the <i>bbk46</i> translation are indicated with arrows. Amino acid sequences were aligned using the CLUSTAL W algorithm in the MEGALIGN program from the DNASTAR Lasergene suite.</p

<i>B. burgdorferi in vivo</i>-expressed candidate genes organized by functional category.

a<p>In some cases two <i>Bbive</i> clones shared overlapping, non-identical sequence, as indicated by two <i>Bbive</i> clone numbers.</p>b<p>ORF, open reading frame that maps just downstream and in the same orientation to the <i>Bbive</i> sequence.</p>c<p>Annotation described by Fraser <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003567#ppat.1003567-Fraser1" target="_blank">[45]</a>.</p

In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice

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