Polymorphisms Influencing Expression of Dermonecrotic Toxin in <i>Bordetella bronchiseptica</i>

<div><p><i>Bordetella bronchiseptica</i> is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of <i>B. bronchiseptica</i> S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of <i>dnt</i> encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of <i>dnt</i> and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for <i>dnt</i>. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.</p></div

SNP type of the <i>dnt</i> promoter in <i>Bordetella bronchiseptica</i>.

<p>^<i>a</i> SNP types of the region upstream of <i>dnt</i>.</p><p>^<i>b</i> no <i>dnt</i> in genomic DNA or shotgun DNA sequences.</p><p>^<i>c</i> undefined sequence types as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116604#pone.0116604.s002" target="_blank">S2 Fig</a>.</p><p>^<i>d</i> sequence types according to the <i>Bordetella</i> MLST database (<a href="http://pubmlst.org/bordetella" target="_blank">http://pubmlst.org/bordetella</a>).</p><p>^<i>e</i> not designated.</p><p>^<i>f</i> Four complete genome sequences and 58 whole-genome shotgun sequences were obtained from the NCBI website (<a href="http://www.ncbi.nlm.nih.gov/nuccore" target="_blank">http://www.ncbi.nlm.nih.gov/nuccore</a>).</p><p>SNP type of the <i>dnt</i> promoter in <i>Bordetella bronchiseptica</i>.</p

The putative BvgA-recognition sites upstream of <i>dnt</i>.

<p>(A) Table to evaluate the fitness of heptad sequences for BvgA-recognition site according to the algorithm of Merkel <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116604#pone.0116604.ref033" target="_blank">33</a>]. The first row shows the heptad sequences of typical high-affinity binding sites for BvgA. Nucleotide substitutions shown in the table result in decreases of scores by the penalty values indicated in the first column. (B) Heptad sequences presumed as BvgA-recognition sites upstream of <i>dnt</i>. Arrows with numbers indicate the heptad sequences and scores calculated by the algorithm.</p

SNPs upstream of <i>dnt</i> influence the expression level of DNT.

<p>(A) Alignments of SNPs upstream of <i>dnt</i>. The positions +1 and +47 indicate the start sites of transcription and translation, respectively. The strains indicated by boldface are hereafter used as representatives for each SNP type. BP and BB indicate <i>B</i>. <i>pertussis</i> and <i>B</i>. <i>bronchiseptica</i>, respectively. (B) Detection of DNT in whole-cell lysates by immunoblotting. Whole-cell lysates of indicated strains that were cultivated for 12 h were prepared and subjected to SDS-PAGE, followed by immunoblotting, as described in Materials and Methods. The intensity of each band was measured using Image Gauge Version 4.1 (FUJIFILM Corporation, Tokyo, Japan) and the obtained values of DNT signals were normalized by those of FtsZ signals in each lane, and are expressed below the upper panel as relative intensities when DNT in the sample from RB50 is 1.0. Purified DNT was subjected to SDS-PAGE at 50 ng/lane to indicate the position of the toxin. The results of strains R033, TYA13, and STY137 were omitted but showed similar amounts of DNT as other D2-type strains. (C) Measurement of <i>dnt</i> mRNA level by quantitative RT-PCR. Total bacterial RNA was prepared from S798 with D3-type promoter, RB50 with D1-type promoter, AFUY13 with D2-type promoter, and S798P<i>dnt</i>-72C, three independent derivatives of S798 having a nucleotide substitution of C for T at position -72 and subjected to quantitative RT-PCR, as described in Materials and Methods. The relative levels of <i>dnt</i> mRNA normalized to <i>recA</i> mRNA are shown. Each bar represents the mean + standard deviation of three samples. (D) The reporter plasmids harboring a promoter region of D1, D2, or D3 type were introduced into <i>B</i>. <i>bronchiseptica</i> RB50 and the β-galactosidase reporter assay was carried out after incubation of the bacteria for the indicated periods. Dashed black lines represent growth curves of host strain (No-plasmid) as representative data monitored by OD₆₅₀. Each plot represents the mean ± standard deviation of four samples.</p

<i>dnt</i> is missing in mouse isolates.

<p>(A) Detection of DNT in whole-cell lysates of mouse isolates by immunoblotting. Whole-cell lysates were prepared and subjected to SDS-PAGE and immunoblotting as mentioned in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116604#pone.0116604.g001" target="_blank">Fig. 1B</a> and Materials and Methods. The amount of DNT in the samples was estimated as mentioned in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116604#pone.0116604.g001" target="_blank">Fig. 1B</a>. (B) PCR analysis of <i>dnt</i> and surrounding genes. Agarose gel electrophoresis of DNA fragments amplified by PCR targeting <i>dnt</i> and the surrounding genes. The genetic locus and genes amplified are indicated to the left. The PCR for the locus BB3975-BB3979 was carried out with primers corresponding to the locations indicated by thick black arrows in (C), which generates approximately ~9-kbp fragments from strains RB50 (a rabbit isolate) and S798 (a pig isolate). Note that the amplified fragments of ~4-kbp were obtained when mouse isolates were used as templates. (C) Schematic representation of the region surrounding <i>dnt</i>. White and shaded arrows indicate the open-reading frames with their relative lengths and translational directions. The shaded arrows are genes amplified by PCR shown in (B). The thick black arrows indicate the positions of primers used in PCR for amplification of the BB3975–3979 locus shown in (B). In mouse isolates, <i>dnt</i> is replaced by the indicated sequence. The translational stop codons of BB3977 and BB3979 are underlined. Thin arrows indicate the inverted repeat.</p

Activities of <i>dnt</i> promoters, in which nucleotides are substituted at positions -129, -72, +22, and/or +38.

<p>(A) Transcriptional activities of the promoter regions of <i>dnt</i> with or without nucleotide substitutions as indicated in the left column were estimated by the β-galactosidase reporter assay. The nucleotide substitutions were made on the basis of three typical promoter sequences, D1, D2, and D3, which are exemplified by RB50, AFUY13, and S798 strains, respectively. *, P < 0.01; **, P < 0.001; ***, P < 0.0001, compared between indicated samples. Each bar represents the mean + standard deviation of six samples. (B) Detection of DNT in whole-cell lysates by immunoblotting. RB50 (D1 type), AFUY13 (D2), S798 (D3), and an S798 derivative S798P<i>dnt</i>-72C having a nucleotide substitution of C for T at position -72 were cultivated for 12 h and whole-cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting, as described in Materials and Methods. The amount of DNT in the samples was estimated as mentioned in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116604#pone.0116604.g001" target="_blank">Fig. 1B</a>. Purified DNT was subjected to SDS-PAGE at 50 ng/lane to indicate the position of the toxin.</p

The rabbit ileum loop test for the rCPILE of <i>Clostridium perfringens</i> strain W5052.

<p>Fluid accumulation was observed in several ileal loops (arrows) in response to the injected rCPILE. Loop 1, saline; Loop 2, rCPIILE-a (0.1 μg) and trypsin-digested rCPILE-b (0.9 μg); Loop 3, rCPILE-a (1 μg) and trypsin-treated rCPILE-b (9 μg); Loop 4, rCPILE-a (10 μg) and trypsin-treated rCPILE-b (90 μg); and Loop 5, cholera toxin (1 μg).</p

The morphological changes of the L929 cells after treatment with the rCPILE of <i>Clostridium perfringens</i> strain W5052.

<p>The L929 cell cells were treated for 16 hours with various concentrations of the rCPILE-a, trypsin-treated rCPILE-b, and a mixture of rCPILE-a and trypsin-treated rCPILE-b. The morphology of the L929 cells was observed by optical microscopy.</p

The nucleotide and amino acid sequences of the new enterotoxin components, CPILE-a and CPILE-b, of <i>Clostridium perfringens</i> strain W5052.

<p>A -10 region promoter sequence and the putative Shine Dalgarno (S. D.) sequence were presented by single and double underlining, respectively. The in-frame stop-codon is indicated by asterisks.</p

The NADase activity of the rCPILE-a of <i>Clostridium perfringens</i> strain W5052.

<p>Various concentrations of the recombinant component a homologue were incubated at room temperature for 6 hr with 1 mM NAD+, and the NA in the products was quantified by the MS system.</p