<i>c</i>. <i>crescentus</i> ArfB rescues human cells from ICT1 silencing.

<p>Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.</p

ICT1 ribosome release activity supports viability in <i>c</i>. <i>crescentus</i>.

<p>A co-transduction experiment was used to test whether ICT1 complements the synthetic lethal phenotype of deleting <i>ARFB</i> and <i>SSRA</i>. (A) Cartoon depicting the co-transduction experiment and predicted frequency of the outcomes if <i>ARFB</i> were not essential. (B) Column graph indicating the average co-transduction frequency from 3 independent experiments, with error bars indicating the standard deviation.</p

ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.

<p>In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.</p

ICT1 and ArfB share conserved residues that are required for release activity.

<p>Clustal Omega alignment of human ICT1 and ArfB proteins from <i>E</i>. <i>COLI</i> and <i>C</i>. <i>CRESCENTUS</i>. Blue stars indicate residues required for ≥ 60% ICT1 peptidyl-tRNA hydrolysis activity on non-stop ribosomes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005964#pgen.1005964.ref022" target="_blank">22</a>]. Red stars indicate residues required for ≥ 60% <i>E</i>. <i>COLI</i> ArfB hydrolysis activity on non-stop ribosomes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005964#pgen.1005964.ref022" target="_blank">22</a>]. The N-terminal extension of ICT1 contains the mitochondrial localization signal. The remaining 143 C-terminal residues, thought to constitute the active portion of ICT1, share 26% sequence identity with <i>C</i>. <i>CRESCENTUS</i> ArfB.</p

expression of ICT1 partially complements the growth defect of δ<i>ssra</i> cells.

<p>Growth of wild-type cells, Δ<i>SSRA</i> cells, or Δ<i>SSRA</i> cells expressing ArfB or ICT1 from a plasmid was monitored during exponential phase. The average doubling times (± standard deviation) from ≥3 experiments are shown.</p

Strains, plasmids, primers and RNAs used in this study.

<p>Strains, plasmids, primers and RNAs used in this study.</p

Host Specificity of Ovine <i>Bordetella parapertussis</i> and the Role of Complement

<div><p>The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, <i>Bordetella parapertussis</i>, is composed of two distinct clades that have specialized to different hosts: one to humans (<i>Bpp_hu</i>), and the other to sheep (<i>Bpp_ov</i>). While <i>Bpp_hu</i> and the other classical bordetellae can efficiently colonize mice, <i>Bpp_ov</i> strains are severely defective in their ability to colonize the murine respiratory tract. <i>Bpp_ov</i> genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on <i>B</i>. <i>bronchiseptica</i> and <i>Bpp_hu</i> strains. <i>Bpp_ov</i> lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing <i>in vitro</i>. Based on these results, we hypothesized that <i>Bpp_ov</i> failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the <i>Bpp_ov</i> defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, <i>Bpp_ov</i> strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of <i>Bpp_ov</i> to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.</p></div

Sheep serum does not kill Bpp5.

<p><i>B</i>. <i>bronchiseptica</i> RB50 (diamond), RB50Δ<i>wbm</i> (square), <i>B</i>. <i>parapertussis</i>_<i>hu</i> 12822 (empty triangle) or <i>B</i>. <i>parapertussis</i>_<i>ov</i> Bpp5 (filled triangle) or HI (circle) were incubated with PBS (solid lines) or CVF treated (dashed lines) sheep serum for 1 hour at the indicated concentrations. The average percent survival of three independent experiments is shown +/- standard error. * indicates a <i>p</i> value of ≤0.05 between <i>B</i>. <i>parapertussis</i>_<i>ov</i> strains and <i>B</i>. <i>bronchiseptica</i> strain RB50.</p

Maximum likelihood genome-wide SNP tree and murine lung colonization of the classical bordetellae.

<p>(A) Genomes of bordetellae <i>B</i>. <i>pertussis</i> strains Tohama I, <i>B</i>. <i>parapertussis</i> strains Bpp5, 12822, and <i>B</i>. <i>bronchiseptica</i> strains 253, 1289, MO149 were mapped against reference genome RB50. The ML tree (100 bootstrap replicates, 50% bootstrap cut-off) was estimated with RAxML. (B) Bacterial numbers are represented as percent change in the average CFU from day 0 to 7 post-inoculation (100% recovery cut-off) of three to four mice per time point.</p

Complement efficiently deposits onto the cell surface and kills Bpp5.

<p>(A) <i>B</i>. <i>bronchiseptica</i> RB50 (diamond), RB50Δ<i>wbm</i> (square) or <i>B</i>. <i>parapertussis</i>_<i>ov</i> Bpp5 (triangle) were incubated with complement active (solid lines) or complement inactive serum (dashed lines) at the indicated concentrations for 1 hour. The average percent survival of three independent experiments is shown +/- standard error. (B) Flow cytometry analysis of C3b deposition onto RB50, RB50Δ<i>wbm</i> or Bpp5 incubated with complement sufficient (solid line) or deficient (dashed line) serum. Samples were unstained or stained with FITC-anti-mouse C3 antibodies and analyzed and a representative image was shown. The average percentage of FITC-positive cells of three replicates is indicated +/- standard error. * indicates <i>p</i> value <0.05 in comparison to RB50.</p