Horizontally acquired divergent O-antigen contributes to escape from cross-immunity in the classical bordetellae

BACKGROUND: Horizontal gene transfer (HGT) allows for rapid spread of genetic material between species, increasing genetic and phenotypic diversity. Although HGT contributes to adaptation and is widespread in many bacteria, others show little HGT. This study builds on previous work to analyze the evolutionary mechanisms contributing to variation within the locus encoding a prominent antigen of the classical bordetellae. RESULTS: We observed amongst classical bordetellae discrete regions of the lipopolysaccharide O-antigen locus with higher sequence diversity than the genome average. Regions of this locus had less than 50% sequence similarity, low dN/dS ratios and lower GC content compared to the genome average. Additionally, phylogenetic tree topologies based on genome-wide SNPs were incongruent with those based on genes within these variable regions, suggesting portions of the O-antigen locus may have been horizontally transferred. Furthermore, several predicted recombination breakpoints correspond with the ends of these variable regions. To examine the evolutionary forces that might have selected for this rare example of HGT in bordetellae, we compared in vitro and in vivo phenotypes associated with different O-antigen types. Antibodies against O1- and O2-serotypes were poorly cross-reactive, and did not efficiently kill or mediate clearance of alternative O-type bacteria, while a distinct and poorly immunogenic O-antigen offered no protection against colonization. CONCLUSIONS: This study suggests that O-antigen variation was introduced to the classical bordetellae via HGT through recombination. Additionally, genetic variation may be maintained within the O-antigen locus because it can provide escape from immunity to different O-antigen types, potentially allowing for the circulation of different Bordetella strains within the same host population

Release of nonstop ribosomes is essential

Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-based trans-translation pathway to release these "nonstop" ribosomes and maintain protein synthesis capacity. trans-translation is essential in some species, but in others, such as Caulobacter crescentus, trans-translation can be inactivated. To determine why trans-translation is dispensable in C. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lacking ssrA, the gene encoding tmRNA. One of these genes, CC1214, was essential in ΔssrA cells. Purified CC1214 protein could release nonstop ribosomes in vitro. CC1214 is a homolog of the Escherichia coli ArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in which ssrA has been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria

Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans

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

<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 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

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

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