Transferred interbacterial antagonism genes augment eukaryotic innate immune function

Abstract

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits1. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option2. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system3. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

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Figure 1: Recurrent horizontal gene transfer of tae genes into diverse eukaryotic lineages.
Figure 2: Eukaryotic dae genes encode differentially expressed PG amidases with conserved specificity.
Figure 3: Dae2 is a bacteriolytic toxin that restricts the proliferation of B. burgdorferi in the tick I. scapularis.

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Acknowledgements

We thank L. Holland for assistance with transcriptome analysis, D. Vollmer and C. Aldridge for PG preparation, J. Parrish for microinjection assistance, J. Young for assistance with phylogenetic analyses, H. Merrikh for sharing equipment, and T. Alber, C. Fuqua, K. Clay, E. Rynkiewicz, U. Pal, C. Grundner, G. Nester, P. Singh and members of the Malik and Mougous laboratories for helpful discussions. This work was funded by the National Institutes of Health (AI080609 to J.D.M. and AI083640 to X.F.Y.), the Defense Threat Reduction Agency (HDTRA-1-13-014 to J.D.M.) and the BBSRC (BB/I020012/1 to W.V.). S.C. was supported by a Howard Hughes Medical Institute (HHMI)-sponsored Life Sciences Research Foundation fellowship, M.A.F. by the American Society for Microbiology Undergraduate Research Fellowship, and M.D.D. by an Irvington Institute Fellowship from the Cancer Research Institute. C.J.-W. and H.S.M. are investigators of the HHMI. J.D.M. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

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S.C., M.D.D., H.S.M. and J.D.M. designed the study. S.C., M.D.D., S.B.P., J.B., Y.Y., B.L.J., L.K.F.-L., M.A.F., B.N.H., C.J.-W., X.F.Y., W.V., H.S.M. and J.D.M. performed experiments, analysed data and provided intellectual input into aspects of this study. S.C., M.D.D., S.B.P., H.S.M. and J.D.M. wrote the manuscript; all authors contributed to its editing.

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Correspondence to Joseph D. Mougous.

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Extended data figures and tables

Extended Data Figure 1 Genomic evidence for validated eukaryotic dae genes.

Eukaryotic dae genes from the indicated organisms are listed adjacent to schematic representations of available predicted open reading frames (colour-coded according to family as in Fig. 1a) and corresponding genomic context of dae genes. Flanking genes are colour-coded according to organisms that homologues of these genes are found in (broadly, in eukaryotes, black; only closely related eukaryotic species, grey; both bacteria and eukaryotes, white). Diagonal lines denote ends of genomic contigs. In the right column, splice sites (red vertical lines) and conserved intron positions (red dashed circles) are shown. In Oxytricha trifallax, the somatic nucleus (macronucleus) contains 16,000 chromosomes and is a rearranged form of the germline nucleus (micronucleus)28. The complete dae3 gene in Oxytricha is found in the macronucleus on a chromosome with three characteristic GGGGTTTT telomere sequences. Three fragments comprising the dae3 gene are found in the micronuclear genome (http://oxy.ciliate.org/). In Nematostella vectensis and Branchiostoma floridae, lineage-specific duplication events have resulted in two adjacent dae4 paralogues with gene names labelled (numbers). In Capitella teleta and Lottia gigantea, shared synteny on both sides of the dae4 gene is indicated (red dashed circles).

Extended Data Figure 2 Phylogenetic tree of bacterial tae2 and eukaroytic dae2 genes.

A phylogenetic tree was constructed using Bayesian methods in MrBayes34 to compare to the maximum likelihood tree shown in Fig. 1b. Branch support >0.7 is indicated by asterisks or by numbers. The scale bar shows estimated divergence in amino acid changes per residue. Eukaryotic dae2 genes are indicated by dashed boxes, which highlight two separate HGT events. In both phylogenetic trees, the two eukaryotic dae2 clades are well supported as monophyletic clades, supporting our conclusion of two HGT events. Likewise, many major bacterial groups are well supported in both trees. Differences in the overall topology of the trees, mostly owing to changes in deep branches that are not well supported in either phylogenetic tree, reflect uncertainty in the ancient history of these genes and should therefore be treated with caution.

Extended Data Figure 3 Phylogenetic tree of bacterial tae3 and eukaryotic dae3 genes.

a, b, Phylogenetic trees were constructed using either maximum likelihood methods (a) or Bayesian methods (b). Branch support >0.7 is indicated by asterisks or by numbers. The scale bar shows estimated divergence in amino acid changes per residue. Eukaryotic dae3 genes are indicated by dashed boxes, which highlight two separate HGT events. In both trees, the two eukaryotic dae3 clades are well supported as monophyletic clades, supporting our conclusion of two separate HGT events. Likewise, many major bacterial groups are well supported in both trees. Differences in the overall topology of the trees, mostly owing to changes in deep branches that are not well supported in either phylogenetic tree, reflect uncertainty in the ancient history of these genes and should therefore be treated with caution.

Extended Data Figure 4 Phylogenetic tree of bacterial tae4 and eukaryotic dae4 genes.

a, b, Phylogenetic trees were constructed using either maximum likelihood methods (a) or Bayesian methods (b). Branch support >0.7 is indicated by asterisks or by numbers. The scale bar shows estimated divergence in amino acid changes per residue. Eukaryotic dae4 genes are indicated by dashed boxes, which highlight two separate HGT events. In both trees, the two eukaryotic dae4 clades are well supported as monophyletic clades, supporting our conclusion of two separate HGT events. Likewise, many major bacterial groups are well supported in both trees. Differences in the overall topology of the trees, mostly owing to changes in deep branches that are not well supported in either phylogenetic tree, reflect uncertainty in the ancient history of these genes and should therefore be treated with caution.

Extended Data Figure 5 Evidence for dae2 in other chelicerates.

a, Alignment of Dae2 from ticks and mites (I. scapularis and M. occidentalis) with Dae2 sequences from partially assembled genomes of two scorpions (Mesobuthus martensii and Centruroides exilicauda) and the horsehoe crab (Limulus polyphemus). Splice junctions are denoted (vertical red lines). All three alignable partial sequences start (red diagonal slashes) in the same position as the shared splice site in tick and mite dae2 genes, suggesting that this is probably the beginning of the exons in all dae genes shown. A second intron position is shared between the tick, scorpion and horseshoe crab dae genes and is nearby the mite intron position. b, Phylogenetic tree based on partial nucleotide sequences of dae2 from the indicated chelicerate species. Scale bar shows estimated divergence, in substitutions per nucleotide. c, Chelicerate phylogeny with approximate dates of divergence13. The unknown divergence time of sarcoptiform and trombidiform mites is indicated by a question mark. We find no evidence for dae2 in the complete genome of the trombidiform mite Tetranychus urticae nor in the partial (several species) or complete genome (Stegodyphus mimosarum) of any spider. Putative dae2 gene loss events in trombidiform mites and spiders are denoted (dashed lines).

Extended Data Figure 6 Evidence for retention of important catalytic motifs and recurrent eukaroytic-specific addition of secretion signals.

ac, Alignments for the predicted Dae N-terminal signal sequences (shaded blue) and catalytic motifs (right) are shown for each of the families. The consensus sequence logo of residues surrounding the cysteine and histidine positions of catalytic dyads from extant Tae enzymes are shown above alignments from each family. Below are aligned eukaroytic Dae proteins in these same regions. Representatives derived from distinct HGT events are separated by a space. Predicted N-terminal secretion signals (blue) and predicted catalytic residues (red) are coloured. Lowering the cut-off value in SignalP36 from the default value of 0.45 to the ‘sensitive’ value of 0.34 predicted a signal peptide in residues 1–21 of C. gigas Dae4.

Extended Data Figure 7 Dae2 degrades mDAP- but not Lys-type PG.

a, b, Partial HPLC chromatograms of sodium-borohydride-reduced soluble PG fragments (muropeptides) from Bacillus subtilis (a) or Streptococcus pneumoniae (b). PG sacculi products resulting from incubation with buffer (Control) or the indicated Dae2 proteins (wild type (WT) or C43A), followed by cellosyl digestion are shown. Major peaks are labelled. a, Muropeptides from B. subtilis include Tri (GlcNAc–MurNAc(reduced (r))–l-Ala–d-γ-Glu–mDAP(amidated (NH2))), Tetra (GlcNAc–MurNAc(r)–l-Ala–d-γ-Glu–mDAP(NH2)–d-Ala), and TetraTri (GlcNAc–MurNAc–l-Ala–d-γ-Glu–mDAP(NH2)–d-Ala–mDAP(NH2) d-γ-Glu–l-Ala–MurNAc(r)–GlcNAc). b, Muropeptides from S. pneumoniae include Tri (GlcNAc–MurNAc(r)–l-Ala–d-γ-Gln–l-Lys) and TetraTri (GlcNAc–MurNAc–l-Ala–d-γ-Gln–l-Lys–d-Ala–l-Lys–d-γ-Gln–l-Ala–MurNAc(r)–GlcNAc). l-Ser–l-Ala branch is indicated by ‘(SA)’ and deacetylation by ‘(deAc)’.

Extended Data Figure 8 Dae2 is active against B. burgdorferi PG.

HPLC elution profiles of B. burgdorferi sacculi incubated with buffer (Control) or the indicated Dae2 proteins (wild type (WT) or C43A), followed by cellosyl digestion are shown. Discrete peaks lost (red) or produced (green) upon digestion by Dae2 are denoted with arrowheads in control and wild-type chromatograms, respectively. Unresolved peaks, probably corresponding to a complex mixture of multi-crosslinked species cleaved by Dae2, are also highlighted (blue line). B. burgdorferi PG composition is complex and not yet resolved, thus approximate elution times of uncrosslinked versus crosslinked species are based on E. coli muropeptides in the same solvent system.

Extended Data Figure 9 Disruption of dae2 expression does not significantly alter tick physiology at repletion.

a, Knockdown of dae2 does not increase the B. burgdorferi burden in infected nymphs at engorgement. Loads were quantified by qPCR analysis of flaB, a B. burgdorferi-specific gene, and normalized to TROSPA, a tick-specific gene. n = 20. For this and subsequent panels, each data point represents a pool of three nymphs, and horizontal bars represent mean values, which were not significantly different in a two-tailed nonparametric Mann–Whitney test (P > 0.5). b, Disruption of dae2 expression did not affect engorgement weights of nymphal ticks fed on B. burgdorferi-infected mice. Tick weights were measured at repletion and 2 weeks post-repletion. Error bars show ± s.d., n = 8. c, Overall bacterial load was not affected by knockdown of dae2. Bacterial load was assessed by qPCR analysis of the 16S rRNA normalized against the tick-specific gene TROSPA. Load is represented on both a linear (bottom) and log2 (top) scale, which is denoted by a gap on the y-axis.

Extended Data Table 1 Evolutionary analyses of dae and tae gene families

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Chou, S., Daugherty, M., Peterson, S. et al. Transferred interbacterial antagonism genes augment eukaryotic innate immune function. Nature 518, 98–101 (2015). https://doi.org/10.1038/nature13965

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