High rate of adaptation of mammalian proteins that interact with <i>Plasmodium</i> and related parasites

<div><p><i>Plasmodium</i> parasites, along with their Piroplasm relatives, have caused malaria-like illnesses in terrestrial mammals for millions of years. Several <i>Plasmodium</i>-protective alleles have recently evolved in human populations, but little is known about host adaptation to blood parasites over deeper evolutionary timescales. In this work, we analyze mammalian adaptation in ~500 <i>Plasmodium</i>- or Piroplasm- interacting proteins (PPIPs) manually curated from the scientific literature. We show that (i) PPIPs are enriched for both immune functions and pleiotropy with other pathogens, and (ii) the rate of adaptation across mammals is significantly elevated in PPIPs, compared to carefully matched control proteins. PPIPs with high pathogen pleiotropy show the strongest signatures of adaptation, but this pattern is fully explained by their immune enrichment. Several pieces of evidence suggest that blood parasites specifically have imposed selection on PPIPs. First, even non-immune PPIPs that lack interactions with other pathogens have adapted at twice the rate of matched controls. Second, PPIP adaptation is linked to high expression in the liver, a critical organ in the parasite life cycle. Finally, our detailed investigation of alpha-spectrin, a major red blood cell membrane protein, shows that domains with particularly high rates of adaptation are those known to interact specifically with <i>P</i>. <i>falciparum</i>. Overall, we show that host proteins that interact with <i>Plasmodium</i> and Piroplasm parasites have experienced elevated rates of adaptation across mammals, and provide evidence that some of this adaptation has likely been driven by blood parasites.</p></div

Pathogen pleiotropy is linked to adaptation for immune PPIPs.

<p>Each violin plot depicts a set of 1000 ratios comparing the mean proportion of adaptive codons in PPIPs to that in 1000 sets of matched controls. Adaptive codons per gene are an average across all branches. The dashed line indicates the null 1:1 expectation for PPIPs:matched controls. <b>(a)</b> PPIPs are divided into those that are only known to interact with <i>Plasmodium</i>, those that interact with <i>Plasmodium</i> and Piroplasms, and those that also interact with viruses and bacteria. The fraction of PPIPs with annotated immune functions increases with the number of interacting pathogens, as does the ratio of adaptive codons in PPIPs: matched controls. <b>(b)</b> All PPIPs compared to PPIPs without annotated immune functions. The ratio of adaptive codons in PPIPs:matched controls is significantly lower for PPIPs without immune functions than for all PPIPs. <b>(c)</b> Non-immune PPIPs are divided into those that are only known to interact with <i>Plasmodium</i>, those that interact with <i>Plasmodium</i> and Piroplasms, and those that also interact with viruses and bacteria. Unlike for all PPIPs (a), interaction with pathogens beyond <i>Plasmodium</i> is not significantly correlated with additional adaptation in non-immune PPIPs. In all panels, each violin is significantly above the 1:1 expectation of PPIPs:matched controls (all p≤0.02). Horizontal bars indicate differences between violins; * = p<0.05, NS = not significant.</p

Observations of <i>Plasmodium</i> and Piroplasm infection in well-sequenced mammal groups.

<p>References for parasite infection in these species or their close relatives are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007023#pgen.1007023.s005" target="_blank">S1 Table</a>.</p

Tissue expression of PPIPs and control genes.

<p>The GTEx median RPKM + 1 values for each gene and tissue were log₂ transformed and plotted for PPIPs (pink) and one set of control genes (blue), matched for total expression. Whiskers denote the range of values 0.1 times the interquartile range from the box. Significance was determined by permutation within each tissue, i.e., by comparing the mean expression of PPIPs to the means of 1000 sets of matched control genes. *** = p<0.001; * = p<0.05.</p

Top 20 GO functions enriched for PPIPs.

<p>Top 20 GO functions enriched for PPIPs.</p

PPIPs have experienced a significant excess of adaptive substitutions in mammals.

<p>All comparisons are made between PPIPs and carefully matched control proteins (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007023#sec013" target="_blank">Methods</a>, Permutation Tests). <b>(a)</b> Evolutionary constraint across great apes, as measured by the ratio of non-synonymous to synonymous polymorphisms, is equivalent in PPIPs and matched controls. The thick red line represents the cumulative density of pN/(pS+1) for PPIPs, whereas the cloud of thin blue lines represents the cumulative densities of 100 sets of matched controls. <b>(b)</b> Across 24 mammal species, the ratio of non-synonymous to synonymous substitutions is elevated in PPIPs versus matched controls. Lines as in (a). <b>(c)</b> BUSTED detects mammalian adaptation in 37% of PPIPs and, on average, 24% of matched controls. Error bars indicate the 95% range of the proportion of matched control genes with evidence of BUSTED adaptation, over 100 sets of matched controls. <b>(d)</b> Across the mammalian phylogeny (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007023#pgen.1007023.g001" target="_blank">Fig 1</a>), BS-REL tests identify PPIPs as evolving adaptively on a higher number of branches. Lines as in (a). <b>(e)</b> BS-REL tests identify a higher proportion of codons in PPIPs as evolving adaptively. Adaptive codons per gene are given as an average across all branches. Lines as in (a). <b>(f)</b> The ratio of adaptive codons in PPIPs versus matched controls increases (p = 7x10^-5) as the BUSTED threshold for including BS-REL estimates becomes more stringent. The solid line indicates the mean excess; the dashed line indicates the 1:1 expectation; gray shading indicates 95% confidence intervals. In all panels, *** = p<0.001; NS = p>0.05.</p

PPIP adaptation is widespread throughout mammals.

<p>Each violin plot depicts a set of 1000 ratios comparing the mean proportion of adaptive codons in PPIPs to that in 1000 sets of matched controls. Adaptive codons per gene are an average across all branches contained in the specified clade. The dashed line indicates the null 1:1 expectation for PPIPs:matched controls.</p

Highly adaptive PPIPs have high expression in the liver.

<p>For both PPIPs and controls matched for total expression, the 5% of the gene set with the most adaptive codons (averaged across all branches) was compared to the remainder of the gene set. Expression is plotted as log₂(GTEx median RPKM + 1) for each set of genes and tissue. For matched controls, highly adaptive genes are expressed at low levels in all malaria-relevant tissues. This pattern differs for PPIPs, particularly in the liver. Significance was determined with the KS test. *** = p<0.001; ** = p <0.01, * = p <0.05; NS = not significant.</p