Antimicrobial Functions of Lactoferrin Promote Genetic Conflicts in Ancient Primates and Modern Humans

Abstract

Lactoferrin is a multifunctional mammalian immunity protein that limits microbial growth through sequestration of nutrient iron. Additionally, lactoferrin possesses cationic protein domains that directly bind and inhibit diverse microbes. The implications for these dual functions on lactoferrin evolution and genetic conflicts with microbes remain unclear. Here we show that lactoferrin has been subject to recurrent episodes of positive selection during primate divergence predominately at antimicrobial peptide surfaces consistent with long-term antagonism by bacteria. An abundant lactoferrin polymorphism in human populations and Neanderthals also exhibits signatures of positive selection across primates, linking ancient host-microbe conflicts to modern human genetic variation. Rapidly evolving sites in lactoferrin further correspond to molecular interfaces with opportunistic bacterial pathogens causing meningitis, pneumonia, and sepsis. Because microbes actively target lactoferrin to acquire iron, we propose that the emergence of antimicrobial activity provided a pivotal mechanism of adaptation sparking evolutionary conflicts via acquisition of new protein functions.

Fig 1. Dynamic evolution of the lactoferrin N lobe in primates.

A. Paired primate phylograms showing signatures of positive selection in lactoferrin and transferrin. dN/dS ratios along each lineage are shown, with ratios greater than 1 (indicative of positive selection) shown in blue. Branches with no silent or nonsynonymous mutations display ratios in parentheses. *For lactoferrin analyses the sequence of the Taiwanese macaque was used, whereas for transferrin rhesus macaque was included. This difference does not change the topology of the primate phylogram. B. Sites subject to positive selection in lactoferrin and transferrin are shown (blue arrows) along a schematic of the two proteins (phylogenetic analysis by maximum likelihood, posterior probability >0.95 by Naïve and Bayes Empirical Bayes analyses). The relative positions of the N and C lobes are shown. C. Ribbon diagrams for crystal structures of diferric lactoferrin (PDB: 1LFG) and transferrin (PDB: 3V83), with side chains of sites under positive selection calculated in B shown in blue. Iron in the N and C lobes is shown in red.

Fig 2. Diversity and evolution of human lactoferrin.

A. Schematic representation of the lactoferrin protein showing positions of abundant (>1% allele frequency) nonsynonymous polymorphisms found in humans (arrows). Sites previously identified under positive selection across primates are shown as blue bars. The position of one variant, rs1126478 at amino acid position 47, which is also rapidly evolving in primates, is shown in magenta. The position of lysine 47 (K47) is also shown in the lactoferrin crystal structure (bottom panel). B. Relative allele frequencies of the R47 (blue) and K47 (red) lactoferrin variants shown as pie charts across human populations. Data were obtained from the 1000 Genomes Project Phase III. C. Extended haplotype homozygosity (EHH) plot around the lactoferrin for the R47 (blue) and K47 (red) around the variable position site, showing the extended haplotype around the K47 variant. D. Haplotype bifurcation plot showing breakdown of linkage disequilibrium in individuals carrying the lactoferrin R47 (blue) and K47 (red) alleles around the variant position. Thickness of the line corresponds to the number of individuals with shared haplotypes.

Fig 3. Rapid evolution of lactoferrin-derived antimicrobial peptides and pathogen binding interfaces.

A. Amino acid alignment of the lactoferricin and lactoferrampin peptide sequences across primates. Sites under positive selection are denoted with black arrows, with amino acids at these positions color-coded. Conserved tryptophan (red) and cysteine (blue) residues are highlighted, which contribute to target membrane interactions and disulfide bond formation respectively. The reported cleavage sites of the two peptides are denoted with red arrows. B. Left: solution structure of the free human lactoferricin peptide (PDB: 1Z6V), with sites under positive selection (blue), including position 47 (magenta) indicated. Conserved tryptophan and cysteine residues highlighted in A are also shown. Right: enlarged view of the human lactoferrin N lobe highlighting sequences corresponding to lactoferricin (cyan) and lactoferrampin (green) antimicrobial peptides. Sites previously identified under positive selection in primates are shown in blue, with the position 47 variant shown in magenta. C. Crystal structure (PDB: 2PMS) of human lactoferrin N lobe (gray) bound to PspA from Streptococcus pneumoniae (orange). Side chains of sites under positive selection (blue), including position 47 (magenta) are shown.

Fig 4. Model of lactoferrin evolution and genetic conflicts with pathogens.

Following a duplication of the transferrin gene in the ancestor of eutherian mammals, interactions between the transferrin (yellow) C lobe and the bacterial transferrin receptors such as TbpA (green) led to the emergence of a molecular arms race. In contrast, while lactoferrin has likely also been engaged in evolutionary conflicts with pathogen iron acquisition receptors like LbpA (purple), the emergence of antimicrobial peptide activity in the N lobe would have provided novel defense activity against pathogens targeting lactoferrin as an iron source. This function would have led to the emergence of pathogen inhibitors of lactoferrin antimicrobial peptide activity (such as PspA or LbpB), which have dominated subsequent evolutionary conflicts localized to the lactoferrin N lobe.