Escape from bacterial iron piracy through rapid evolution of transferrin

Abstract

Iron sequestration provides an innate defense, termed nutritional immunity, leading pathogens to scavenge iron from hosts. Although the molecular basis of this battle for iron is established, its potential as a force for evolution at host-pathogen interfaces is unknown. We show that the iron transport protein transferrin is engaged in ancient and ongoing evolutionary conflicts with TbpA, a transferrin surface receptor from bacteria. Single substitutions in transferrin at rapidly evolving sites reverse TbpA binding, providing a mechanism to counteract bacterial iron piracy among great apes. Furthermore, the C2 transferrin polymorphism in humans evades TbpA variants from Haemophilus influenzae, revealing a functional basis for standing genetic variation. These findings identify a central role for nutritional immunity in the persistent evolutionary conflicts between primates and bacterial pathogens.

Fig. 1. Primate transferrin has undergone recurrent positive selection at the binding interface with bacterial TbpA

(A) A primate phylogram highlighting rapid evolution of transferrin. dN/dS ratios along each branch of the primate lineage are listed, with dN/dS values >1 highlighted in blue. Branches with no synonymous changes display N:S substitution ratios in parentheses. (B) Schematic of the human transferrin protein (yellow). Amino acid positions showing evidence of positive selection (PAML, posterior probability > 0.95) are denoted by blue arrows. Regions of transferrin spanning the N. meningitidis TbpA binding interface () are highlighted in brown. (C) Ribbons representation of the structure (PDB: 3V8X) of human transferrin (yellow) in complex with N. meningitidis TbpA (gray), with the position of the bacterial outer membrane labeled. Amino acid side-chains in transferrin with strong evidence of positive selection are marked by blue spheres. The position of a ferric ion is modeled as a red dot. (D) Competitive TbpA binding assays performed as dot blot (left) or ELISA (right), using E. coli expressing indicated pathogen TbpA. Samples were incubated with horseradish peroxidase (HRP)-conjugated human transferrin alone (0.5 ug/mL), or HRP-transferrin in the presence of increasing concentrations of recombinant purified transferrin (5, 10, or 20 ug/mL) from indicated primates. Error bars represent standard deviation (SD) of four independent experiments. ** indicates P < 0.01 relative to human transferrin.

Fig. 2. Transferrin divergence in humans and chimpanzees impairs TbpA binding

(A) Schematic representation (top) showing divergent amino acid positions between human and chimpanzee transferrin. Blue arrows indicate amino acids that also display signatures of positive selection. Amino acid alignment (bottom) around position 591, highlighting the chimpanzee-specific E591 to K substitution. (B) Sites of positive selection in transferrin (blue) proximal to loop 3 of TbpA. Position 589, which is variable in human populations, is highlighted in green. (C) Competitive binding dot blots and ELISAs using recombinant human and chimpanzee transferrin, along with human E591K and chimpanzee K591E mutant proteins. Error bars represent SD of four independent experiments. ** denotes P < 0.01. (D) Distribution of the transferrin C2 polymorphism (green) across human populations. (E) Primate phylogeny and amino acid alignment displaying toggling of transferrin position 589 across primates. Colors denote variable amino acids at each position. Variability at position 591 is also highlighted. (F) Competitive binding dot blot and ELISAs using the major transferrin variant (C1), the transferrin P589S variant (C2), as well as chimpanzee and gorilla transferrin. Error bars represent SD of four independent experiments. ** indicates P <0.01 relative to human (C1) transferrin.

Fig. 3. Rapid evolution and functional variation of TbpA among human pathogens

(A) Competitive binding dot blots and ELISAs assessing TbpA binding to the human transferrin C2 variant. Error bars represent SD of four independent experiments. ** indicates P < 0.01 relative to human (C1) transferrin, or C2 transferrin (vertical line). * indicates P = 0.05. (B) Gene tree of TbpA from H. influenzae isolates. TbpA from strain Eagan (magenta) as well as two divergent TbpA variants, strains 11 and 15 (green) are highlighted. (C) Structure of human transferrin (yellow) in complex with N. meningitidis TbpA (gray). Side-chains of amino acids under positive selection in transferrin (blue), H. influenzae TbpA (purple), and Neisseria TbpA (orange) are denoted by colored spheres. * indicates position under positive selection in both Haemophilus and Neisseria TbpA. A “top” view of TbpA (right) shows exposure of rapidly evolving sites at the transferrin binding interface. (D) Schematic representation of sites among either H. influenzae or Neisseria TbpA showing evidence of positive selection, overlaid on TbpA from N. meningitidis. Arrows denote sites that pass multiple tests of positive selection incorporating PhyML TbpA gene trees as well as phylogenies that account for recombination break points. Predicted extracellular loops are indicated in dark gray. * indicates a single amino acid position showing evidence of positive selection in both Haemophilus and Neisseria. (E) Indicated E.coli strains expressing mutations of TbpA were tested for interactions with human transferrin-HRP (left panel). A control blot was stained with ponceau (middle panel) as a loading control. Western blots using antibodies against N. gonorrhoeae TbpA or total E. coli. were performed with cell lysates from indicated strains (right panels).