Evolutionary conflicts for nutrient iron are revealing potential new genetic mechanisms of disease resistance as well as avenues for therapeutic development.

Iron acquisition plays an important role in evolutionary interactions between microbes, both in the environment and within the host. Competition for iron can prevent infection by pathogens, while genetic changes in iron acquisition systems can enhance microbial virulence.

Molecular arms races can emerge between host iron-binding proteins and microbial ‘iron piracy’ factors that steal this nutrient for growth. Such rapid evolution may also contribute to the host range of pathogenic microbes.

The battle between microbes and their hosts for nutrient iron is emerging as a new front of evolutionary genetic conflict.

Host–pathogen interactions provide valuable systems for the study of evolutionary genetics and natural selection. The sequestration of essential iron has emerged as a crucial innate defense system termed nutritional immunity, leading pathogens to evolve mechanisms of ‘iron piracy’ to scavenge this metal from host proteins. This battle for iron carries numerous consequences not only for host–pathogen evolution but also microbial community interactions. Here we highlight recent and potential future areas of investigation on the evolutionary implications of microbial iron piracy in relation to molecular arms races, host range, competition, and virulence. Applying evolutionary genetic approaches to the study of microbial iron acquisition could also provide new inroads for understanding and combating infectious disease.

The outcome of an infection can have profound consequences for both host and pathogen populations. Intense selective pressures make host–pathogen interactions an attractive biological model to study evolutionary genetics over relatively short intervals of time. To date, much work has focused on rapid evolution involving canonical host immune defenses or antibiotic resistance []. However, we now know that hosts possess numerous additional means to restrict pathogens, including factors engaged in other core physiological functions. Nutrient iron sequestration provides one such alternative mode of host defense against bacteria and eukaryotic pathogens []. Iron is an essential micronutrient for microbes, as well as their hosts, due to its ability to readily shift between ferrous (Fe) and ferric (Fe) oxidative states for redox catalysis or electron transport. This ability to readily accept and donate electrons also makes iron highly volatile, necessitating a well-coordinated iron transport and storage system in metazoans to prevent the production of toxic free radicals []. The sequestration of free iron by host proteins simultaneously prevents acquisition by microbes, a protective effect termed(see Glossary ) []. While appreciation has grown for the role of nutrient metals in infection, these ‘battles for iron’ and other trace metals provide intriguing cases for investigation from an evolutionary perspective. Here we discuss emerging questions on the control of iron in microbial infection and highlight recent and potential future insights regarding the evolution of molecular arms races, host range, microbial competition, and pathogen virulence.

Microbes respond to iron starvation by actively scavenging this nutrient from host proteins to meet their metabolic requirements ( Figure 1 ) []. One of the most common microbial iron acquisition strategies involves the secretion of, small molecule chelators, which possess an affinity for iron unmatched even by proteins such as transferrin []. Microbes then recover iron–siderophore complexes via cell surface receptors. Obviating the need for siderophores, several microbes also express receptors that directly recognize and extract iron from host proteins including transferrin and lactoferrin []. Additional mechanisms involve the acquisition of heme, the iron-containing porphyrin cofactor, from abundant host proteins such as hemoglobin []. Ferric reductases are an important class of iron acquisition systems in fungal pathogens, which convert transferrin or lactoferrin-bound ferric iron into a soluble ferrous form []. The identification of iron acquisition genes as pathogenfurther underscores the role of iron in infection, as well as the potential for evolutionary conflicts to arise in the struggle for this precious nutrient.

Nutritional Immunity and Microbial Iron Piracy. Illustration highlighting major components of bacterial iron acquisition, including surface receptors as well as secreted siderophores and hemophores. Host nutritional immunity proteins are denoted in bold.

Figure 1 Nutritional Immunity and Microbial Iron Piracy. Illustration highlighting major components of bacterial iron acquisition, including surface receptors as well as secreted siderophores and hemophores. Host nutritional immunity proteins are denoted in bold.

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

In the decades following Schade and Caroline's initial discoveries, Eugene Weinberg proposed that withholding iron from microbial pathogens provided an important cornerstone of host defense, which he termed nutritional immunity []. Weinberg's theory explained previous observations that human iron overload disorders such as hereditary hemochromatosis and thalassemia render affected individuals highly susceptible to bacterial and fungal infections. The theory of nutritional immunity was also consistent with George Cartwright's earlier observations that infection induces an acute reduction in circulating iron levels []. Subsequent microbiology and molecular genetic studies established that nutritional immunity plays a pivotal role in defense against an array of pathogens, including bacteria, fungi, and parasites []. Owing to the iron-binding properties of proteins, such as transferrin, circulating levels of free iron in the body are orders of magnitude below the requirements for optimal microbial growth.

A potential role for iron in immunity became apparent following an elegant series of experiments by Arthur Schade and Leona Caroline in the early 1940s []. While attempting to develop a vaccine against Shigella, the researchers observed that addition of raw egg white to their culture media severely inhibited the growth of diverse bacteria as well as fungi. The antiseptic properties of egg white have in fact been recognized since the days of Shakespeare, where it was applied to wounds during Act III of King Lear. While various nutrient supplements failed to reverse the antimicrobial effect of egg whites, incinerated yeast extract did, suggesting that the limiting component was elemental in nature. Of 31 individual elements tested, supplementation with iron alone was sufficient to restore microbial growth in the presence of egg white. Adding to the fortuitous nature of their discovery, the authors posited that an iron-binding component present in the egg white prevented acquisition of this nutrient by microbes, which could have important implications for immunity. Two years later the scientists reported similar activity present in human blood serum []. The factor responsible for this activity in both cases was later revealed to be the protein, which plays a central role in animal iron metabolism by binding and transporting this metal to target cells [].

Raw hen egg white and the role of iron in growth inhibition of Shigella dysenteriae, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae.

New Perspectives on Ancient Evolutionary Arms Races

positive selection. Recurrent bouts of positive selection at such interfaces can give rise to so-called ‘molecular arms races’, in which the host and pathogen must continually adapt to maintain comparative fitness [ 1 Daugherty M.D.

Malik H.S. Rules of engagement: molecular insights from host–virus arms races. Red Queen Hypothesis, which proposed that antagonistic coevolution leads to a perpetual cycle of adaptation in which neither opponent gains a permanent advantage [ 28 Van Valen L. A new evolutionary law. 29 Sawyer S.L.

et al. Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G. 30 Sawyer S.L.

et al. Positive selection of primate TRIM5α identifies a critical species-specific retroviral restriction domain. 31 Elde N.C.

et al. Protein kinase R reveals an evolutionary model for defeating viral mimicry. 32 Mitchell P.S.

et al. Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA. 33 Patel M.R.

et al. Convergent evolution of escape from hepaciviral antagonism in primates. 34 Demogines A.

et al. Dual host–virus arms races shape an essential housekeeping protein. 35 Sironi M.

et al. Evolutionary insights into host–pathogen interactions from mammalian sequence data. Novel mutations that alter host–pathogen interactions can provide a substantial fitness advantage and spread in a population through. Recurrent bouts of positive selection at such interfaces can give rise to so-called ‘molecular arms races’, in which the host and pathogen must continually adapt to maintain comparative fitness []. Genes subject to such evolutionary conflicts are often characterized by an increased rate of nonsynonymous to synonymous substitutions (termed dN/dS or ω), reflecting recurrent selection for novel amino acid substitutions that alter protein interaction surfaces. Instances of such molecular arms races also exemplify Leigh Van Valen's, which proposed that antagonistic coevolution leads to a perpetual cycle of adaptation in which neither opponent gains a permanent advantage []. Several core components of the vertebrate immune system have subsequently been shown to engage in such conflicts, some of which are able to dictate the outcome of an infection [].

36 Barber M.F.

Elde N.C. Escape from bacterial iron piracy through rapid evolution of transferrin. 21 Cornelissen C.N.

et al. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors. 37 Schryvers A.B. Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae. 38 Cornelissen C.N.

et al. The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers. 39 Yu R.H.

Schryvers A.B. The interaction between human transferrin and transferrin binding protein 2 from Moraxella (Branhamella) catarrhalis differs from that of other human pathogens. 40 Moraes T.F.

et al. Insights into the bacterial transferrin receptor: the structure of transferrin-binding protein B from Actinobacillus pleuropneumoniae. 41 Noinaj N.

et al. Structural basis for iron piracy by pathogenic Neisseria. Figure 2 36 Barber M.F.

Elde N.C. Escape from bacterial iron piracy through rapid evolution of transferrin. Evolutionary Conflict at the Transferrin–Transferrin-binding protein A (TbpA) interface. (A) Cocrystal structure (Protein Data Bank: 3V8X) of human transferrin bound to TbpA from Neisseria meningitidis. Side chains of rapidly evolving amino acid positions in primate transferrin are shown in blue, with rapidly evolving TbpA sites among human pathogens shown in red (as described in Our recent work highlighted the battle for iron as a new interface for Red Queen evolutionary conflicts []. As described earlier, transferrin was among the first vertebrate proteins to be implicated in nutritional immunity and is also a frequent target of iron acquisition by microbes. Reasoning that transferrin could be a focal point for genetic conflicts with pathogens, we performed phylogenetic analyses of transferrin gene divergence in the primate lineage. Not only has transferrin been subject to strong positive selection in primates but also rapidly evolving sites almost entirely overlap with the binding interface of a bacterial surface receptor, transferrin-binding protein A (TbpA), an important virulence factor in several human pathogens including Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, and Moraxella catarrhalis, as well as a number of agricultural pathogens ( Figure 2 A) []. Single amino acid substitutions at rapidly evolving sites in transferrin were sufficient to control TbpA-binding specificities between related primates as well as for an abundant human transferrin variant, termed C2 ( Figure 2 B). Genetic signatures of positive selection at the transferrin–TbpA binding interface suggest that this interaction has been a key determinant of infection during millions of years of primate divergence. More broadly, these results demonstrate that nutritional immunity, similar to more established immune pathways, has strongly impacted host fitness during our long and intertwined history with microbes.

37 Schryvers A.B. Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae. 42 García-Montoya I.A.

et al. Lactoferrin a multiple bioactive protein: an overview. 43 Beddek A.J.

Schryvers A.B. The lactoferrin receptor complex in Gram negative bacteria. 44 Noinaj N.

et al. Structural insight into the lactoferrin receptors from pathogenic Neisseria. 45 Yamauchi K.

et al. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. 46 Haney E.F.

et al. Novel lactoferrampin antimicrobial peptides derived from human lactoferrin. 47 Hammerschmidt S.

et al. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae. 48 Senkovich O.

et al. Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein A provides insight into microbial defense mechanism. 49 Deka R.K.

et al. Crystal structure of the Tp34 (TP0971) lipoprotein of Treponema pallidum: implications of its metal-bound state and affinity for human lactoferrin. Evidence for an evolutionary arms race between transferrin and TbpA raises the question as to whether other host nutritional immunity factors may be subject to similar conflicts. Many pathogens encode receptors for other host iron-binding proteins including lactoferrin, a transferrin paralog expressed in milk, saliva, tears, mucus, and the secondary granules of neutrophils []. The evolution of lactoferrin introduces a fascinating twist; in addition to sequestering iron, lactoferrin has acquired mutations to generate antimicrobial peptide (AMP) domains that bind and disrupt pathogen membranes []. Many pathogens in turn encode factors that either scavenge lactoferrin-bound iron or inhibit associated AMP activity []. How these distinct functions have shaped lactoferrin evolution or potential arms races with pathogens remains to be determined.

50 Flo T.H.

et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. 51 Bachman M.A.

et al. Interaction of lipocalin 2, transferrin, and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia. 52 Hantke K.

et al. Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN. 53 Abergel R.J.

et al. Anthrax pathogen evades the mammalian immune system through stealth siderophore production. Genetic conflicts in nutritional immunity may also unfold by means other than simple point mutation and selection at protein interaction sites. For example, the vertebrate protein lipocalin 2 (also known as siderocalin or NGAL) is a potent innate immunity factor that functions in part through binding and sequestration of siderophores, preventing their uptake by microbes []. Some pathogens evade this defense through production of modified ‘stealth siderophores’, which are not recognized by lipocalin 2 []. Whether lipocalin 2 in turn has undergone adaptation resulting in enhanced or modified siderophore recognition is unknown. Understanding the extent to which molecular arms races have influenced other nutritional immunity factors beyond transferrin could reveal additional modes of adaptation underlying host–pathogen evolutionary conflicts.

54 Posey J.E.

Gherardini F.C. Lack of a role for iron in the Lyme disease pathogen. 54 Posey J.E.

Gherardini F.C. Lack of a role for iron in the Lyme disease pathogen. 55 Aguirre J.D.

et al. A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi. 56 Blount Z.D.

et al. Genomic analysis of a key innovation in an experimental Escherichia coli population. 57 Leiby N.

Marx C.J. Metabolic erosion primarily through mutation accumulation, and not tradeoffs, drives limited evolution of substrate specificity in Escherichia coli. 58 Chou H.H.

et al. Fast growth increases the selective advantage of a mutation arising recurrently during evolution under metal limitation. The barrier imposed by nutritional immunity has seemingly produced an even more drastic evasion strategy by one pathogen – giving up iron altogether. Previous work has demonstrated that the bacterial spirochete Borrelia burgdorferi, the causative agent of Lyme disease, lacks a requirement for iron shared by nearly all other organisms []. This was an astounding discovery given that iron serves as a cofactor for numerous metalloproteins involved in essential cellular processes including the electron transport chain and DNA metabolism. How then has B. burgdorferi managed such an evolutionary feat? Closer inspection of the B. burgdorferi genome revealed that numerous genes encoding iron-binding proteins have been lost, and remaining enzymes that normally bind iron have undergone modification to bind manganese in its place []. Beyond these general observations, we are only beginning to unravel the stepwise genetic mechanisms that lead to such major evolutionary innovations []. Identifying other microbes that have foregone the requirement for iron could provide useful comparison points to understand the mechanics of complex evolutionary transitions.