Metal Sequestration by S100 Proteins in Chemically Diverse Environments

Abstract

During infection, the mammalian host initiates a metal-withholding response against invading microbial pathogens to inhibit their growth and survival, a process often termed “nutritional immunity”. The host-defense S100 proteins calprotectin (S100A8/S100A9 oligomer), S100A12, and S100A7 play key roles in the innate immune response by sequestrating essential transition metal nutrients from microbes in the extracellular space. Accumulating evidence suggests that the antimicrobial activity of these proteins varies between infection sites and may be affected by the local chemical environment. Herein we discuss the interplay between host metal-withholding proteins and microbial pathogens in the context of the chemical complexity of infection sites and highlight recent advances in our understanding of how chemically diverse conditions affect the properties and functions of S100 proteins.

Nutritional Immunity: Metal Withholding to Combat Microbial Pathogens

Transition metals play many important roles in biology. All organisms rely on transition metals to carry out a tremendous array of processes that are crucial for growth and survival. More than one-third of known enzymes contain metal cofactors, which serve diverse structural and catalytic roles [1]. Transition metals can also be harmful when present in elevated levels, generating damaging molecules such as reactive oxygen species (ROS) (see Glossary) or disrupting cellular processes upon mis-metalation of proteins [2, 3]. Consequently, organisms tightly regulate metal levels to meet their physiological needs and avoid potential damage [4, 5]. At the host–pathogen interface, invading microbial pathogens must acquire metal nutrients from the host to colonize and cause disease. In response to microbial infection, the mammalian host employs several mechanisms to starve microbes of essential metal nutrients, a process often termed “nutritional immunity” [6, 7]. Early examples of nutritional immunity include inhibition of microbial growth through Fe(III) sequestration by lactoferrin (LF) and transferrin (TF). Since then, additional metal-chelating host-defense proteins have been identified and include members of the S100 protein family—key players in the metal-withholding innate immune response that expand the repertoire of transition metals that are sequestered from pathogens by the host (Figure 1A) [7–10]. These proteins are deployed throughout the host in a diversity of locales that are expected to exhibit distinct and oftentimes heterogeneous chemical environments [11]. The consequences of the chemical and molecular complexity of the host on the functional properties of metal-sequestering S100 proteins and their interplay with microbes have become increasingly appreciated in recent years. In this review, we consider the molecular complexity of infection sites and highlight recent advances that address how the chemical environment can modulate the host metal-withholding response in the extracellular space.

Figure 1. The Extracellular Role of S100 Proteins in Metal Sequestration.

(a) CP is produced as the heterodimer and released by white blood cells (e.g., neutrophils) and undergoes Ca(II)-induced self-association in the extracellular space. The resulting (S100A8/S100A9)₂ heterotetramer is protease resistant, and exhibits enhanced transition metal affinities and enhanced antimicrobial activity compared to the S100A8/S100A9 heterodimer. White blood cells also produce and release S100A12, whereas S100A7 is expressed by epithelial cells. The functional properties of S100A7 and S100A12 are also modulated by Ca(II) ions. In the extracellular space, the three S100 proteins compete with microbes for bioavailable divalent first-row transition-metal ions. (b) Effect of the chemical environment and microbial metabolites on Fe(II) sequestration by CP. CP can sequester Fe(II) under aerobic conditions that favor the oxidation of Fe to Fe(III) by shifting the redox speciation of the metal. Fe(II) is prevalent in infections sites that display reducing and acidic conditions. Microbial metabolites can inhibit (e.g., siderophores) or promote (e.g., phenazines) Fe binding to CP.

Metal Withholding by S100 Host-Defense Proteins

S100 proteins are Ca(II)-binding proteins that are expressed throughout the human body and participate in a wide range of biological processes [11]. Each S100 polypeptide (~10 kDa) has a characteristic α-helical structure and harbors two helix-loop-helix Ca(II)-binding EF-hand motifs (Figure 2). Ca(II) binding at these sites promotes conformational change that modulates the functional properties of the protein [12]. At sites of infection, metal availability is restricted by a subset of S100 proteins that are deployed during the innate immune response (Figure 1A) [7–9]. These proteins include calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer, calgranulin A/B oligomer), S100A7 (psoriasin), and S100A12 (calgranulin C).

Figure 2. Structures and Transition Metal Binding Motifs of Human CP, S100A7 and S100A12.

(a) Crystal structure of Ni(II)-, Ca(II)- and Na(I)-bound CP (PDB 5W1F) [28]. A heterodimer unit of S100A8 (green) and S100A9 (blue) is taken from the structure of the heterotetramer. (b) Crystal structure of Zn(II)- and Ca(II)-bound S100A7 (PDB 2PSR) [37]. (c) Crystal structure of Zn(II)- and Na(I)-bound S100A12 (PDB 2WC8) [42]. (d) Zn(II)-bound His₃Asp motif (PDB 2WC8) [42] found in S100A7, S100A12 and CP (left), and Ni(II)-bound His₆ motif (PDB 5W1F) [28] found exclusively in CP (right). For all structures, the N- and C-termini are labeled, and the His₃Asp and His₆ binding sites are marked in gray and yellow circles, respectively. Zn(II) is shown in gray, Ni(II) is shown in teal, Ca(II) is shown in yellow, and Na(I) is shown in purple. The transition-metal binding residues are shown in orange.

CP has been studied extensively for its role in metal sequestration at the host–pathogen interface [9]. It is produced and released by white blood cells and constitutes ~40% of the total cytoplasmic protein in neutrophils; thus, CP is a major component of the neutrophil response to microbial pathogens [13, 14]. Unlike other S100 proteins that typically form symmetric dimers, CP consists of a heterooligomer of S100A8 (α subunit, 10.8 kDa) and S100A9 (β subunit, 13.2 kDa) (Figure 2A) [15]. Under low Ca(II) ion levels (e.g., nanomolar concentrations in the cytoplasm of neutrophils), apo CP exists as a heterodimer (αβ) [15, 16]. Upon release into the extracellular space, CP encounters high Ca(II) levels (~2 mM) that promote the self-association of two heterodimers to form a (αβ)₂ heterotetramer [16–18]. Ca(II) binding and subsequent tetramerization are pivotal for the extracellular function of CP, resulting in a protease-resistant form that exhibits enhanced transition metal affinities and antimicrobial activity compared to the apo αβ heterodimer [9, 19, 20]. Two transition metal-binding sites, commonly termed site 1 and site 2, form at the heterodimer interface and include metal-binding residues from each subunit. Site 1 is a His₃Asp motif that exhibits high affinity for Zn(II), lower affinities for Mn(II), Fe(II), and Ni(II), and readily selects for Zn(II) over these metal ions [21–25]. Consequently, the His₃Asp site is described as being Zn(II) selective. Site 2 is a functionally versatile His₆ motif that exhibits high affinity for Mn(II), Fe(II), Ni(II), and Zn(II) and sequesters these four metal ions (Figure 2D) [22, 23, 26–28]. CP also binds Cu at both sites [29], but the details of this coordination chemistry and how sites 1 and 2 contribute to Cu withholding requires elucidation. Each subunit also contains a single cysteine residue, which can form disulfide bonds within and between CP heterodimers [15, 30, 31].

S100A7 and S100A12 also contribute to the host metal withholding response [32–34]; however, both human proteins have received less attention than CP, partially because they are not as highly conserved as CP in mammals. For example, S100A12 is not present in mice, and the primary structure of murine S100A7 is substantially different from that of the human protein [35]. As a result, murine infection models have not provided insight into how S100A12 and S100A7 affect metal availability and the host–pathogen interaction. S100A7 is a homodimer (22 kDa) that is constitutively expressed in epithelial cells (Figure 2B) [36, 37]. The protein contains two His₃Asp metal-binding sites at the homodimer interface that are highly selective for Zn(II) binding [37, 38]. In addition, each S100A7 polypeptide contains two cysteine residues which can form intra-subunit disulfide bonds within homodimers [38, 39]. The S100A12 homodimer (21 kDa) is expressed mainly in neutrophils [40] and exhibits complex oligomerization tendencies, including self-association into higher-order oligomers in the presence of metal ions (Figure 2C) [41–43]. Like S100A7, S100A12 binds Zn(II) with high affinity and selectivity at two His₃Asp sites at the homodimer interface [42, 44]. Similar to CP, Ca(II) ions influence the functional properties and Zn(II) affinities of S100A7 and S100A12 [38, 44].

Infection Sites are Chemically Heterogeneous

Infection sites are inherently complex and vary widely in microbial abundance and diversity, composition of host defense factors, and availability of metabolites and metal ions [45, 46]. Moreover, a given infection site may be highly heterogeneous, consisting of microenvironments with distinct chemical properties, including different oxygen concentrations, redox potentials, and pH levels [45, 47]. Exploring the chemical composition of infection sites is important for gaining molecular understanding of host–microbe interactions; however, few experimental approaches have the sensitivity and spatial resolution to decipher the subtle differences in nutrient and metabolite concentrations in tissues and infection microenvironments [48]. In the context of nutritional immunity, initial insights into the composition of infection sites were provided from investigations of Staphylococcus aureus infection [49]. Following colonization, S. aureus induces inflammatory responses that result in tissue abscesses, confined pockets of staphylococci surrounded by necrotic and healthy innate immune cells [50]. Imaging mass spectrometry (IMS) analyses of kidney and liver tissues from infected mice revealed that high levels of CP localize with S. aureus abscesses [49, 51]. Integration of elemental bio-imaging of the abscessed tissues with the IMS results revealed that these CP-rich areas were also enriched in Ca(II) but devoid of Zn(II) and Mn(II). More recently, the abundance and spatial distribution of nutrients as well as microbial and host factors in staphylococcal abscesses were resolved to a higher degree using a multi-faceted approach that included bioluminescent imaging (BLI), magnetic resonance imaging (MRI) and targeted proteomics [46, 52]. By evaluating the Fe stress response of S. aureus throughout infected mice, heterogeneities in Fe starvation conditions that varied among infected organs as well as within distinct abscesses in the same tissue were observed [46]. In response to Fe starvation, S. aureus produces the siderophores staphyloferrin A and B. Heterogeneous distributions of these siderophores were detected in staphylococcal abscesses isolated from various tissues of infected mice [53]. Elemental bio-imaging further supported these observations, demonstrating high variability in metal distributions within individual tissues [53]. Recent studies also indicate that the interaction between S. aureus and host defense factors varies in different infection sites. For example, whereas CP penetrates staphylococcal abscesses in kidney and liver infections [49, 51], abscesses from staphylococcal infection in the heart were found to be devoid of CP [54].

Biofilms represent another heterogeneous environment; individual cells in relatively close proximity to each other can exhibit distinct transcriptional profiles and virulence potentials [55]. Elemental bio-imaging of Pseudomonas aeruginosa biofilms revealed Zn and Mn gradients across the biofilm surface that ended at nutrient-depleted edges, and IMS analysis demonstrated distinct expression patterns of proteins throughout the biofilm [56, 57]. For example, general metabolic proteins such as cytochrome cbb₃ oxidase were uniformly distributed across the biofilm, whereas small ribosomal proteins where enriched within the inner section. The proportion of antimicrobials produced by P. aeruginosa, including anti-staphylococcal factors and proteases that interfere with S. aureus growth and metabolism, also varied in different regions of the biofilm. Proteomic analysis of the Zn- and Mn-depleted edges of the P. aeruginosa biofilm revealed decreased levels of anti-staphylococcal biosynthetic proteins compared to the nutrient-rich inner section of the biofilm [56]. These results suggest a link between metal limitation and the production of anti-staphylococcal factors by P. aeruginosa, and thus may indicate conditions in which polymicrobial infections of P. aeruginosa and S. aureus can occur. Both pathogens have been isolated together from infection sites, including burn and surgical wounds and chronic respiratory infections in cystic fibrosis (CF) lungs [58, 59]. Moreover, P. aeruginosa exhibited lower anti-staphylococcal activity when grown in the presence of CP, further suggesting a link between metal limitation and commensalism between these two pathogens [56]. Recently, a study reported that Zn(II) sequestration by CP inhibited the activity of the pseudomonal protease LasA that contributes to S. aureus lysis [60]. Collectively, these results suggest that CP promotes co-colonization of P. aeruginosa and S. aureus and illustrate the complex interplay between metal nutrient availability and microbial responses.

Microbial and Host Metabolites Affect Metal Sequestration by Calprotectin

CP sequesters Fe in the reduced ferrous (Fe(II)) form at the His₆ site. Recent studies of Fe(II) withholding by CP indicate that the redox environment and presence of bacterial and host metabolites affect the ability of CP to sequester Fe(II) (Figure 1B) [27, 61–63]. Fe(II) binding at the His₆ site of CP occurs under both anaerobic conditions that favor this reduced form of Fe and aerobic conditions that favor the Fe(III) oxidation state. Sequestering Fe(II) under aerobic conditions involves a shift in the redox speciation of Fe from Fe(III) to Fe(II) and can be accelerated by the presence of an exogenous reductant (e.g., β-mercaptoethanol) [27, 61]. Some infection sites also favor Fe(II) over Fe(III), particularly those characterized by reducing, micro-aerobic, anaerobic, and low pH environments [64–67]. For example, an airway infection of P. aeruginosa during late-stage CF infection can develop biofilm-like structures. Combined with an overall decrease in oxygen tension, hypoxic microenvironments result that allow Fe to persist in its reduced form in the CF lung [68].

Recent investigations also highlight that bacterial and host metabolites can have marked effect on the interplay between CP and bacterial pathogens. The ability of CP to sequester Fe was found to be enhanced in the presence of phenazines [61, 69], redox-active metabolites produced by P. aeruginosa that reduce Fe(III) to Fe(II) (Figure 1B) [68]. In contrast, Fe binding to CP can be attenuated in the presence of Fe(III)-scavenging siderophores including pyoverdine, staphyloferrin B, enterobactin, acinetobactin, and ferrioxamine B [61, 69, 70]. Other Fe sources that are accessible to pathogens during infection can decrease the ability of CP to induce Fe starvation stress. For example, heme was recently demonstrated to protect P. aeruginosa and S. aureus from CP-mediated inhibition of Fe uptake and Fe-starvation responses [63]. Thus, the contribution of CP to the host Fe-withholding response is likely determined by a complex interplay between the protein and host/microbial metabolites.

Additional metallophores have been proposed to affect the metal sequestering activity of CP. For example, S. aureus produces the broad-spectrum metallophore staphylopine [71]. Recent work suggested that staphylopine enables S. aureus to compete with CP for Zn(II) in vitro, and to resist host-imposed metal starvation during infection [72]. Putative biosynthetic gene clusters for analogous secondary metabolites have been identified in a wide range of gram-positive and -negative bacteria, suggesting that broad-spectrum metallophores are widely spread among pathogens [72, 73]. For example, the staphylopine-like metallophore pseudopaline is produced by P. aeruginosa and has been demonstrated to promote Zn(II) uptake under metal-limited conditions [74]. Yersiniabactin, a siderophore produced by Gram-negative bacteria including Yersinia spp. and Escherichia coli, chelates and transports Fe(III) as well as Cu(II) and Ni(II) [75–77]. Yersiniabactin has also been implicated in Zn(II) uptake by Yersinia pestis and E. coli [78–80]. A thorough evaluation of the interplay of these microbial metallophores with metal-sequestering host-defense proteins, including how they compete for metal nutrients, is an important area for future exploration.

Oxidative Environments Modulate the Functions of Calprotectin and S100A7

During the innate immune response, neutrophils generate and release ROS as a defense strategy that promotes oxidative damage in invading pathogens [81]. This oxidative burst is proposed to function synergistically with the metal-withholding innate immune response, as microbial superoxide dismutases (SODs) that are crucial for antioxidant defense have essential metallocofactors [82, 83]. An additional consequence of the neutrophil oxidative burst is the oxidation of host molecules. Several reports identified oxidized species of CP, including methionine sulfoxide (MetO) and disulfide crosslinking modifications, in murine abscesses and various clinical samples from humans [30, 31, 84–88]. These oxidative post-translational modifications have structural and functional consequences on CP (Figure 3A). For instance, methionine oxidation disrupts Ca(II)-induced heterotetramer formation and consequently enhances the susceptibility of Ca(II)-bound CP to proteolytic degradation [30]. Remarkably, coordination of a transition metal at the His₆ site of methionine-oxidized CP restores the heterotetramer and confers protease resistance. In addition, the formation of disulfide bonds either within or between CP heterodimers results in increased protease sensitivity [30, 31]. Collectively, these findings highlight that an oxidative environment and the presence of ROS can affect the structure and overall lifetime of CP in the extracellular milieu.

Figure 3. The Effects of Post-Translational Oxidation on CP and S100A7.

(a) CP can undergo methionine sulfoxide (MetO) and disulfide bond formation, and these oxidized CP species are more susceptible to proteolysis than the Ca(II)-bound CP heterotetramer. The enhanced protease susceptibility is reversed upon binding of transition metal at the His₆ site for oxidized CP. (b) Oxidation of cysteine residues in S100A7 results in intra-subunit disulfide bonds. The disulfides can be reduced by the thioredoxin system (Trx/TrxR). Oxidized S100A7 exhibits lower tendency to be reduced in the presence of Ca(II) ions, which depress the midpoint potential by ~40 mV. Zn(II) binding further depresses the midpoint potential, and prevents reduction of S100A7 by Trx/TrxR.

Each S100A7 subunit contains two cysteine residues that oxidize to form intra-subunit disulfide bonds, and both the reduced (S100A7_red) and oxidized (S100A7_ox) forms of the protein have been identified in samples from human skin and the female genital tract [39, 89]. A study of S100A7 isolated from psoriasis lesions suggested that the thioredoxin system may reduce the protein in vivo [39]. The cysteine redox behavior of S100A7 was later demonstrated to be modulated by Ca(II) and Zn(II) binding [38]. Ca(II) binding lowered the reduction potential of the protein by >40 mV and conditions that allowed disulfide bond reduction for the apo protein afforded negligible reduction of the disulfide bonds for Ca(II)- and Zn(II)-bound S100A7_ox (Figure 3B). Metal-binding studies indicate that both S100A7_red and S100A7_ox bind Zn(II) with similar affinity at the His₃Asp sites; however, S100A7_ox exhibited slower metal substitution at the His₃Asp sites and was found to be more effective at inhibiting bacterial growth [38]. Further studies are required to determine how disulfide bond formation and reduction affect the structural and functional properties of S100A7, including its stability and susceptibility to proteolytic degradation.

Metal Sequestration under Mildly Acidic Conditions

Acidic environments normally occur in the gastrointestinal, genital and urinary tracts, skin and intracellular endocytic vesicles [90–93]. Moreover, extracellular acidification is associated with sites of chronic infection and inflammation, including CF airways, the gut during inflammatory bowel disease (IBD), and biofilm-related infections [90, 94]. Acidic conditions have been shown to trigger human neutrophil activation and to induce a transient increase in intracellular Ca(II) concentration [95], and thus may have consequences on CP and S100A12 production and release. Invading pathogens may encounter acidic environments during colonization, or generate such conditions, and mechanisms to sense and initiate appropriate responses to extracellular acidic pH have been identified in various bacterial and fungal species [96]. The relationship between low pH and metal ion homeostasis in microbes has been addressed in several studies. For example, transcriptional analyses of Helicobacter pylori, Salmonella enterica serovar Typhimurium and S. aureus revealed differential expression of metal acquisition systems in response to low pH, including prioritizing proton-driven metal transporters and shifting from Fe(III) to Fe(II) uptake [97–99]. Metal-chelating host-defense proteins are also affected by acidic conditions. For instance, TF releases bound Fe(III) under acidic conditions, a process considered to be important for Fe delivery to host cells following TF endocytosis [100]. LF is capable of binding and sequestering Fe(III) at low pH [101], although its antimicrobial activity has been shown to decrease under these conditions [102].

CP is prevalent at infection and inflammation sites that are expected to be acidic, including the CF lung and IBD colon [103, 104]. The biochemical properties and antimicrobial activity of CP at low-pH regimes have been evaluated recently (Figure 4) [105]. Mildly acidic pH perturbed the oligomerization process induced by Ca(II) binding, requiring 5-fold more Ca(II) ions for complete tetramerization as well as promoting heterotetramer dissociation. Thus, in addition to oxidative post-translational modifications, the local pH likely modulates the speciation of CP oligomers in the extracellular space. The antimicrobial activity of CP against several bacterial pathogens, including S. aureus, Klebsiella pneumoniae, and S. Typhimurium, was attenuated under mildly acidic pH [105]. Lastly, the Mn(II) affinity of CP decreases significantly at mildly acidic pH, rendering the protein ineffective in Mn(II) sequestration [105]. CP is currently the only known Mn(II)-sequestering host-defense protein and these results suggest that this role may be limited to near-neutral pH environments. A recent study demonstrated that S100A12 is also affected by low pH, suggesting that the Zn(II) affinity of the protein decreases at low pH unless excess Ca(II) ions are present [106]. Nuclear magnetic resonance (NMR) studies of S100A12 revealed protonation of histidine residues involved in Zn(II) binding at mildly acidic pH, and demonstrated how Ca(II) binding reverts pH-induced conformational changes and restores the high Zn(II) high affinity [106].

Figure 4. The Functional Properties of CP are pH-Dependent.

Mildly acidic conditions impair Ca(II)-induced tetramerization CP, an important process for extracellular CP that promotes protease stability and modulates the functional properties of the protein. CP displays reduced Mn(II) binding affinity at low pH, as well as attenuated antimicrobial activity against microbial pathogens such as S. aureus and S. Typhimurium.

Concluding Remarks

Our understanding of nutritional immunity and its roles in the host-microbe interaction and infectious disease has significantly expanded over the last years. Recent work has provided new insights into both the mechanisms used by the host to withhold metal nutrients from invading pathogens, and the strategies employed by microbes to evade host-imposed metal starvation. Considerable attention has been given to CP, a key player in the host metal-withholding response that exhibits broad-spectrum antimicrobial activity by sequestering multiple transition metal ions at sites of infections. The current working model of metal sequestration by CP is based on numerous biochemical, biophysical and functional studies that have been reported over many years, and its antimicrobial activity against various bacterial and fungal pathogens is established [9]. Nevertheless, a growing body of observations from animal infection models deviate from the results obtained using bacterial cultures grown under standard conditions, indicating that the antimicrobial activity of CP is likely to be niche-and pathogen-specific. For example, S. aureus is susceptible to CP-imposed Zn(II) and Mn(II) starvation in planktonic culture. While this behavior was found to be recapitulated in liver and kidney abscesses of a murine model [49, 51], S. aureus exhibited higher fitness in heart infection in the presence of CP [54]. Likewise, CP was demonstrated to inhibit the growth S. Typhimurium cultures by sequestering Zn(II) and Mn(II), whereas in a murine model of colitis, the pathogen thrived in the presence of high levels of CP [107, 108]. The ability of pathogens to resist metal starvation is typically attributed to the expression of high-affinity metal acquisition systems that can outcompete the host’s metal withholding response [7]. Recent studies provide a more nuanced picture in which the local chemical environment may also favor microbial metal acquisition. For example, acidic environments may benefit pathogens by disrupting the ability of CP and TF to sequester Mn(II) and Fe(III), respectively [105, 109]. We note that factors other than metal sequestration can account for different outcomes in various infection models. For example, CP deficiency may result in differences in neutrophil trafficking and neutrophil extracellular traps (NETs) formation [54, 110]. Comprehensive evaluation of how various physicochemical conditions affect the interplay between host-defense factors like CP and pathogenic microbes is an important area for future exploration.

Infection and inflammation sites likely contain multiple host-defense factors that contribute synergistically to the innate immune response. While S100A12 and S100A7 have received less attention than CP, their function in Zn(II) withholding during infection is accepted and as noted above several recent studies indicate their metal-sequestering ability is affected by the local environment [38, 39, 106]. Since S100A12 and S100A7 can be deployed at the same sites as CP, the combined contributions of these S100s are worthy of consideration and may be particularly important for Zn(II) withholding. In particular, by lowering Zn(II) availability, Zn(II) withholding by S100A12 and S100A7 may allow CP to more readily sequester other divalent metals like Mn(II) and Fe(II) [44]. The contribution of S100 proteins and other host defense factors to nutritional immunity has so far been evaluated mostly on a case-by-case basis, which may not fully recapitulate their combined effort. Further consideration should be given to the possible outcomes of this multi-protein response to the development of disease. Lastly, another important future direction is understanding how the ability of CP to elicit a multi-metal starvation response in microbial pathogens is affected by the presence of various metabolites at a given host niche. For example, a thorough evaluation of the metal competition between CP and broad-spectrum bacterial metallophores will inform the contribution of CP to the metal-withholding response during infection, and illuminate the importance of these metallophores to metal homeostasis at the host–pathogen interface. Along these lines, recent developments in the ability to characterize and visualize the chemical heterogeneity in infection sites provide new opportunities to decipher the interplay between microbial responses to metal starvation and metal-sequestering host defense proteins [48], making headway towards understanding the competition over metal nutrients between host and microbes. We look forward to seeing the outcomes of future efforts that would allow further refinement of current models of nutritional immunity.

Outstanding Questions.

• How do various environmental conditions affect the interplay between the host metal-withholding response and microbial pathogens?

• How do microbial responses to CP change in combination with other metal-sequestering host defense proteins such as S100A7, S100A12 and TF?

• How do various metallophores compete with CP and other S100 proteins for nutrient metals?

Highlights.

• Calprotectin, S100A7, and S100A12 sequester first row transition metal ions in the extracellular space and thereby starve invading pathogens of essential nutrients.

• Infection sites are chemically complex and can vary in pH, redox conditions, and the distribution of proteins, metabolites, and nutrients.

• The chemical environment at infection sites affects the structure and metal-withholding function of S100 proteins.

Glossary

Bioluminescent imaging (BLI)

a non-invasive imaging technique based on sensitive detection of light-emitting reporters that are incorporated into living organisms.

Elemental bio-imaging

a method for in situ analysis of metals at very low concentrations.

Imaging mass spectrometry (IMS)

a method to map the spatial distribution of biomolecules across a sample according to their mass-to-charge ratios.

Metallophore

a secondary metabolite that binds transition metals with high affinity and contributes to their uptake into cells. A siderophore is a secondary metabolite that has a high propensity to scavenge Fe(III).

Mis-metalation

the incorporation of an incorrect metal into metalloprotein that may result in defective protein structure and/or function.

Neutrophil extracellular traps (NETs)

net-like structures consisting of DNA embedded with antimicrobial peptides and proteins. NETs are released by activated neutrophils to combat extracellular pathogens.

Nuclear magnetic resonance (NMR)

a spectroscopic method to identify organic molecules and biomolecules and determine their structure, dynamics, and chemical environment.

Phenazines

redox-active bacterial metabolites implicated in metabolism, quorum sensing, and virulence.

Reactive oxygen species (ROS)

reactive oxygen-containing molecules and radicals including hydrogen peroxide, hydroxyl radical, and superoxide that can damage various biomolecules.

Redox potential

the oxidizing or reducing conditions in the environment that determine the oxidation state of different chemical species.

Superoxide dismutase (SOD)

an enzyme that catalyze the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide.