Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

Lipocalin-2 is an essential component of the innate immune response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

TLR3 agonism augments CD47 inhibition in acute myeloid leukemia.

CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

Altered Mitochondrial Homeostasis during Systemic Lupus Erythematosus Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus.

Inflammation involves a delicate balance between pathogen clearance and limiting host tissue damage, and perturbations in this equilibrium promote disease. Patients suffering from autoimmune diseases, such as systemic lupus erythematosus (SLE), have higher levels of serum S100A9 protein and increased risk for infection. S100A9 is highly abundant within neutrophils and modulates antimicrobial activity in response to bacterial pathogens. We reasoned that increased serum S100A9 in SLE patients reflects accumulation of S100A9 protein in neutrophils and may indicate altered neutrophil function. In this study, we demonstrate elevated S100A9 protein within neutrophils from SLE patients, and MRL/lpr mice associates with lower mitochondrial superoxide, decreased suicidal neutrophil extracellular trap formation, and increased susceptibility to Staphylococcus aureus infection. Furthermore, increasing mitochondrial superoxide production restored the antibacterial activity of MRL/lpr neutrophils in response to S. aureus These results demonstrate that accumulation of intracellular S100A9 associates with impaired mitochondrial homeostasis, thereby rendering SLE neutrophils inherently less bactericidal.

Increased Dietary Manganese Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus.

Dietary metals can modify the risk to infection. Previously, we demonstrated that heightened dietary manganese (Mn) during systemic Staphylococcus aureus infection increases S. aureus virulence. However, immune cells also operate in these same environments and the effect of dietary Mn on neutrophil function in vivo has not been assessed. This study reveals that increased concentrations of Mn impairs mitochondrial respiration and superoxide production in neutrophils responding to S. aureus. As a result, high Mn accelerates primary degranulation, while impairing suicidal neutrophil extracellular trap (NET) formation, which decreases bactericidal activity. In vivo, elevated dietary Mn accumulated extracellularly in the heart, indicating that excess Mn may be more bioavailable in the heart. Coinciding with this phenotype, neutrophil function in the heart was most impacted by a high Mn diet, as neutrophils produced lower levels of mitochondrial superoxide and underwent less suicidal NET formation. Consistent with an ineffective neutrophil response when mice are on a high Mn diet, S. aureus burdens were increased in the heart and mice were more susceptible to systemic infection. Therefore, elevated dietary Mn not only affects S. aureus but also renders neutrophils less capable of restricting staphylococcal infection.

Mitochondrial Calcium Uniporter Affects Neutrophil Bactericidal Activity during Staphylococcus aureus Infection.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

mTORC2 Activity Disrupts Lysosome Acidification in Systemic Lupus Erythematosus by Impairing Caspase-1 Cleavage of Rab39a.

Lysosomes maintain immune homeostasis through the degradation of phagocytosed apoptotic debris; however, the signaling events regulating lysosomal maturation remain undefined. In this study, we show that lysosome acidification, key to the maturation process, relies on mTOR complex 2 (mTORC2), activation of caspase-1, and cleavage of Rab39a. Mechanistically, the localization of cofilin to the phagosome recruits caspase-11, which results in the localized activation of caspase-1. Caspase-1 subsequently cleaves Rab39a on the phagosomal membrane, promoting lysosome acidification. Although caspase-1 is critical for lysosome acidification, its activation is independent of inflammasomes and cell death mediated by apoptosis-associated speck-like protein containing a caspase recruitment domain, revealing a role beyond pyroptosis. In lupus-prone murine macrophages, chronic mTORC2 activity decouples the signaling pathway, leaving Rab39a intact. As a result, the lysosome does not acidify, and degradation is impaired, thereby heightening the burden of immune complexes that activate FcγRI and sustain mTORC2 activity. This feedforward loop promotes chronic immune activation, leading to multiple lupus-associated pathologies. In summary, these findings identify the key molecules in a previously unappreciated signaling pathway that promote lysosome acidification. It also shows that this pathway is disrupted in systemic lupus erythematosus.

A Phenome-Wide Association Study Uncovers a Pathological Role of Coagulation Factor X during Acinetobacter baumannii Infection.

Coagulation and inflammation are interconnected, suggesting that coagulation plays a key role in the inflammatory response to pathogens. A phenome-wide association study (PheWAS) was used to identify clinical phenotypes of patients with a polymorphism in coagulation factor X. Patients with this single nucleotide polymorphism (SNP) were more likely to be hospitalized with hemostatic and infection-related disorders, suggesting that factor X contributes to the immune response to infection. To investigate this, we modeled infections by human pathogens in a mouse model of factor X deficiency. Factor X-deficient mice were protected from systemic Acinetobacter baumannii infection, suggesting that factor X plays a role in the immune response to A. baumannii Factor X deficiency was associated with reduced cytokine and chemokine production and alterations in immune cell population during infection: factor X-deficient mice demonstrated increased abundance of neutrophils, macrophages, and effector T cells. Together, these results suggest that factor X activity is associated with an inefficient immune response and contributes to the pathology of A. baumannii infection.

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus.

Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG-immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus.

Apoptotic Debris Accumulates on Hematopoietic Cells and Promotes Disease in Murine and Human Systemic Lupus Erythematosus.

Apoptotic debris, autoantibody, and IgG-immune complexes (ICs) have long been implicated in the inflammation associated with systemic lupus erythematosus (SLE); however, it remains unclear whether they initiate immune-mediated events that promote disease. In this study, we show that PBMCs from SLE patients experiencing active disease, and hematopoietic cells from lupus-prone MRL/lpr and NZM2410 mice accumulate markedly elevated levels of surface-bound nuclear self-antigens. On dendritic cells (DCs) and macrophages (MFs), the self-antigens are part of IgG-ICs that promote FcγRI-mediated signal transduction. Accumulation of IgG-ICs is evident on ex vivo myeloid cells from MRL/lpr mice by 10 wk of age and steadily increases prior to lupus nephritis. IgG and FcγRI play a critical role in disease pathology. Passive transfer of pathogenic IgG into IgG-deficient MRL/lpr mice promotes the accumulation of IgG-ICs prior to significant B cell expansion, BAFF secretion, and lupus nephritis. In contrast, diminishing the burden IgG-ICs in MRL/lpr mice through deficiency in FcγRI markedly improves these lupus pathologies. Taken together, our findings reveal a previously unappreciated role for the cell surface accumulation of IgG-ICs in human and murine lupus.

Receptor cross-talk spatially restricts p-ERK during TLR4 stimulation of autoreactive B cells.

To maintain tolerance, autoreactive B cells must regulate signal transduction from the BCR and TLRs. We recently identified that dendritic cells and macrophages regulate autoreactive cells during TLR4 activation by releasing IL-6 and soluble CD40 ligand (sCD40L). These cytokines selectively repress Ab secretion from autoreactive, but not antigenically naive, B cells. How IL-6 and sCD40L repress autoantibody production is unknown. In this work, we show that IL-6 and sCD40L are required for low-affinity/avidity autoreactive B cells to maintain tolerance through a mechanism involving receptor cross-talk between the BCR, TLR4, and the IL-6R or CD40. We show that acute signaling through IL-6R or CD40 integrates with chronic BCR-mediated ERK activation to restrict p-ERK from the nucleus and represses TLR4-induced Blimp-1 and XBP-1 expression. Tolerance is disrupted in 2-12H/MRL/lpr mice where IL-6 and sCD40L fail to spatially restrict p-ERK and fail to repress TLR4-induced Ig secretion. In the case of CD40, acute signaling in B cells from 2-12H/MRL/lpr mice is intact, but the chronic activation of p-ERK emanating from the BCR is attenuated. Re-establishing chronically active ERK through retroviral expression of constitutively active MEK1 restores tolerance upon sCD40L, but not IL-6, stimulation, indicating that regulation by IL-6 requires another signaling effector. These data define the molecular basis for the regulation of low-affinity autoreactive B cells during TLR4 stimulation; they explain how autoreactive but not naive B cells are repressed by IL-6 and sCD40L; and they identify B cell defects in lupus-prone mice that lead to TLR4-induced autoantibody production.

mTOR signaling is required for phagocyte free radical production, GLUT1 expression, and control of Staphylococcus aureus infection.

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.

Lactate Utilization Enables Metabolic Escape to Confer Resistance to BET Inhibition in Acute Myeloid Leukemia.

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment. SIGNIFICANCE: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950.

Bacterial hydrophilins promote pathogen desiccation tolerance.

Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.

The impact of metal availability on immune function during infection.

Nutrient transition metals are required cofactors for many proteins to perform functions necessary for life. As such, the concentration of nutrient metals is carefully maintained to retain critical biological processes while limiting toxicity. During infection, invading bacterial pathogens must acquire essential metals, such as zinc, manganese, iron, and copper, from the host to colonize and cause disease. To combat this, the host exploits the essentiality and toxicity of nutrient metals by producing factors that limit metal availability, thereby starving pathogens or accumulating metals in excess to intoxicate the pathogen in a process termed 'nutritional immunity'. As a result of inflammation, a heterogeneous environment containing both metal-replete and -deplete niches is created, in which nutrient metal availability may have an underappreciated role in regulating immune cell function during infection. How the host manipulates nutrient metal availability during infection, and the downstream effects that nutrient metals and metal-sequestering proteins have on immune cell function, are discussed in this review.

Neutrophil extracellular traps enhance macrophage killing of bacterial pathogens.

Neutrophils and macrophages are critical to the innate immune response, but cooperative mechanisms used by these cells to combat extracellular pathogens are not well understood. This study reveals that S100A9-deficient neutrophils produce higher levels of mitochondrial superoxide in response to Staphylococcus aureus and, as a result, form neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not improve neutrophil killing of S. aureus in isolation but augments macrophage killing. NET formation enhances antibacterial activity by increasing phagocytosis by macrophages and by transferring neutrophil-specific antimicrobial peptides to them. Similar results were observed in response to other phylogenetically distinct bacterial pathogens including Streptococcus pneumoniae and Pseudomonas aeruginosa, implicating this as an immune defense mechanism that broadly enhances antibacterial activity. These results demonstrate that achieving maximal bactericidal activity through NET formation requires macrophages and that accelerated and more robust suicidal NETosis makes neutrophils adept at increasing antibacterial activity, especially when A9 deficient.

Spatially Targeted Proteomics of the Host-Pathogen Interface during Staphylococcal Abscess Formation.

Staphylococcus aureus is a common cause of invasive and life-threatening infections that are often multidrug resistant. To develop novel treatment approaches, a detailed understanding of the complex host-pathogen interactions during infection is essential. This is particularly true for the molecular processes that govern the formation of tissue abscesses, as these heterogeneous structures are important contributors to staphylococcal pathogenicity. To fully characterize the developmental process leading to mature abscesses, temporal and spatial analytical approaches are required. Spatially targeted proteomic technologies such as micro-liquid extraction surface analysis offer insight into complex biological systems including detection of bacterial proteins and their abundance in the host environment. By analyzing the proteomic constituents of different abscess regions across the course of infection, we defined the immune response and bacterial contribution to abscess development through spatial and temporal proteomic assessment. The information gathered was mapped to biochemical pathways to characterize the metabolic processes and immune strategies employed by the host. These data provide insights into the physiological state of bacteria within abscesses and elucidate pathogenic processes at the host-pathogen interface.

S100 Proteins in the Innate Immune Response to Pathogens.

S100 proteins are distinct dimeric EF-hand Ca2+-binding proteins that can bind Zn2+, Mn2+, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.

Arachidonic Acid Kills Staphylococcus aureus through a Lipid Peroxidation Mechanism.

Staphylococcus aureus infects every niche of the human host. In response to microbial infection, vertebrates have an arsenal of antimicrobial compounds that inhibit bacterial growth or kill bacterial cells. One class of antimicrobial compounds consists of polyunsaturated fatty acids, which are highly abundant in eukaryotes and encountered by S. aureus at the host-pathogen interface. Arachidonic acid (AA) is one of the most abundant polyunsaturated fatty acids in vertebrates and is released in large amounts during the oxidative burst. Most of the released AA is converted to bioactive signaling molecules, but, independently of its role in inflammatory signaling, AA is toxic to S. aureus Here, we report that AA kills S. aureus through a lipid peroxidation mechanism whereby AA is oxidized to reactive electrophiles that modify S. aureus macromolecules, eliciting toxicity. This process is rescued by cotreatment with antioxidants as well as in a S. aureus strain genetically inactivated for lcpA (USA300 ΔlcpA mutant) that produces lower levels of reactive oxygen species. However, resistance to AA stress in the USA300 ΔlcpA mutant comes at a cost, making the mutant more susceptible to ß-lactam antibiotics and attenuated for pathogenesis in a murine infection model compared to the parental methicillin-resistant S. aureus (MRSA) strain, indicating that resistance to AA toxicity increases susceptibility to other stressors encountered during infection. This report defines the mechanism by which AA is toxic to S. aureus and identifies lipid peroxidation as a pathway that can be modulated for the development of future therapeutics to treat S. aureus infections.IMPORTANCE Despite the ability of the human immune system to generate a plethora of molecules to control Staphylococcus aureus infections, S. aureus is among the pathogens with the greatest impact on human health. One class of host molecules toxic to S. aureus consists of polyunsaturated fatty acids. Here, we investigated the antibacterial properties of arachidonic acid, one of the most abundant polyunsaturated fatty acids in humans, and discovered that the mechanism of toxicity against S. aureus proceeds through lipid peroxidation. A better understanding of the molecular mechanisms by which the immune system kills S. aureus, and by which S. aureus avoids host killing, will enable the optimal design of therapeutics that complement the ability of the vertebrate immune response to eliminate S. aureus infections.