The siderophore yersiniabactin (Ybt) produced by a subset of intestinal adherent-invasive Escherichia coli (AIEC) drive intestinal fibrosis in murine model of Crohn's disease (CD). This is linked to the Ybt-induced disruption of host metal homeostasis and activation of the hypoxia-inducible factor 1-alpha (HIF-1α) in macrophages. Elevated glycolytic activity has been documented in both intestinal tissues and macrophages from patients with CD, indicating that metabolic reprogramming is a characteristic feature of the disease. Here, we show that HIF-1α stabilization by Ybt+ AIEC requires active host glycolysis. This effect is independent of Hif1a transcription and lipopolysaccharide stimulation and is not solely explained by intracellular bacterial load but instead relies on host metabolic activity. Mechanistically, Ybt+ AIEC activated the Akt-mTOR pathway to support HIF-1α translation. Inhibition of glycolysis suppressed this signaling axis, reducing HIF-1α translation and nuclear localization. Given the association between Ybt+ AIEC and fibrosis in CD, these findings suggest that targeting host glycolysis may limit AIEC-driven macrophage HIF-1α activation and fibrotic progression in CD patients.

Individuals with poorly controlled diabetes mellitus often develop multispecies skin and soft tissue infections, with Staphylococcus aureus and Pseudomonas aeruginosa among the most prevalent bacteria isolated from infection sites worldwide. Diabetic infections are recalcitrant to conventional antibiotic regimens and may be a reservoir for emergent antibiotic-resistant bacterial strains. Supporting this, we have previously shown that rifampicin treatment elicits the emergence and expansion of rifampicin-resistant (Rif-r) S. aureus only in diabetic mice, potentially due to greater bacterial outgrowth increasing the frequency of resistance-conferring mutations. However, whether S. aureus exhibits altered resistance outcomes during multispecies diabetic infections is unclear. During co-infection with P. aeruginosa under normoglycemic conditions, S. aureus exhibits reduced growth and altered susceptibility to several antibiotics. In contrast, we previously observed that glucose availability allows S. aureus to largely overcome P. aeruginosa-mediated growth inhibition. Here, we explored S. aureus resistance outcomes under hyperglycemic conditions in the context of co-infection with P. aeruginosa during antibiotic challenge. We found that P. aeruginosa exoproducts regulated by the Pseudomonas quinolone signal quorum sensing system inhibit the emergence but not the expansion of Rif-r S. aureus in vitro under glucose-replete conditions. In contrast, we recovered equivalent Rif-r S. aureus burdens from diabetic mice during mono- and co-infection with P. aeruginosa. These results demonstrate that the diabetic infection microenvironment is conducive to emergent Rif-r S. aureus despite external pressures elicited by P. aeruginosa.IMPORTANCEPoorly controlled diabetes mellitus confers an increased susceptibility to bacterial infections, with Staphylococcus aureus and Pseudomonas aeruginosa frequently isolated from diabetic skin wounds. S. aureus readily develops antibiotic resistance during diabetic mono-infection under antibiotic pressure, but whether this occurs during diabetic co-infection is unclear. Under normoglycemic conditions, secreted P. aeruginosa factors alter S. aureus tolerance to several antibiotics. Here, we show that these P. aeruginosa exoproducts further inhibit the emergence of antibiotic-resistant S. aureus regardless of glucose availability in vitro, but this does not occur during subcutaneous co-infection in diabetic mice. These results provide initial insights regarding conditions that may inhibit S. aureus resistance development in hyperglycemic environments but underscore the influence of the host infection microenvironment in shaping resistance outcomes.

Individuals with diabetes are at increased risk for severe outcomes from vaccine-preventable infections and often mount weaker immune responses to vaccination. The diabetes-related factors underlying this impaired immunity remain unclear, but defining them is critical for improving vaccine strategies in this vulnerable population. Here, we isolated insulin deficiency as a contributing factor to decreased vaccine-mediated immune responses using a mouse model. Following immunization with an alum-adjuvanted protein subunit vaccine, insulin-deficient mice exhibited reduced antigen-specific IgG antibody responses, decreased B-cell and T-cell numbers, and lower germinal center B-cell counts within the vaccine-draining lymph node. Three-dimensional whole-organ light sheet microscopy combined with virtual reality-assisted analysis further revealed significantly smaller germinal center volumes in insulin-deficient mice compared with controls. These findings indicate that insulin deficiency can significantly constrain germinal center responses and impair antibody production from vaccination. Our results provide foundational evidence that diabetes-associated metabolic changes can significantly and negatively influence the quality of vaccine-induced immunity and highlight insulin deficiency as a potential physiological factor in this process. This work establishes a framework for defining the mechanisms of diabetes-related immune suppression and guiding the design of more effective vaccines tailored to the unique immunological requirements of people with diabetes.

Individuals with diabetes mellitus frequently develop severe skin and soft tissue infections (SSTIs) that are recalcitrant to antibiotic treatment. We examined how diabetes affects the emergence of antibiotic resistance in a Staphylococcus aureus SSTI. We determined that S. aureus evolves antibiotic resistance rapidly in diabetic mice, while resistance did not occur in nondiabetic mice over the course of infection. Diabetes-associated immune cell dysfunction plays a minor role in the emergence of resistance, while hyperglycemia plays a dominant role facilitating the expansion and takeover of resistant mutants in diabetic infections. Furthermore, vancomycin intermediate resistant isolates display a pronounced fitness defect in nondiabetic mice but not in diabetic mice. Together, these data suggest that the diabetic infection environment represents an ideal reservoir for the emergence and proliferation of antibiotic resistance. Controlling the blood sugar of diabetic mice with insulin resulted in significantly decreased incidence of antibiotic-resistant S. aureus.

Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) pose a major healthcare burden. Distinct inflammatory and resolution phases comprise the host immune response to SSTIs. Resolution is a myeloid PPARγ-dependent anti-inflammatory phase that is essential for the clearance of MRSA. However, the signals activating PPARγ to induce resolution remain unknown. Here, we demonstrate that myeloid glucose transporter 1 (GLUT-1) is essential for the onset of resolution. MRSA-challenged macrophages are unsuccessful in generating an oxidative burst or immune radicals in the absence of GLUT-1 due to a reduction in the cellular NADPH pool. This translates in vivo as a significant reduction in lipid peroxidation products required for the activation of PPARγ in MRSA-infected mice lacking myeloid GLUT-1. Chemical induction of PPARγ during infection circumvents this GLUT-1 requirement and improves resolution. Thus, GLUT-1-dependent oxidative burst is essential for the activation of PPARγ and subsequent resolution of SSTIs.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS- CoV- 2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID- 19), with amplified immune activation in diverse cell types, including cells without angiotensin- converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS- CoV- 2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self- organization in the infected host environment. Interestingly, a machine learning analysis to SARS- CoV- 2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL- 37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS- CoV- 2 relative to low- pathogenicity coronaviruses. Moreover, xenoAMPs from SARS- CoV- 2 but not low- pathogenicity homologs assemble double- stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll- like receptor (TLR)- 3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID- 19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin- 6 and CXCL1 levels as observed in COVID- 19 patients.

Diabetes mellitus, characterized by impaired insulin signaling, is associated with increased incidence and severity of infections. Various diabetes-related complications contribute to exacerbated bacterial infections, including hyperglycemia, innate immune cell dysfunction, and infection with antibiotic-resistant bacterial strains. One defining symptom of diabetes is hyperglycemia, resulting in elevated blood and tissue glucose concentrations. Glucose is the preferred carbon source of several bacterial pathogens, and hyperglycemia escalates bacterial growth and virulence. Hyperglycemia promotes specific mechanisms of bacterial virulence known to contribute to infection chronicity, including tissue adherence and biofilm formation. Foot infections are a significant source of morbidity in individuals with diabetes and consist of biofilm-associated polymicrobial communities. Bacteria perform complex interspecies behaviors conducive to their growth and virulence within biofilms, including metabolic cross-feeding and altered phenotypes more tolerant to antibiotic therapeutics. Moreover, the metabolic dysfunction caused by diabetes compromises immune cell function, resulting in immune suppression. Impaired insulin signaling induces aberrations in phagocytic cells, which are crucial mediators for controlling and resolving bacterial infections. These aberrancies encompass altered cytokine profiles, the migratory and chemotactic mechanisms of neutrophils, and the metabolic reprogramming required for the oxidative burst and subsequent generation of bactericidal free radicals. Furthermore, the immune suppression caused by diabetes and the polymicrobial nature of the diabetic infection microenvironment may promote the emergence of novel strains of multidrug-resistant bacterial pathogens. This review focuses on the "triple threat" linked to worsened bacterial infections in individuals with diabetes: (i) altered nutritional availability in diabetic tissues, (ii) diabetes-associated immune suppression, and (iii) antibiotic treatment failure.

ABSTRACT 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. IMPORTANCE Sirolimus, 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.

It is estimated that over 11% of the U.S. population has diabetes and that 38% of the adult population is pre-diabetic. Diabetes carries increased risk and severity of many infections, which has been attributed to defects in the innate immune system. Despite the high impact of diabetes on infection, the underlying mechanisms of immune dysfunction in the diabetic host have yet to be fully elucidated. We have recently shown that innate immune cells in a diabetic host environment cannot undergo respiratory burst of reactive oxygen species and nitric oxide in response to infection with Staphylococcus aureus, the most common pathogen associated with frequently severe and invasive skin and soft tissue infection (SSTI) in patients with diabetes. The inhibition of respiratory burst was due to defects in the activation-dependent expression of the glucose transporter GLUT1, which is required for respiratory burst in neutrophils and macrophages. We therefore sought to further elucidate the specific mechanisms by which phagocytes in a diabetic environment are impaired in their ability to control S. aureusSSTI. We observed that mTOR signaling is impaired in a diabetic mouse model of S. aureusSSTI, which is upstream of both respiratory burst and GLUT-1 expression. Inhibition of mTOR signaling with rapamycin phenocopies the higher bacterial burden during diabetic infection, as well as the inhibition of respiratory burst and GLUT-1 expression. We further observed that mTOR activity upon macrophage activation is impaired in the absence of insulin and growth factor signaling. These findings begin to elucidate a role for mTOR signaling in the mechanism of immune suppression caused by diabetes.

ABSTRACT Diabetes is associated with several health consequences, including increased susceptibility to more frequent and severe infections. Bacterial infections associated with diabetes are typically polymicrobial, with Staphylococcus aureus and Pseudomonas aeruginosa frequently isolated from the same infection site. S. aureus and P. aeruginosa are frequently found in diabetic skin and soft tissue infections, in the lungs of people with cystic fibrosis, and in indwelling device infections. Numerous studies have investigated interactions between these two pathogens primarily using in vitro systems. These models have several limitations as they do not accurately reflect the complexities of an immune response nor the nutrient dynamics in a diabetic infection microenvironment. Here, we describe a novel murine indwelling device co-infection model that allows us to study the interactions between S. aureus and P. aeruginosa within the context of an immune response during both normal and diabetic infections. Our data shows that P. aeruginosa significantly inhibits S. aureus growth during co-infection in a normal mouse and that inhibition is not dependent on the P. aeruginosa PQS quorum sensing system. Conversely, in a diabetic co-infection, S. aureus overcomes inhibition by P. aeruginosa and this phenotype is reliant on S. aureus glycolysis. We also demonstrate that both organisms display increased virulence potential in a diabetic co-infection as we observe increased dissemination to peripheral tissues. This study revealed novel in vivo interactions between S. aureus and P. aeruginosa and advances our understanding of the complex interactions between microorganisms in polymicrobial infections in clinically relevant infection microenvironments. IMPORTANCE Individuals with diabetes are prone to more frequent and severe infections, with many of these infections being polymicrobial. Polymicrobial infections are frequently observed in skin infections and in individuals with cystic fibrosis, as well as in indwelling device infections. Two bacteria frequently co-isolated from infections are Staphylococcus aureus and Pseudomonas aeruginosa. Several studies have examined the interactions between these microorganisms. The majority of these studies use in vitro model systems that cannot accurately replicate the microenvironment of diabetic infections. We employed a novel murine indwelling device model to examine interactions between S. aureus and P. aeruginosa. Our data show that competition between these bacteria results in reduced growth in a normal infection. In a diabetic infection, we observe increased growth of both microbes and more severe infection as both bacteria invade surrounding tissues. Our results demonstrate that diabetes changes the interaction between bacteria resulting in poor infection outcomes.

Staphylococcus aureus is a human skin pathogen capable of causing invasive infections in many tissues in the human body. The host of virulence factors, such as toxins and proteases, available to S. aureus contribute to its diverse disease presentations. The majority of these virulence factors are under the control of the Agr quorum sensing system. The interaction between the Agr system and some well-established metabolic regulators has long been noted, but no mechanism has been provided as to these indirect interactions. In this study, we examine the connection between Agr and CcpA, a regulator of central carbon metabolism with a known positive impact on Agr function. We further investigated the interaction of Agr and CodY, a regulator of amino acid metabolism and a member of the stringent response with a known negative impact on Agr function. We show that though there are alterations in intracellular amino acid levels in each of these mutants that are consistent with their effect on Agr, there does not seem to be a direct impact on the translation of the Agr system itself that contributes to the altered expression observed in these mutants. Given the changes in cellular metabolism in a DccpA mutant, we find reduced levels of intracellular ATP even in the presence of glucose. This reduction in ATP, combined with the reduced affinity of the AgrC sensor kinase for ATP, explains the reduction in Agr activity long observed in DccpA strains. IMPORTANCE The human pathogen Staphylococcus aureus produces a great number of virulence factors that contribute to the pathogen's ability to cause dangerous, invasive infections. Understanding the full scope of the regulation of these virulence factors can provide us with new information about how to target virulence factor production. For years, researchers in the field have observed an impact of metabolic regulators on virulence factor production with no mechanistic explanation. Here, we describe the role of two of these regulators, CcpA and CodY, in virulence factor expression and provide evidence of indirect mechanisms contributing to the control of the Agr system and virulence factor production by these two metabolic regulators. Our study sheds light on the interplay between metabolism and virulence in S. aureus and provides an explanation as to how these concepts are linked.

Elevated blood/tissue glucose is a hallmark feature of advanced diabetes, and people with diabetes are prone to more frequent and invasive infections with Staphylococcus aureus. Phagocytes must markedly increase glucose consumption during infection to generate and oxidative burst and kill invading bacteria. Similarly, glucose is essential for S. aureus survival in an infection and competition with the host, for this limited resource is reminiscent of nutritional immunity. Here, we show that infiltrating phagocytes do not express their high-efficiency glucose transporters in modeled diabetic infections, resulting in a diminished respiratory burst and increased glucose availability for S. aureus We show that excess glucose in these hyperglycemic abscesses significantly enhances S. aureus virulence potential, resulting in worse infection outcomes. Last, we show that two glucose transporters recently acquired by S. aureus are essential for excess virulence factor production and the concomitant increase in disease severity in hyperglycemic infections.

The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD- scid IL2Rγ null (NSG) mouse and Sprague–Dawley-Rag2 tm2hera Il2rγ tm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus . The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.

Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) represent serious healthcare burdens worldwide. The host initially controls these infections with a pro-inflammatory infiltrate. However, once established, MRSA viability remains constant. To clear established MRSA SSTIs, the host must transition into the post-inflammatory resolution phase marked by infiltration of alternatively activated macrophages. Here we show that the host nuclear receptor, peroxisome proliferation activator receptor γ (PPARγ), is essential for this transition and MRSA clearance. Chemical PPARγ inhibition or genetic ablation of PPARγ in myeloid cells results in an extended inflammatory phase and exacerbated MRSA SSTIs. Conversely, treating mice with PPARγ agonists hastens the onset of the resolution phase and improves MRSA clearance in a myeloid-dependent fashion. The resolving fibrotic abscess lacks abundant glucose and oxygen but is replete with antimicrobial peptides, which together contribute to MRSA clearance. Thus, PPARγ agonists may serve as viable treatment options for complicated MRSA SSTIs.

Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.

Bacterial pneumonia is a leading cause of death late after burn injury due to the severe immune dysfunction that follows this traumatic injury. The Mechanistic/Mammalian Target of Rapamycin (mTOR) pathway drives many effector functions of innate immune cells required for bacterial clearance. Studies have demonstrated alterations in multiple cellular processes in patients and animal models following burn injury in which mTOR is a central component. Goals of this study were to (1) investigate the importance of mTOR signaling in antimicrobial activity by neutrophils and (2) therapeutically target mTOR to promote normalization of the immune response. We utilized a murine model of 20% total body surface area burn and the mTOR-specific inhibitor rapamycin. Burn injury led to innate immune hyperresponsiveness in the lung including recruitment of neutrophils with greater ex vivo oxidative activity compared with neutrophils from sham-injured mice. Elevated oxidative function correlated with improved clearance of Pseudomonas aeruginosa, despite down-regulated expression of the bacterial-sensing TLR molecules. Rapamycin administration reversed the burn injury-induced lung innate immune hyperresponsiveness and inhibited enhanced bacterial clearance in burn mice compared with untreated burn mice, resulting in significantly higher mortality. Neutrophil ex vivo oxidative burst was decreased by rapamycin treatment. These data indicate that (1) neutrophil function within the lung is more important than recruitment for bacterial clearance following burn injury and (2) mTOR inhibition significantly impacts innate immune hyperresponsiveness, including neutrophil effector function, allowing normalization of the immune response late after burn injury.

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for Abs in protecting against recurring infections, but S. aureus modulates the B cell response through expression of staphylococcus protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of costimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs. The absence of long-lived PCs was associated with a rapid decline in Ag-specific class-switched Ab. In contrast, when previously inoculated mice were challenged with an isogenic SpA-deficient S. aureus mutant, cells proliferated in the BM survival niches and sustained long-term Ab titers. The effects of SpA on PC fate were limited to the secondary response, because Ab levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with wild-type or SpA-deficient S. aureus mutant. Thus, failure to establish long-term protective Ab titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool.

Critically ill patients typically present with hyperglycemia. Treatment with conventional insulin therapy (targeting 144-180 mg/dl) improves patient survival; however, intensive insulin therapy (IIT) targeting normal blood glucose levels (81-108 mg/dl) increases the incidence of moderate and severe hypoglycemia, and increases mortality. Septic patients are especially prone to IIT-induced hypoglycemia, but the mechanism remains unknown. Here, we show that codelivery of insulin with otherwise sublethal doses of LPS induced hypoglycemic shock in mice within 1-2 h. LPS impaired clearance of insulin, which amplified insulin receptor signaling. These effects were mediated by caspase-11, TLR4, and complement, each of which trigger eicosanoid production that potentiates insulin signaling. Finally, in an animal model of sepsis, we observed that Salmonella typhimurium-infected mice exhibited simultaneous impaired insulin clearance coexisting with insulin resistance. Our results raise the possibility that septic patients have impaired insulin clearance, which could increase their susceptibility to hypoglycemia during IIT, contraindicating its use.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4+ T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease.

Summary Staphylococcus aureus is a Gram‐positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO‐resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l ‐lactate and peptides promotes S. aureus growth at moderate NO‐levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate‐quinone and l ‐lactate‐quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO‐stress for the combined utilization of peptides and l ‐lactate. We address the specific contributions of Lqo‐mediated l ‐lactate utilization and Mqo‐dependent amino acid consumption during NO‐stress. We show that Lqo conversion of l ‐lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate‐binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.