Glucose transporter 1 is essential for the resolution of methicillin-resistant S. aureus skin and soft tissue infections.

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.

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy.

Coronavirus disease 2019 (COVID-19) has claimed millions of lives since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and lung disease appears the primary cause of death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors. Two cytokines in particular, IP-10 and IL-8, were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei, not only inhibited virus replication but also production of pro-inflammatory cytokines, and thus ameliorated the histopathological changes caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs. IMPORTANCE: Here, established an ex vivo human precision-cut lung slice platform for assessing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, viral replication kinetics, innate immune response, disease progression, and antiviral drugs. Using this platform, we identified early induction of specific cytokines, especially IP-10 and IL-8, as potential predictors for severe coronavirus disease 2019 (COVID-19), and uncovered a hitherto unrecognized phenomenon that while infectious virus disappears at late times of infection, viral RNA persists and lung histopathology commences. This finding may have important clinical implications for both acute and post-acute sequelae of COVID-19. This platform recapitulates some of the characteristics of lung disease observed in severe COVID-19 patients and is therefore a useful platform for understanding mechanisms of SARS-CoV-2 pathogenesis and for evaluating the efficacy of antiviral drugs.

Specialized phosphate transport is essential for Staphylococcus aureus nitric oxide resistance.

Staphylococcus aureus is a major human pathogen capable of causing a variety of diseases ranging from skin and soft tissue infections to systemic presentations such as sepsis, endocarditis, and osteomyelitis. For S. aureus to persist as a pathogen in these environments, it must be able to resist the host immune response, including the production of reactive oxygen and nitrogen species (e.g., nitric oxide, NO·). Extensive work from our lab has shown that S. aureus is highly resistant to NO·, especially in the presence of glucose. RNA-seq performed on S. aureus exposed to NO· in the presence and absence of glucose showed a new system important for NO· resistance-phosphate transport. The phosphate transport systems pstSCAB and nptA are both upregulated upon NO·-exposure, particularly in the presence of glucose. Both are key for phosphate transport at an alkaline pH, which the cytosol of S. aureus becomes under NO· stress. Accordingly, the ΔpstSΔnptA mutant is attenuated under NO stress in vitro as well as in macrophage and murine infection models. This work defines a new role in infection for two phosphate transporters in S. aureus and provides insight into the complex system that is NO· resistance in S. aureus.IMPORTANCEStaphylococcus aureus is a bacterial pathogen capable of causing a wide variety of disease in humans. S. aureus is unique in its ability to resist the host immune response, including the antibacterial compound known as nitric oxide (NO·). We used an RNA-sequencing approach to better understand the impact of NO· on S. aureus in different environments. We discovered that inorganic phosphate transport is induced by the presence of NO·. Phosphate is important for the generation of energy from glucose, a carbon source favored by S. aureus. We show that the absence of these phosphate transporters causes lowered energy levels in S. aureus. We find that these phosphate transporters are essential for S. aureus to grow in the presence of NO· and to cause infection. Our work here contributes significantly to our understanding of S. aureus NO· resistance and provides a new context in which S. aureus phosphate transporters are essential.

The contribution of DNA repair pathways to Staphylococcus aureus fitness and fidelity during nitric oxide stress.

Staphylococcus aureus is a major human pathogen that causes a variety of illnesses, ranging from minor skin and soft tissue infections to more severe systemic infections. Although the primary host immune response can typically clear bacterial infections, S. aureus is uniquely resistant to inflammation. For instance, our laboratory has determined that S. aureus is highly resistant to nitric oxide (NO⋅), an important component of the innate immune response that plays a role in both immunomodulatory and antibacterial processes. Additionally, NO⋅ and its derivatives can cause damage to S. aureus DNA, more specifically, deamination and/or oxidation of DNA bases; however, regulation and repair mechanisms of DNA in S. aureus are understudied. Thus, we hypothesize that several DNA repair mechanisms may account for the replication fidelity of S. aureus and may contribute to fitness in the presence of NO⋅. Here, we show the role of several DNA repair mechanisms in S. aureus. More specifically, we found that recombinational repair genes recJ, recG, and polA may play a role in the repair of NO⋅-induced replication fork collapses. We also show the role of the base excision repair pathway protein, MutY, in reducing NO⋅-mediated mutagenesis. Overall, our results suggest that NO⋅ leads to DNA damage, which subsequently induces the activity of several DNA repair pathways, contributing to the replication fidelity and fitness of S. aureus.IMPORTANCEPathogenic bacteria must evolve various mechanisms in order to evade the host immune response that they are infecting. One aspect of the primary host immune response to an infection is the production of an inflammatory effector component, nitric oxide (NO⋅). Staphylococcus aureus has uniquely evolved a diverse array of strategies to circumvent the inhibitory activity of nitric oxide. One such mechanism by which S. aureus has evolved allows the pathogen to survive and maintain its genomic integrity in this environment. For instance, here, our results suggest that S. aureus employs several DNA repair pathways to ensure replicative fitness and fidelity under NO⋅ stress. Thus, our study presents evidence of an additional strategy that allows S. aureus to evade the cytotoxic effects of host NO⋅.

Pulmonary Expression of Interleukin-17 Contributes to Neutrophil Infiltration into the Lungs during Pneumonic Plague.

Inhalation of respiratory droplets infected with Yersinia pestis results in a rapidly progressing and lethal necrotic pneumonia called primary pneumonic plague. Disease manifests as biphasic, with an initial preinflammatory phase with rapid bacterial replication in the lungs absent readily detectable host immune responses. This is followed by the onset of a proinflammatory phase that sees the dramatic upregulation of proinflammatory cytokines and extensive neutrophil accumulation in the lungs. The plasminogen activator protease (Pla) is an essential virulence factor that is responsible for survival of Y. pestis in the lungs. Our lab recently showed that Pla functions as an adhesin that promotes binding to alveolar macrophages to facilitate translocation of effector proteins called Yops into the cytosol of target host cells via a type 3 secretion system (T3SS). Loss of Pla-mediated adherence disrupted the preinflammatory phase of disease and resulted in early neutrophil migration to the lungs. While it is established that Yersinia broadly suppresses host innate immune responses, it is not clear precisely which signals need to be inhibited to establish a preinflammatory stage of infection. Here, we show that early Pla-mediated suppression of Interleukin-17 (IL-17) expression in alveolar macrophages and pulmonary neutrophils limits neutrophil migration to the lungs and aids in establishing a preinflammatory phase of disease. In addition, IL-17 ultimately contributes to neutrophil migration to the airways that defines the later proinflammatory phase of infection. These results suggest that the pattern of IL-17 expression contributes to the progression of primary pneumonic plague.

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy.

COVID-19 has claimed millions of lives since the emergence of SARS-CoV-2, and lung disease appears the primary cause of the death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where the human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors, reflecting the heterogeneity of human populations. In particular, two cytokines (IP-10 and IL-8) were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei , not only inhibited virus replication but also production of pro-inflammatory cytokines, and ameliorated the histopathological changes of the lungs caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs. SIGNIFICANCE: Here we established an ex vivo human precision-cut lung slice platform for assessing SARS-CoV-2 infection, viral replication kinetics, innate immune response, disease progression, and antiviral drugs. Using this platform, we identified early induction of specific cytokines, especially IP-10 and IL-8, as potential predictors for severe COVID-19, and uncovered a hitherto unrecognized phenomenon that while infectious virus disappears at late times of infection, viral RNA persists and lung histopathology commences. This finding may have important clinical implications for both acute and post-acute sequelae of COVID-19. This platform recapitulates some of the characteristics of lung disease observed in severe COVID-19 patients and is therefore a useful platform for understanding mechanisms of SARS-CoV-2 pathogenesis and for evaluating the efficacy of antiviral drugs.

Activation and Regulation of NLRP3 by Sterile and Infectious Insults.

Nod-Like Receptor (NLR) is the largest family of Pathogen Recognition Receptors (PRRs) that patrols the cytosolic environment. NLR engagement drives caspase-1 activation that cleaves pro-IL-1B which then gets secreted. Released IL-1B recruits immune cells to the site of infection/injury. Caspase-1 also cleaves Gasdermin-D (GSDM-D) that forms pores within the plasma membrane driving inflammatory cell death called pyroptosis. NLRP3 is the most extensively studied NLR. The NLRP3 gene is encoded by 9 exons, where exon 1 codes for pyrin domain, exon 3 codes for NACHT domain, and Leucine Rich Repeat (LRR) domain is coded by exon 4-9. Exon 2 codes for a highly disorganized loop that connects the rest of the protein to the pyrin domain and may be involved in NLRP3 regulation. The NLRP3 inflammasome is activated by many structurally divergent agonists of microbial, environmental, and host origin. Activated NLRP3 interacts with an adaptor protein, ASC, that bridges it to pro-Caspase-1 forming a multi-protein complex called inflammasome. Dysregulation of NLRP3 inflammasome activity is a hallmark of pathogenesis in several human diseases, indicating its highly significant clinical relevance. In this review, we summarize the existing knowledge about the mechanism of activation of NLRP3 and its regulation during activation by infectious and sterile triggers.

Treatment with Fluticasone Propionate Increases Antibiotic Efficacy during Treatment of Late-Stage Primary Pneumonic Plague.

Severe and late-stage pneumonias are often difficult to treat with antibiotics alone due to overwhelming host inflammatory responses mounted to clear infection. These host responses contribute to pulmonary damage leading to acute lung injury, acute respiratory distress syndrome, and death. In order to effectively treat severe and late-stage pneumonias, use of adjunctive therapies must be considered to reduce pulmonary damage when antimicrobial agents can be administered. Pneumonic plague, a severe pneumonia caused by inhalation of Yersinia pestis, is a fatal disease that causes death within 6 days without antibiotic intervention. Late-stage pneumonic plague is difficult to treat, as antibiotics must be delivered within 24 h after onset of symptoms to be effective. Here, we use a murine model of primary pneumonic plague to examine how host inflammatory responses impact antibiotic treatment of late-stage pneumonic plague. We developed a murine infection model demonstrating the poor outcomes associated with delayed delivery of antibiotics. We show that pretreatment of mice with intranasal fluticasone propionate increased the efficacy of delayed antibiotic delivery and enhanced murine survival. Mice receiving fluticasone propionate also showed decreased bacterial burden and reduced inflammatory pathology in the lungs. Further, we show that treatment and survival correlated with decreased levels of interleukin-6 (IL-6) and reduced neutrophil infiltration to the lungs. This work demonstrates how host inflammatory responses complicate treatment of late-stage pneumonic plague and suggests that targeting of host inflammatory responses may improve treatment of severe, late-stage pneumonia.

The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals.

Staphylococcus aureus is the most pathogenic member of the Staphylococcaceae. While it acquired an arsenal of canonical virulence determinants that mediate pathogenicity, it has also metabolically adapted to thrive at sites of inflammation. Notably, it has evolved to grow in the presence of nitric oxide (NO·). To this end, we note that the Rex regulon, composed of genes encoding dehydrogenases, metabolite transporters, and regulators, is much larger in S. aureus than other Staphylococcus species. Here, we demonstrate that this expanded Rex regulon is necessary and sufficient for NO· resistance. Preventing its expression results in NO· sensitivity, and the closely related species, Staphylococcus simiae, also possesses an expanded Rex regulon and exhibits NO· resistance. We hypothesize that the expanded Rex regulon initially evolved to provide efficient anaerobic metabolism but that S. aureus has co-opted this feature to thrive at sites of inflammation where respiration is limited. One distinguishing feature of the Rex regulon in S. aureus is that it contains the srrAB two-component system. Here, we show that Rex blocks the ability of SrrA to auto-induce the operon, thereby preventing maximal SrrAB expression. This results in NO·-responsive srrAB expression in S. aureus but not in other staphylococci. Consequently, higher expression of cytochromes and NO· detoxification are also observed in S. aureus alone, allowing for continued respiration at NO· concentrations beyond that of S. simiae. We therefore contend that the intersection of the Rex and SrrAB regulons represents an evolutionary event that allowed S. aureus to metabolically adapt to host inflammatory radicals during infection. IMPORTANCE Pathogens must evolve virulence potential to improve transmission to new hosts as well as evolve metabolically to thrive within their current host. Staphylococcus aureus has achieved both of these, and here, we show that one such metabolic adaptation was the expansion of the Rex regulon. First, it affords S. aureus with efficient respiration-independent growth critical to surviving the inflammatory environment replete with respiration-inhibiting immune radicals. Second, it includes the srrAB operon encoding a two-component system critical to maximizing respiratory capacity in the face of host nitric oxide (NO·), a potent respiratory inhibitor. This second facet is only apparent in S. aureus and not in other closely related species. Thus, evolutionarily, it must have occurred relatively recently. The intertwining of the Rex and SrrAB regulons represents an important evolutionary event that affords S. aureus the metabolic flexibility required to thrive within inflamed tissue and cause disease.

The Yersinia pestis GTPase BipA Promotes Pathogenesis of Primary Pneumonic Plague.

Yersinia pestis is a highly virulent pathogen and the causative agent of bubonic, septicemic, and pneumonic plague. Primary pneumonic plague caused by inhalation of respiratory droplets contaminated with Y. pestis is nearly 100% lethal within 4 to 7 days without antibiotic intervention. Pneumonic plague progresses in two phases, beginning with extensive bacterial replication in the lung with minimal host responsiveness, followed by the abrupt onset of a lethal proinflammatory response. The precise mechanisms by which Y. pestis is able to colonize the lung and survive two very distinct disease phases remain largely unknown. To date, a few bacterial virulence factors, including the Ysc type 3 secretion system, are known to contribute to the pathogenesis of primary pneumonic plague. The bacterial GTPase BipA has been shown to regulate expression of virulence factors in a number of Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhi. However, the role of BipA in Y. pestis has yet to be investigated. Here, we show that BipA is a Y. pestis virulence factor that promotes defense against early neutrophil-mediated bacterial killing in the lung. This work identifies a novel Y. pestis virulence factor and highlights the importance of early bacterial/neutrophil interactions in the lung during primary pneumonic plague.

A Dual Role for the Plasminogen Activator Protease During the Preinflammatory Phase of Primary Pneumonic Plague.

Early after inhalation, Yersinia pestis replicates to high numbers in the airways in the absence of disease symptoms or notable inflammatory responses to cause primary pneumonic plague. The plasminogen activator protease (Pla) is a critical Y. pestis virulence factor that is important for early bacterial growth in the lung via an unknown mechanism. In this article, we define a dual role for Pla in the initial stages of pulmonary infection. We show that Pla functions as an adhesin independent of its proteolytic function to suppress early neutrophil influx into the lungs, and that Pla enzymatic activity contributes to bacterial resistance to neutrophil-mediated bacterial killing. Our results suggest that the fate of Y. pestis infection of the lung is decided extremely early during infection and that Pla plays a dual role to tilt the balance in favor of the pathogen.

Modeling Pneumonic Plague in Human Precision-Cut Lung Slices Highlights a Role for the Plasminogen Activator Protease in Facilitating Type 3 Secretion.

Pneumonic plague is the deadliest form of disease caused by Yersinia pestis Key to the progression of infection is the activity of the plasminogen activator protease Pla. Deletion of Pla results in a decreased Y. pestis bacterial burden in the lung and failure to progress into the lethal proinflammatory phase of disease. While a number of putative functions have been attributed to Pla, its precise role in the pathogenesis of pneumonic plague is yet to be defined. Here, we show that Pla facilitates type 3 secretion into primary alveolar macrophages but not into the commonly used THP-1 cell line. We also establish human precision-cut lung slices as a platform for modeling early host/pathogen interactions during pneumonic plague and solidify the role of Pla in promoting optimal type 3 secretion using primary human tissue with relevant host cell heterogeneity. These results position Pla as a key player in the early host/pathogen interactions that define pneumonic plague and showcase the utility of human precision-cut lung slices as a platform to evaluate pulmonary infection by bacterial pathogens.