Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Ketogenesis promotes tolerance to Pseudomonas aeruginosa pulmonary infection.

Pseudomonas aeruginosa is a common cause of pulmonary infection. As a Gram-negative pathogen, it can initiate a brisk and highly destructive inflammatory response; however, most hosts become tolerant to the bacterial burden, developing chronic infection. Using a murine model of pneumonia, we demonstrate that this shift from inflammation to disease tolerance is promoted by ketogenesis. In response to pulmonary infection, ketone bodies are generated in the liver and circulate to the lungs where they impose selection for P. aeruginosa strains unable to display surface lipopolysaccharide (LPS). Such keto-adapted LPS strains fail to activate glycolysis and tissue-damaging cytokines and, instead, facilitate mitochondrial catabolism of fats and oxidative phosphorylation (OXPHOS), which maintains airway homeostasis. Within the lung, P. aeruginosa exploits the host immunometabolite itaconate to further stimulate ketogenesis. This environment enables host-P. aeruginosa coexistence, supporting both pathoadaptive changes in the bacteria and the maintenance of respiratory integrity via OXPHOS.

Immunometabolic control by Klebsiella pneumoniae.

Klebsiella pneumoniae is a common Gram-negative pathogen associated with community-acquired and healthcare-associated infections. Its ability to acquire genetic elements resulted in its rapid development of resistance to virtually all antimicrobial agents. Once infection is established, K. pneumoniae is able to evade the host immune response and perhaps more importantly, undergo metabolic rewiring to optimize its ability to maintain infection. K. pneumoniae lipopolysaccharide and capsular polysaccharide are central factors in the induction and evasion of immune clearance. Less well understood is the importance of immunometabolism, the intersection between cellular metabolism and immune function, in the host response to K. pneumoniae infection. Bacterial metabolism itself is perceived as a metabolic stress to the host, altering the microenvironment at the site of infection. In this review, we will discuss the metabolic responses induced by K. pneumoniae, particularly in response to stimulation with the metabolically active bacteria versus pathogen-associated molecular patterns alone, and their implications in shaping the nature of the immune response and the infection outcome. A better understanding of the immunometabolic response to K. pneumoniae may help identify new targets for therapeutic intervention in the treatment of multidrug-resistant bacterial infections.

Staphylococcus aureus stimulates neutrophil itaconate production that suppresses the oxidative burst.

Neutrophils are critical in the host defense against Staphylococcus aureus, a major human pathogen. However, even in the setting of a robust neutrophil response, S. aureus can evade immune clearance. Here, we demonstrate that S. aureus impairs neutrophil function by triggering the production of the anti-inflammatory metabolite itaconate. The enzyme that synthesizes itaconate, Irg1, is selectively expressed in neutrophils during S. aureus pneumonia. Itaconate inhibits neutrophil glycolysis and oxidative burst, which impairs survival and bacterial killing. In a murine pneumonia model, neutrophil Irg1 expression protects the lung from excessive inflammation but compromises bacterial clearance. S. aureus is thus able to evade the innate immune response by targeting neutrophil metabolism and inducing the production of the anti-inflammatory metabolite itaconate.

Anti-Inflammatory Metabolites in the Pathogenesis of Bacterial Infection.

Host and pathogen metabolism have a major impact on the outcome of infection. The microenvironment consisting of immune and stromal cells drives bacterial proliferation and adaptation, while also shaping the activity of the immune system. The abundant metabolites itaconate and adenosine are classified as anti-inflammatory, as they help to contain the local damage associated with inflammation, oxidants and proteases. A growing literature details the many roles of these immunometabolites in the pathogenesis of infection and their diverse functions in specific tissues. Some bacteria, notably P. aeruginosa, actively metabolize these compounds, others, such as S. aureus respond by altering their own metabolic programs selecting for optimal fitness. For most of the model systems studied to date, these immunometabolites promote a milieu of tolerance, limiting local immune clearance mechanisms, along with promoting bacterial adaptation. The generation of metabolites such as adenosine and itaconate can be host protective. In the setting of acute inflammation, these compounds also represent potential therapeutic targets to prevent infection.

Immunometabolic crosstalk during bacterial infection.

Following detection of bacteria, macrophages switch their metabolism from oxidative respiration through the tricarboxylic acid cycle to high-rate aerobic glycolysis. This immunometabolic shift enables pro-inflammatory and antimicrobial responses and is facilitated by the accumulation of fatty acids, tricarboxylic acid-derived metabolites and catabolism of amino acids. Recent studies have shown that these immunometabolites are co-opted by pathogens as environmental cues for expression of virulence genes. We review mechanisms by which host immunometabolites regulate bacterial pathogenicity and discuss opportunities for the development of therapeutics targeting metabolic host-pathogen crosstalk.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

Staphylococcus aureus adaptive evolution: Recent insights on how immune evasion, immunometabolic subversion and host genetics impact vaccine development.

Despite meritorious attempts, a S. aureus vaccine that prevents infection or mitigates severity has not yet achieved efficacy endpoints in prospective, randomized clinical trials. This experience underscores the complexity of host-S. aureus interactions, which appear to be greater than many other bacterial pathogens against which successful vaccines have been developed. It is increasingly evident that S. aureus employs strategic countermeasures to evade or exploit human immune responses. From entering host cells to persist in stealthy intracellular reservoirs, to sensing the environmental milieu and leveraging bacterial or host metabolic products to reprogram host immune responses, S. aureus poses considerable challenges for the development of effective vaccines. The fact that this pathogen causes distinct types of infections and can undergo transient genetic, transcriptional or metabolic adaptations in vivo that do not occur in vitro compounds challenges in vaccine development. Notably, the metabolic versatility of both bacterial and host immune cells as they compete for available substrates within specific tissues inevitably impacts the variable repertoire of gene products that may or may not be vaccine antigens. In this respect, S. aureus has chameleon phenotypes that have alluded vaccine strategies thus far. Nonetheless, a number of recent studies have also revealed important new insights into pathogenesis vulnerabilities of S. aureus. A more detailed understanding of host protective immune defenses versus S. aureus adaptive immune evasion mechanisms may offer breakthroughs in the development of effective vaccines, but at present this goal remains a very high bar. Coupled with the recent advances in human genetics and epigenetics, newer vaccine technologies may enable such a goal. If so, future vaccines that protect against or mitigate the severity of S. aureus infections are likely to emerge at the intersection of precision and personalized medicine. For now, the development of S. aureus vaccines or alternative therapies that reduce mortality and morbidity must continue to be pursued.

Immunometabolites Drive Bacterial Adaptation to the Airway.

Pseudomonas aeruginosa and Staphylococcus aureus are both opportunistic pathogens that are frequently associated with chronic lung infections. While bacterial virulence determinants are critical in initiating infection, the metabolic flexibility of these bacteria promotes their persistence in the airway. Upon infection, these pathogens induce host immunometabolic reprogramming, resulting in an airway milieu replete with immune-signaling metabolites. These metabolites are often toxic to the bacteria and create a steep selection pressure for the emergence of bacterial isolates adapted for long-term survival in the inflamed lung. In this review, we discuss the main differences in the host immunometabolic response to P. aeruginosa and S. aureus, as well as how these pathogens alter their own metabolism to adapt to airway metabolites and cause persistent lung infections.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Pseudomonas aeruginosa Consumption of Airway Metabolites Promotes Lung Infection.

Prevailing dogma indicates that the lung of cystic fibrosis (CF) individuals is infected by multiple pathogens due to the abundant accumulation of mucus, which traps most of inhaled organisms. However, this hypothesis does not explain how specific opportunists, like Pseudomonas aeruginosa, are selected in the CF lung to cause chronic disease. This strongly suggests that other factors than mucus are accrued in the human airway and might predispose to bacterial disease, especially by P. aeruginosa. In this review we discuss the role of macrophage metabolites, like succinate and itaconate, in P. aeruginosa pneumonia. We analyze how dysfunction of the CF transmembrane conductance regulator (CFTR) favors release of these metabolites into the infected airway, and how P. aeruginosa exploits these elements to induce transcriptomic and metabolic changes that increase its capacity to cause intractable disease. We describe the host and pathogen pathways associated with succinate and itaconate catabolism, mechanisms of bacterial adaptation to these determinants, and suggest how both experimental settings and future therapies should consider macrophage metabolites abundance to better study P. aeruginosa pathogenesis.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection.

Klebsiella pneumoniae ST258 is a human pathogen associated with poor outcomes worldwide. We identify a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of these organisms in vivo. Comparison of a wild-type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by CRISPR-Cas9 targeting reveals greater NADH:ubiquinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. The acquisition of atf3 induces changes in the bacterial acetylome, promoting lysine acetylation of multiple proteins involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost leads to greater consumption of glucose in the host airway and increased bacterial burden in the lung, independent of cytokine levels and immune cell recruitment. Acquisition of this acyltransferase enhances fitness of a K. pneumoniae ST258 isolate and may contribute to the success of this clonal complex as a healthcare-associated pathogen.

Staphylococcus aureus induces an itaconate-dominated immunometabolic response that drives biofilm formation.

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate. The itaconate synthetic enzyme, IRG1, is activated by host mitochondrial stress, which is induced by staphylococcal glycolysis. Itaconate inhibits S. aureus glycolysis and selects for strains that re-direct carbon flux to fuel extracellular polysaccharide (EPS) synthesis and biofilm formation. Itaconate-adapted strains, as illustrated by S. aureus isolates from chronic airway infection, exhibit decreased glycolytic activity, high EPS production, and proficient biofilm formation even before itaconate stimulation. S. aureus thus adapts to the itaconate-dominated immunometabolic response by producing biofilms, which are associated with chronic infection of the human airway.

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.

Airway immunometabolites fuel Pseudomonas aeruginosa infection.

Pulmonary infections are associated with a brisk inflammatory reaction to bacterial surface components. Lipopolysaccharides (LPS) trigger macrophage activation and release of mitochondrial metabolites that control the intensity of the immune response. Whereas succinate induces oxidative stress (ROS), HIF1α stabilization, glycolysis and IL-1ß release, itaconate suppresses inflammation by inhibiting succinate oxidation, glycolytic flux and promoting anti-oxidant Nrf2-HO-1 functions. P. aeruginosa is a major pathogen associated with acute and chronic lung infection. Although both secreted toxins, LPS and proteases are key factors to establish acute P. aeruginosa pneumonia, lack of these components in chronic P. aeruginosa isolates suggest these organisms exploit other mechanisms to adapt and persist in the lung. Upon inhalation, P. aeruginosa strains trigger airway macrophage reprograming and bacterial variants obtained from acutely and chronically infected subjects exhibit metabolic adaptation consistent with succinate and itaconate assimilation; namely, high expression of extracellular polysaccharides (EPS), reduced lptD-LPS function, increased glyoxylate shunt (GS) activity and substantial biofilm production. In this review we discuss recent findings illustrating how P. aeruginosa induces and adapts to macrophage metabolites in the human lung, and that catabolism of succinate and itaconate contribute to their formidable abilities to tolerate oxidative stress, phagocytosis and immune clearance.

Consequences of Metabolic Interactions during Staphylococcus aureus Infection.

Staphylococcus aureus is a metabolically flexible pathogen that causes infection in diverse settings. An array of virulence factors, including the secreted toxins, enables S. aureus to colonize different environmental niches and initiate infections by any of several discrete pathways. During these infections, both S. aureus and host cells compete with each other for nutrients and remodel their metabolism for survival. This metabolic interaction/crosstalk determines the outcome of the infection. The reprogramming of metabolic pathways in host immune cells not only generates adenosine triphosphate (ATP) to meet the cellular energy requirements during the infection process but also activates antimicrobial responses for eventual bacterial clearance, including cell death pathways. The selective pressure exerted by host immune cells leads to the emergence of bacterial mutants adapted for chronicity. These host-adapted mutants are often characterized by substantial changes in the expression of their own metabolic genes, or by mutations in genes involved in metabolism and biofilm formation. Host-adapted S. aureus can rewire or benefit from the metabolic activities of the immune cells via several mechanisms to cause persistent infection. In this review, we discuss how S. aureus activates host innate immune signaling, which results in an immune metabolic pressure that shapes S. aureus metabolic adaptation and determines the outcome of the infection.

Participation of the IL-10RB Related Cytokines, IL-22 and IFN-λ in Defense of the Airway Mucosal Barrier.

The airway epithelial barrier is a major barrier protecting against clinically significant infections of the lung. Its integrity is often compromised due to mechanical, chemical, or infectious causes. Opportunistic bacterial pathogens are poised to cause parenchymal infection and become difficult to eradicate due to adaptive metabolic changes, biofilm formation, and the acquisition of antimicrobial resistance and fitness genes. Enhancing mucosal defenses by modulating the cytokines that regulate barrier functions, such as interleukin-22 (IL-22) and interferon-λ (IFN-λ), members of the IL-10 family of cytokines, is an attractive approach to prevent these infections that are associated with high morbidity and mortality. These cytokines both signal through the cognate receptor IL-10RB, have related protein structures and common downstream signaling suggesting shared roles in host respiratory defense. They are typically co-expressed in multiple models of infections, but with differing kinetics. IL-22 has an important role in the producing antimicrobial peptides, upregulating expression of junctional proteins in the airway epithelium and working in concert with other inflammatory cytokines such as IL-17. Conversely, IFN-λ, a potent antiviral in influenza infection with pro-inflammatory properties, appears to decrease junctional integrity allowing for bacterial and immune cell translocation. The effects of these cytokines are pleotropic, with pathogen and tissue specific consequences. Understanding how these cytokines work in the mucosal defenses of the respiratory system may suggest potential targets to prevent invasive infections of the damaged lung.

Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm Formation.

The bacterium Pseudomonas aeruginosa is especially pathogenic, often being associated with intractable pneumonia and high mortality. How P. aeruginosa avoids immune clearance and persists in the inflamed human airway remains poorly understood. In this study, we show that P. aeruginosa can exploit the host immune response to maintain infection. Notably, unlike other opportunistic bacteria, we found that P. aeruginosa alters its metabolic and immunostimulatory properties in response to itaconate, an abundant host-derived immunometabolite in the infected lung. Itaconate induces bacterial membrane stress, resulting in downregulation of lipopolysaccharides (LPS) and upregulation of extracellular polysaccharides (EPS). These itaconate-adapted P. aeruginosa accumulate lptD mutations, which favor itaconate assimilation and biofilm formation. EPS, in turn, induces itaconate production by myeloid cells, both in the airway and systemically, skewing the host immune response to one permissive of chronic infection. Thus, the metabolic versatility of P. aeruginosa needs to be taken into account when designing therapies.

Pulmonary Pathogens Adapt to Immune Signaling Metabolites in the Airway.

A limited number of pulmonary pathogens are able to evade normal mucosal defenses to establish acute infection and then adapt to cause chronic pneumonias. Pathogens, such as Pseudomonas aeruginosa or Staphylococcus aureus, are typically associated with infection in patients with underlying pulmonary disease or damage, such as cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). To establish infection, bacteria express a well-defined set of so-called virulence factors that facilitate colonization and activate an immune response, gene products that have been identified in murine models. Less well-understood are the adaptive changes that occur over time in vivo, enabling the organisms to evade innate and adaptive immune clearance mechanisms. These colonizers proliferate, generating a population sufficient to provide selection for mutants, such as small colony variants and mucoid variants, that are optimized for long term infection. Such host-adapted strains have evolved in response to selective pressure such as antibiotics and the recruitment of phagocytes at sites of infection and their release of signaling metabolites (e.g., succinate). These metabolites can potentially function as substrates for bacterial growth and but also generate oxidant stress. Whole genome sequencing and quantified expression of selected genes have helped to explain how P. aeruginosa and S. aureus adapt to the presence of these metabolites over the course of in vivo infection. The serial isolation of clonally related strains from patients with cystic fibrosis has provided the opportunity to identify bacterial metabolic pathways that are altered under this immune pressure, such as the anti-oxidant glyoxylate and pentose phosphate pathways, routes contributing to the generation of biofilms. These metabolic pathways and biofilm itself enable the organisms to dissipate oxidant stress, while providing protection from phagocytosis. Stimulation of host immune signaling metabolites by these pathogens drives bacterial adaptation and promotes their persistence in the airways. The inherent metabolic flexibility of P. aeruginosa and S. aureus is a major factor in their success as pulmonary pathogens.