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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.

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.

Summary Staphylococcus aureus is the most common cause of skin and soft tissue infections, yet the bacterial genetic changes associated with adaptation to human skin are not well characterized. S. aureus strains isolated from patients with chronic skin colonization and intermittent infection were used to determine the staphylococcal genotypes or phenotypes associated with adaptation to human skin. We demonstrate that polymorphisms in metabolic genes, particularly those involved in the tricarboxylic acid cycle, the fumarate-succinate axis, and the generation of terminal electron transporters, are unexpectedly common. These skin-adapted strains activated glycolysis and hypoxia-inducible factor-1α, interleukin (IL)-1β, and IL-18 release from keratinocytes and promoted dermatopathology equivalent to a methicillin-resistant Staphylococcus aureus USA300 control in a murine model of infection. However, in contrast to USA300, a skin-adapted isolate failed to generate protection from a secondary infectious challenge. Within the context of human skin, there appears to be selection for S. aureus metabolic adaptive changes that promote glycolysis and maintain pathogenicity., Graphical Abstract, Highlights • Staphylococcus aureus is a metabolically adaptive organism • Skin-adapted isolates harbor mutations in fumC and other metabolic genes • Novel metabolic gene variants were identified in skin-adapted strains, Bacteriology; Microbial Genetics; Microbiome

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., 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.

SUMMARY 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., Graphical Abstract, In brief Klebsiella pneumoniae ST258 is a successful human pathogen. Here, Ahn et al. identify an acyltransferase of the superfamily 3 (atf3) concentrated within one of the ST258 clades. Acquisition of this gene leads to enhanced bioenergetic properties, giving the pathogen a competitive advantage in vivo.

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-1β 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.

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.

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.

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 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., The authors show that the pathogen Staphylococcus aureus induces a distinct airway immunometabolic response, dominated by release of itaconate. This metabolite, in turn, potentiates extracellular polysaccharide synthesis and biofilm formation in S. aureus, which may facilitate chronic infection.

Many different species of gram-negative bacteria are associated with infection in the lung, causing exacerbations of chronic obstructive pulmonary disease, cystic fibrosis (CF), and ventilator-associated pneumonias. These airway pathogens must adapt to common host clearance mechanisms that include killing by antimicrobial peptides, antibiotics, oxidative stress, and phagocytosis by leukocytes. Bacterial adaptation to the host is often evident phenotypically, with increased extracellular polysaccharide production characteristic of some biofilm-associated organisms. Given the relatively limited repertoire of bacterial strategies to elude airway defenses, it seems likely that organisms sharing the same ecological niche might also share common strategies to persistently infect the lung. In this review, we will highlight some of the major factors responsible for the adaptation of Pseudomonas aeruginosa to the lung, addressing how growth in biofilms enables persistent infection, relevant to, but not limited to, the pathogenesis of infection in CF. In contrast, we will discuss how carbapenem-resistant Klebsiella pneumoniae evade immune clearance, an organism often associated with ventilator-associated pneumonia and health-care-acquired pneumonias, but not a typical pathogen in CF.

Human skin is commonly colonized and infected by Staphylococcus aureus. Exactly how these organisms are sensed by keratinocytes has not been clearly delineated. Using a combination of metabolic and transcriptomic methodologies, we found that S. aureus infection is sensed as a metabolic stress by the hypoxic keratinocytes. This induces HIF1a signaling, which promotes IL-1 β production and stimulates aerobic glycolysis to meet the metabolic requirements of infection. We demonstrate that staphylococci capable of glycolysis, including WT and agr mutants, readily induce HIF1a responses. In contrast, Δpyk glycolytic mutants fail to compete with keratinocytes for their metabolic needs. Suppression of glycolysis using 2-DG blocked keratinocyte production of IL-1 β in vitro and significantly exacerbated the S. aureus cutaneous infection in a murine model. Our data suggest that S. aureus impose a metabolic stress on keratinocytes that initiates signaling necessary to promote both glycolysis and the proinflammatory response to infection.

Glycolytic Staphylococcus aureus generate lactate that targets specific histone deacetylases to stimulate the production of IL-10 and enable biofilm formation.

Staphylococcus aureus small colony variants (SCVs) are frequently associated with chronic infection, yet they lack expression of many virulence determinants associated with the pathogenicity of wild-type strains. We found that both wild-type S. aureus and a ΔhemB SCV prototype potently activate glycolysis in host cells. Glycolysis and the generation of mitochondrial reactive oxygen species were sufficient to induce necroptosis, a caspase-independent mechanism of host cell death that failed to eradicate S. aureus and instead promoted ΔhemB SCV pathogenicity. To support ongoing glycolytic activity, the ΔhemB SCV induced over a 100-fold increase in the expression of fumC, which encodes an enzyme that catalyses the degradatin of fumarate, an inhibitor of glycolysis. Consistent with fumC-dependent depletion of local fumarate, the ΔhemB SCV failed to elicit trained immunity and protection from a secondary infectious challenge in the skin. The reliance of the S. aureus SCV population on glycolysis accounts for much of its role in the pathogenesis of S. aureus skin infection.

Klebsiella pneumoniae ST258 are human pathogens associated with poor outcomes in patients worldwide. We identified a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of this group of organisms in vivo. Comparison of a wild type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by Crispr-Cas9 targeting, revealed increased NADH:quinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. Acquisition of atf3 induced changes in the bacterial acetylome, promoting lysine acetylation of multiple gene products involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost led 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 a promiscuous acyltransferase enhances K. pneumoniae ST258 fitness and promotes its emergence as a major health care associated pathogen.

Carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258) can cause chronic infections in lungs and airways, with repeated episodes of bacteremia. In this report we addressed whether the recruitment of myeloid cells producing the anti-inflammatory cytokine interleukin-10 (IL-10) modulates the clearance of CKRP-ST258 in the lungs and establishes bacterial persistence. Our data demonstrate that during pneumonia caused by a clinical isolate of CRKP-ST258 (KP35) there is an early recruitment of monocyte-myeloid-derived suppressor cells (M-MDSCs) and neutrophils that actively produce IL-10. However, M-MDSCs were the cells that sustained the production of IL-10 over the time of infection evaluated. Using mice unable to produce IL-10 (IL-10(−/−)), we observed that the production of this cytokine during the infection caused by KP35 is important to control bacterial burden, to prevent lung damage, to modulate cytokine production, and to improve host survival. Importantly, intranasal transfer of bone marrow-derived M-MDSCs from mice able to produce IL-10 at 1 day prior to infection improved the ability of IL-10(−/−) mice to clear KP35 in the lungs, decreasing their mortality. Altogether, our data demonstrate that IL-10 produced by M-MDSCs is required for bacterial clearance, reduction of lung tissue damage, and host survival during KP35 pneumonia.

Common pulmonary pathogens, such as Streptococcus pneumoniae and Staphylococcus aureus, as well as the host-adapted pathogens responsible for health care-associated pneumonias, such as the carbapenem-resistant Klebsiella pneumoniae and Serratia marcecsens, are able to activate cell death through the RIPK1/RIPK3/MLKL cascade that causes necroptosis. Necroptosis can influence the pathogenesis of pneumonia through several mechanisms. Activation of this pathway can result in the loss of specific types of immune cells, especially macrophages, and, in so doing, contribute to host pathology through the loss of their critical immunoregulatory functions. However, in other settings of infection, necroptosis promotes pathogen removal and the eradication of infected cells to control excessive proinflammatory signaling. Bacterial production of pore-forming toxins provides a common mechanism to activate necroptosis by diverse bacterial species, with variable consequences depending upon the specific pathogen. Included in this brief review are data demonstrating the ability of the carbapenem-resistant ST258 K. pneumoniae to activate necroptosis in the setting of pneumonia, which is counterbalanced by their suppression of CYLD expression. Exactly how necroptosis and other mechanisms of cell death are coregulated in the response to specific pulmonary pathogens remains a topic of active investigation, and it may provide potential therapeutic targets in the future.

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.

IFN-λ and IL-22, cytokines that share the coreceptor IL-10RB, are both induced over the course of Klebsiella pneumoniae ST258 (KP35) pneumonia. IL-22 is known to protect mucosal barriers, whereas the effects of IFN-λ on the mucosa are not established. We postulated that IFN-λ plays a role in regulating the airway epithelial barrier to facilitate cellular trafficking to the site of infection. In response to IFN-λ, the transmigration of neutrophils across a polarized monolayer of airway epithelial cells was increased, consistent with diminished epithelial integrity. KP35 infection increased epithelial permeability, and pretreatment with IFN-λ amplified this effect and facilitated bacterial transmigration. These effects of IFN-λ were confirmed in vivo, in that mice lacking the receptor for IFN-λ (Ifnlr1(−/−)) were protected from bacteremia in a murine model of KP35 pneumonia. Conversely, the integrity of the epithelial barrier was protected by IL-22, with subsequent impairment of neutrophil and bacterial transmigration in vitro. Maximal expression of IL-22 in vivo was observed later in the course of infection than IFN-λ production, with high levels of IL-22 produced by recruited immune cells at 48 hours, consistent with a role in epithelial barrier recovery. The divergent and opposing expression of these two related cytokines suggests a regulated interaction in the host response to KP35 infection. A major physiological effect of IFN-λ signaling is a decrease in epithelial barrier integrity, which facilitates immune cell recruitment but also enables K. pneumoniae invasion.

Staphylococcus aureus is the most common cause of skin and soft tissue infection, yet the genetic changes in these organisms associated with adapation to human skin are not well characterized. Using S. aureus strains isolated from patients with atopic dermatitis (AD) we demonstrate that polymorphisms in metabolic genes, particularly those involved in the tricarboxylic acid (TCA) cycle, the fumarate/succinate axis, and the generation of terminal electron transporters are unexpectedly common. These AD strains activated glycolysis, HIF-α, and IL-1β and IL-18 release from keratinocytes and promoted dermatopathology equivalent to an MRSA USA300 control in a murine model of infection. However, in contrast to USA300, a typical AD isolate failed to generate protection from a secondary infectious challenge. Within the context of human skin, there is selection for S. aureus metabolic adaptive changes that both promote glycolysis and maintain pathogenicity.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group’s specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.