An oral commensal attenuates Pseudomonas aeruginosa-induced airway inflammation and modulates nitrite flux in respiratory epithelium.

IMPORTANCE: Respiratory infections are a leading cause of morbidity and mortality in people with cystic fibrosis (CF). These infections are polymicrobial in nature with overt pathogens and other colonizing microbes present. Microbiome data have indicated that the presence of oral commensal bacteria in the lungs is correlated with improved outcomes. We hypothesize that one oral commensal, Streptococcus parasanguinis, inhibits CF pathogens and modulates the host immune response. One major CF pathogen is Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterium with intrinsic drug resistance and an arsenal of virulence factors. We have previously shown that S. parasanguinis inhibits P. aeruginosa in vitro in a nitrite-dependent manner through the production of reactive nitrogen intermediates. In this study, we demonstrate that while this mechanism is evident in a cell culture model of the CF airway, an alternative mechanism by which S. parasanguinis may improve outcomes for people with CF is through immunomodulation.

Commensal colonization reduces Pseudomonas aeruginosa burden and subsequent airway damage.

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Interestingly, oral streptococcal colonization has been associated with stable CF lung function. The most abundant streptococcal species found in stable patients, Streptococcus salivarius, has been shown to downregulate pro-inflammatory cytokines in multiple colonization models. However, no studies have demonstrated how S. salivarius potentially improves lung function. Our lab previously demonstrated that the P. aeruginosa exopolysaccharide Psl promotes S. salivarius biofilm formation in vitro, suggesting a possible mechanism by which S. salivarius is incorporated into the CF airway microbial community. In this study, we demonstrate that co-infection of rats leads to enhanced S. salivarius colonization and reduced P. aeruginosa colonization. Histological scores for tissue inflammation and damage are lower in dual-infected rats compared to P. aeruginosa infected rats. Additionally, pro-inflammatory cytokines IL-1ß, IL-6, CXCL2, and TNF-α are downregulated during co-infection compared to P. aeruginosa single-infection. Lastly, RNA sequencing of cultures grown in synthetic CF sputum revealed that P. aeruginosa glucose metabolism genes are downregulated in the presence of S. salivarius, suggesting a potential alteration in P. aeruginosa fitness during co-culture. Overall, our data support a model in which S. salivarius colonization is promoted during co-infection with P. aeruginosa, whereas P. aeruginosa airway bacterial burden is reduced, leading to an attenuated host inflammatory response.

Drosophila melanogaster as a model to study polymicrobial synergy and dysbiosis.

The fruit fly Drosophila melanogaster has emerged as a valuable model for investigating human biology, including the role of the microbiome in health and disease. Historically, studies involving the infection of D. melanogaster with single microbial species have yielded critical insights into bacterial colonization and host innate immunity. However, recent evidence has underscored that multiple microbial species can interact in complex ways through physical connections, metabolic cross-feeding, or signaling exchanges, with significant implications for healthy homeostasis and the initiation, progression, and outcomes of disease. As a result, researchers have shifted their focus toward developing more robust and representative in vivo models of co-infection to probe the intricacies of polymicrobial synergy and dysbiosis. This review provides a comprehensive overview of the pioneering work and recent advances in the field, highlighting the utility of Drosophila as an alternative model for studying the multifaceted microbial interactions that occur within the oral cavity and other body sites. We will discuss the factors and mechanisms that drive microbial community dynamics, as well as their impacts on host physiology and immune responses. Furthermore, this review will delve into the emerging evidence that connects oral microbes to systemic conditions in both health and disease. As our understanding of the microbiome continues to evolve, Drosophila offers a powerful and tractable model for unraveling the complex interplay between host and microbes including oral microbes, which has far-reaching implications for human health and the development of targeted therapeutic interventions.

Oral Commensal Streptococci: Gatekeepers of the Oral Cavity.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

A Commensal Streptococcus Dysregulates the Pseudomonas aeruginosa Nitrosative Stress Response.

Chronic infections in the cystic fibrosis (CF) airway are composed of both pathogenic and commensal bacteria. However, chronic Pseudomonas aeruginosa infections are the leading cause of lung deterioration in individuals with CF. Interestingly, oral commensals can translocate to the CF lung and their presence is associated with improved lung function, presumably due to their ability to antagonize P. aeruginosa. We have previously shown that one commensal, Streptococcus parasanguinis, produces hydrogen peroxide that reacts with nitrite to generate reactive nitrogen intermediates (RNI) which inhibit P. aeruginosa growth. In this study, we sought to understand the global impact of commensal-mediated RNI on the P. aeruginosa transcriptome. RNA sequencing analysis revealed that S. parasanguinis and nitrite-mediated RNI dysregulated expression of denitrification genes in a CF isolate of P. aeruginosa compared to when this isolate was only exposed to S. parasanguinis. Further, loss of a nitric oxide reductase subunit (norB) rendered an acute P. aeruginosa isolate more susceptible to S. parasanguinis-mediated RNI. Additionally, S. parasanguinis-mediated RNI inactivated P. aeruginosa aconitase activity. Lastly, we report that P. aeruginosa isolates recovered from CF individuals are uniquely hypersensitive to S. parasanguinis-mediated RNI compared to acute infection or environmental P. aeruginosa isolates. These findings illustrate that S. parasanguinis hinders the ability of P. aeruginosa to respond to RNI, which potentially prevents P. aeruginosa CF isolates from resisting commensal and host-induced RNI in the CF airway.

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development.

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.

Nitrite Triggers Reprogramming of the Oral Polymicrobial Metabolome by a Commensal Streptococcus.

Commensal streptococci regulate health and homeostasis within oral polymicrobial communities. Remarkably, high salivary nitrite concentrations have also been associated with improved health in the oral cavity. We previously demonstrated that nitrite assists hydrogen peroxide-producing oral commensal streptococci in regulating homeostasis via the generation of reactive nitrogen species (RNS), which have antimicrobial activity on oral pathogens. However, it is unknown how nitrite and commensal streptococci work in concert to influence the metabolome of oral polymicrobial communities. In this study, we report that nitrite aids commensal streptococci in the inhibition of multi-kingdom pathogens that reside in distinct oral niches, which supports commensal dominance. More importantly, we show that commensal streptococci utilize nitrite to drive the metabolic signature of multispecies biofilms in a manner that supports commensal metabolism and resistance to RNS, and restricts metabolic processes that are required for pathogen virulence. Taken together, our study provides insight into how commensal streptococci use nitrite to trigger shifts in the oral polymicrobial metabolome to support health and homeostasis.

Structure-Function Characterization of Streptococcus intermedius Surface Antigen Pas.

Streptococcus intermedius, an oral commensal bacterium, is found at various sites, including subgingival dental plaque, purulent infections, and cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization, and infection. Among the 19 genomically annotated cell wall-attached surface proteins on S. intermedius, Pas is an adhesin that belongs to the antigen I/II (AgI/II) family. Here, we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of VPas and C123Pas show high similarity with AgI/II of Streptococcus mutans. VPas hosts a conserved metal binding site, and likewise, the C123Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine-rich domains (SRCRs), and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa, the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas-deficient mutant (Δpas) displayed significant reduction in dual-biofilm formation with C. albicans. In similar studies with P. aeruginosa, Pas did not mediate the biofilm formation with either the acute isolate (PAO1) or the chronic isolate (FRD1). However, the sortase A-deficient mutant (ΔsrtA) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. IMPORTANCE Streptococcus intermedius, an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and cystic fibrosis lungs. In this study, we have (i) determined the crystal structure of the V and C regions of Pas; (ii) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the bloodstream from the oral cavity; (iii) characterized Pas's high-affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (iv) most importantly, shown that these surface proteins on the oral commensal S. intermedius enhance biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa.

Disruption of Streptococcus mutans and Candida albicans synergy by a commensal streptococcus.

Polymicrobial interactions in dental plaque play a significant role in dysbiosis and homeostasis in the oral cavity. In early childhood caries, Streptococcus mutans and Candida albicans are often co-isolated from carious lesions and associated with increased disease severity. Studies have demonstrated that metabolic and glucan-dependent synergism between C. albicans and S. mutans contribute to enhanced pathogenesis. However, it is unclear how oral commensals influence pathogen synergy. Streptococcus parasanguinis, a hydrogen peroxide (H2O2) producing oral commensal, has antimicrobial activity against S. mutans. In this study, we utilized a three species biofilm model to understand the impact of S. parasanguinis on S. mutans and C. albicans synergy. We report that S. parasanguinis disrupts S. mutans and C. albicans biofilm synergy in a contact and H2O2-independent manner. Further, metabolomics analysis revealed a S. parasanguinis-driven alteration in sugar metabolism that restricts biofilm development by S. mutans. Moreover, S. parasanguinis inhibits S. mutans glucosyltransferase (GtfB) activity, which is important for glucan matrix development and GtfB-mediated binding to C. albicans mannan. Taken together, our study describes a new antimicrobial role for S. parasanguinis and highlights how this abundant oral commensal may be utilized to attenuate pathogen synergism.

Effects of Pseudomonas aeruginosa on Microglial-Derived Extracellular Vesicle Biogenesis and Composition.

The packaging of molecular constituents inside extracellular vesicles (EVs) allows them to participate in intercellular communication and the transfer of biological molecules, however the role of EVs during bacterial infection is poorly understood. The goal of this study was to examine the effects of Pseudomonas aeruginosa (P. aeruginosa) infection on the biogenesis and composition of EVs derived from the mouse microglia cell line, BV-2. BV-2 cells were cultured in exosome-free media and infected with 0, 1.3 × 104, or 2.6 × 104 colony forming units per milliliter P. aeruginosa for 72 h. The results indicated that compared with the control group, BV-2 cell viability significantly decreased after P. aeruginosa infection and BV-2-derived EVs concentration decreased significantly in the P. aeruginosa-infected group. P. aeruginosa infection significantly decreased chemokine ligand 4 messenger RNA in BV-2-derived infected EVs, compared with the control group (p ≤ 0.05). This study also revealed that heat shock protein 70 (p ≤ 0.05) and heat shock protein 90ß (p ≤ 0.001) levels of expression within EVs increased after P. aeruginosa infection. EV treatment with EVs derived from P. aeruginosa infection reduced cell viability of BV-2 cells. P. aeruginosa infection alters the expression of specific proteins and mRNA in EVs. Our study suggests that P. aeruginosa infection modulates EV biogenesis and composition, which may influence bacterial pathogenesis and infection.

A commensal streptococcus hijacks a Pseudomonas aeruginosa exopolysaccharide to promote biofilm formation.

Pseudomonas aeruginosa causes devastating chronic pulmonary infections in cystic fibrosis (CF) patients. Although the CF airway is inhabited by diverse species of microorganisms interlaced within a biofilm, many studies focus on the sole contribution of P. aeruginosa pathogenesis in CF morbidity. More recently, oral commensal streptococci have been identified as cohabitants of the CF lung, but few studies have explored the role these bacteria play within the CF biofilm. We examined the interaction between P. aeruginosa and oral commensal streptococci within a dual species biofilm. Here we report that the CF P. aeruginosa isolate, FRD1, enhances biofilm formation and colonization of Drosophila melanogaster by the oral commensal Streptococcus parasanguinis. Moreover, production of the P. aeruginosa exopolysaccharide, alginate, is required for the promotion of S. parasanguinis biofilm formation and colonization. However, P. aeruginosa is not promoted in the dual species biofilm. Furthermore, we show that the streptococcal adhesin, BapA1, mediates alginate-dependent enhancement of the S. parasanguinis biofilm in vitro, and BapA1 along with another adhesin, Fap1, are required for the in vivo colonization of S. parasanguinis in the presence of FRD1. Taken together, our study highlights a new association between streptococcal adhesins and P. aeruginosa alginate, and reveals a mechanism by which S. parasanguinis potentially colonizes the CF lung and interferes with the pathogenesis of P. aeruginosa.

Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation.

Fine-tuned production of hydrogen peroxide promotes biofilm formation of Streptococcus parasanguinis by a pathogenic cohabitant Aggregatibacter actinomycetemcomitans.

Balanced bacterial biofilm communities help to maintain host health. Disturbance of such balance can lead to bacterial dysbiosis and pathogenesis. However, complex and dynamic bacterial interactions within the biofilm communities are poorly understood. In this study, we used a dual-species biofilm consisting of the periodontal pathogen Aggregatibacter actinomycetemcomitans, and a commensal Streptococcus parasanguinis to investigate bacterial interactions since the two organisms have been found to coexist during the development of localized aggressive periodontal disease. We report that A. actinomycetemcomitans promoted biofilm formation of S. parasanguinis in vitro and in vivo. Protein profiling of S. parasanguinis co-cultured with A. actinomycetemcomitans revealed a significant decrease in the protein level of pyruvate oxidase(PoxL), an enzyme required for the generation of hydrogen peroxide (H2 O2 ). Consistently, the H2 O2 concentration was concurrently decreased. However, the complete removal of H2 O2 impaired the biofilm formation. H2 O2 at a low concentration range regulated by A. actinomycetemcomitans enhanced the biofilm formation. These results demonstrate that A. actinomycetemcomitans promotes the S. parasanguinis biofilm formation through modulating the production of H2 O2 by fine-tuning the expression of poxL, indicating that H2 O2 functions as a signaling molecule. Taken together, this report revealed a previously unknown bacteria-bacteria interaction mechanism.

Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.

Evaluation of ciprofloxacin and metronidazole encapsulated biomimetic nanomatrix gel on Enterococcus faecalis and Treponema denticola.

BACKGROUND: A triple antibiotic mixture (ciprofloxacin; CF, metronidazole; MN, and minocycline; MC) has been used for dental root canal medicaments in pulp regeneration therapy. However, tooth discolorations, cervical root fractures, and inadequate pulp-dentin formation have been reported due to the triple antibiotic regimen. Therefore, an antibiotic encapsulated biomimetic nanomatrix gel was developed to minimize the clinical limitations and maximize a natural healing process in root canal infections. In this study, minimal bacterial concentrations (MBC) of the selected antibiotics (CF and MN) were tested in 14 representative endodontic bacterial species. Then MBC of each CF and MN were separately encapsulated within the injectable self-assembled biomimetic nanomatrix gel to evaluate antibacterial level on Enterococcus faecalis and Treponema denticola. RESULTS: Antibiotic concentrations lower than 0.2 µg/mL of CF and MN demonstrated antibacterial activity on the 14 endodontic species. Furthermore, 6 different concentrations of CF and MN separately encapsulated with the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial activity on Enterococcus faecalis and Treponema denticola at the lowest tested concentration of 0.0625 µg/mL. CONCLUSIONS: These results suggest that each CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial effects, which could be effective for the root canal disinfection while eliminating MC. In the long term, the antibiotic encapsulated injectable self-assembled biomimetic nanomatrix gel can provide a multifunctional antibiotic delivery method with potential root regeneration. Further studies are currently underway to evaluate the effects of combined CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel on clinical samples.

Oral streptococci and nitrite-mediated interference of Pseudomonas aeruginosa.

The oral cavity harbors a diverse community of microbes that are physiologically unique. Oral microbes that exist in this polymicrobial environment can be pathogenic or beneficial to the host. Numerous oral microbes contribute to the formation of dental caries and periodontitis; however, there is little understanding of the role these microbes play in systemic infections. There is mounting evidence that suggests that oral commensal streptococci are cocolonized with Pseudomonas aeruginosa during cystic fibrosis pulmonary infections and that the presence of these oral streptococci contributes to improved lung function. The goal of this study was to examine the underlying mechanism by which Streptococcus parasanguinis antagonizes pathogenic P. aeruginosa. In this study, we discovered that oral commensal streptococci, including Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus gordonii, inhibit the growth of P. aeruginosa and that this inhibition is mediated by the presence of nitrite and the production of hydrogen peroxide (H2O2) by oral streptococci. The requirement of both H2O2 and nitrite for the inhibition of P. aeruginosa is due to the generation of reactive nitrogenous intermediates (RNI), including peroxynitrite. Transposon mutagenesis showed that a P. aeruginosa mutant defective in a putative ABC transporter permease is resistant to both streptococcus/nitrite- and peroxynitrite-mediated killing. Furthermore, S. parasanguinis protects Drosophila melanogaster from killing by P. aeruginosa in a nitrite-dependent manner. Our findings suggest that the combination of nitrite and H2O2 may represent a unique anti-infection strategy by oral streptococci during polymicrobial infections.

Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits.

Malate synthase expression is deregulated in the Pseudomonas aeruginosa cystic fibrosis isolate FRD1.

Pseudomonas aeruginosa causes chronic pulmonary infections, which can persist for decades, in patients with cystic fibrosis (CF). Current evidence suggests that the glyoxylate pathway is an important metabolic pathway for P. aeruginosa growing within the CF lung. In this study, we identified glcB, which encodes for the second key enzyme of the glyoxylate pathway, malate synthase, as a requirement for virulence of P. aeruginosa on alfalfa seedlings. While expression of glcB in PAO1, an acute isolate of P. aeruginosa, responds to some carbon sources that use the glyoxylate pathway, expression of glcB in FRD1, a CF isolate, is constitutively upregulated. Malate synthase activity is moderately affected by glcB expression and is nearly constitutive in both backgrounds, with slightly higher activity in FRD1 than in PAO1. In addition, RpoN negatively regulates glcB in PAO1 but not in FRD1. In summary, the genes encoding for the glyoxylate-specific enzymes appear to be coordinately regulated, even though they are not located within the same operon on the P. aeruginosa genome. Furthermore, both genes encoding for the glyoxylate enzymes can become deregulated during adaptation of the bacterium to the CF lung.

Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in patients with cystic fibrosis (CF). The metabolic pathways utilized by P. aeruginosa during these infections, which can persist for decades, are poorly understood. Several lines of evidence suggest that the glyoxylate pathway, which utilizes acetate or fatty acids to replenish intermediates of the tricarboxylic acid cycle, is an important metabolic pathway for P. aeruginosa adapted to the CF lung. Isocitrate lyase (ICL) is one of two major enzymes of the glyoxylate pathway. In a previous study, we determined that P. aeruginosa is dependent upon aceA, which encodes ICL, to cause disease on alfalfa seedlings and in rat lungs. Expression of aceA in PAO1, a P. aeruginosa isolate associated with acute infection, is regulated by carbon sources that utilize the glyoxyate pathway. In contrast, expression of aceA in FRD1, a CF isolate, is constitutively upregulated. Moreover, this deregulation of aceA occurs in other P. aeruginosa isolates associated with chronic infection, suggesting that high ICL activity facilitates adaptation of P. aeruginosa to the CF lung. Complementation of FRD1 with a PAO1 clone bank identified that rpoN negatively regulates aceA. However, the deregulation of aceA in FRD1 was not due to a knockout mutation of rpoN. Regulation of the glyoxylate pathway by RpoN is likely to be indirect, and represents a unique regulatory role for this sigma factor in bacterial metabolism.