Pseudomonas Persists by Feeding off Itaconate

Itaconate is an immunometabolite with anti-inflammatory and anti-microbial properties. Riquelme et al., 2020Riquelme S.A. Liimatta K. Lung T.W.F. Fields B. Ahn D. Chen D. Lozano C. Sáenz Y. Uhlemann A.C. Kahl B.C. et al.Pseudomonas aeruginosa utilizes host-derived itaconate to redirect its metabolism to promote biofilm formation.Cell Metab. 2020; 31 (this issue): 1091-1106Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar demonstrate that pathogenic Pseudomonas aeruginosa drives itaconate production by macrophages, which it then uses as a carbon source for biofilm formation, allowing it to persist during infection and suppress inflammation. Itaconate is an immunometabolite with anti-inflammatory and anti-microbial properties. Riquelme et al., 2020Riquelme S.A. Liimatta K. Lung T.W.F. Fields B. Ahn D. Chen D. Lozano C. Sáenz Y. Uhlemann A.C. Kahl B.C. et al.Pseudomonas aeruginosa utilizes host-derived itaconate to redirect its metabolism to promote biofilm formation.Cell Metab. 2020; 31 (this issue): 1091-1106Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar demonstrate that pathogenic Pseudomonas aeruginosa drives itaconate production by macrophages, which it then uses as a carbon source for biofilm formation, allowing it to persist during infection and suppress inflammation. Itaconate is a metabolite that is upregulated during pro-inflammatory activation of macrophages (Hooftman and O'Neill, 2019Hooftman A. O'Neill L.A.J. The Immunomodulatory potential of the metabolite itaconate.Trends Immunol. 2019; 40: 687-698Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The gram-negative bacterial component lipopolysaccharide (LPS) is a potent inducer. The mechanism involves upregulation of Irg1, also known as Acod1, which encodes the enzyme aconitate decarboxylase (CAD) (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (479) Google Scholar), which converts the Krebs cycle intermediate cis-aconitate into itaconate. Itaconate has been reported to have multiple targets in macrophages. It downregulates pro-inflammatory factors, such as IL-1β (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (479) Google Scholar), IL-6 (Bambouskova et al., 2018Bambouskova M. Gorvel L. Lampropoulou V. Sergushichev A. Loginicheva E. Johnson K. Korenfeld D. Mathyer M.E. Kim H. Huang L.H. et al.Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.Nature. 2018; 556: 501-504Crossref PubMed Scopus (216) Google Scholar), and reactive oxygen species (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (479) Google Scholar). A derivative of itaconate (4-octylitaconate) has been shown to protect mice from LPS-induced lethality (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (479) Google Scholar), and deletion of Irg1/Acod1 results in increased inflammation during Mycobacterium tuberculosis infection (Nair et al., 2018Nair S. Huynh J.P. Lampropoulou V. Loginicheva E. Esaulova E. Gounder A.P. Boon A.C.M. Schwarzkopf E.A. Bradstreet T.R. Edelson B.T. et al.Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection.J. Exp. Med. 2018; 215: 1035-1045Crossref PubMed Scopus (76) Google Scholar). Itaconate would therefore appear to be anti-inflammatory. Multiple biological mechanisms have been described, including inhibition of succinate dehydrogenase (which will attenuate the pro-inflammatory effects of succinate that require oxidation) (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Cordes et al., 2016Cordes T. Wallace M. Michelucci A. Divakaruni A.S. Sapcariu S.C. Sousa C. Koseki H. Cabrales P. Murphy A.N. Hiller K. Metallo C.M. Immunoresponsive gene 1 and itaconate inhibit succinate dehydrogenase to modulate intracellular succinate levels.J. Biol. Chem. 2016; 291: 14274-14284Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, Németh et al., 2016Németh B. Doczi J. Csete D. Kacso G. Ravasz D. Adams D. Kiss G. Nagy A.M. Horvath G. Tretter L. et al.Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.FASEB J. 2016; 30: 286-300Crossref PubMed Scopus (68) Google Scholar). In addition, itaconate has been shown to modify cysteine residues on target proteins, including KEAP1, leading to activation of the antioxidant transcription factor NRF2 (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (479) Google Scholar). Itaconate has also been shown to activate ATF3, which limits the production of cytokines such as IL-6 (Bambouskova et al., 2018Bambouskova M. Gorvel L. Lampropoulou V. Sergushichev A. Loginicheva E. Johnson K. Korenfeld D. Mathyer M.E. Kim H. Huang L.H. et al.Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.Nature. 2018; 556: 501-504Crossref PubMed Scopus (216) Google Scholar). There is also separate literature on itaconate as an antimicrobial agent, which predates the findings on itaconate as an immunomodulator (Luan and Medzhitov, 2016Luan H.H. Medzhitov R. Food fight: role of itaconate and other metabolites in antimicrobial defense.Cell Metab. 2016; 24: 379-387Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Itaconate has been shown to target Staphylococcus aureus and Legionella pneumophila, inhibiting the enzyme isocitrate lyase, thereby limiting bacterial growth. However, in other bacterial species, notably Pseudomonas aeruginosa, Yersinia pestis, and Mycobacterium tuberculosis, upregulation of itaconate-metabolizing enzymes leads to resistance, with itaconate being converted into pyruvate and acetyl-CoA, acting as a fuel source (Sasikaran et al., 2014Sasikaran J. Ziemski M. Zadora P.K. Fleig A. Berg I.A. Bacterial itaconate degradation promotes pathogenicity.Nat. Chem. Biol. 2014; 10: 371-377Crossref PubMed Scopus (91) Google Scholar). Riquelme and colleagues now add to this literature with a report demonstrating that P. aeruginosa can use itaconate to promote biofilm formation, an event that is especially damaging in the context of diseases of the airways; notably, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (Riquelme et al., 2020Riquelme S.A. Liimatta K. Lung T.W.F. Fields B. Ahn D. Chen D. Lozano C. Sáenz Y. Uhlemann A.C. Kahl B.C. et al.Pseudomonas aeruginosa utilizes host-derived itaconate to redirect its metabolism to promote biofilm formation.Cell Metab. 2020; 31 (this issue): 1091-1106Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The study builds on previous work from this group that demonstrated that P. aeruginosa could use succinate from the host as a fuel source, as well as boosting itaconate to limit the inflammatory process, and thereby promote bacterial survival (Riquelme et al., 2019Riquelme S.A. Lozano C. Moustafa A.M. Liimatta K. Tomlinson K.L. Britto C. Khanal S. Gill S.K. Narechania A. Azcona-Gutiérrez J.M. et al.CFTR-PTEN-dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection.Sci. Transl. Med. 2019; 11: eaav4634Crossref PubMed Scopus (22) Google Scholar). The study began with an analysis of clinical isolates of P. aeruginosa prepared from sputum from individuals with CF, and also from infected wounds. Genes involved in alginate synthesis (which is a critical component of biofilm, a highly resistant layer of bacteria that can coat surfaces, including in tissues) and in the production of protective extracellular polysaccharides (EPSs) were upregulated in these isolates. There was a downregulation of genes required for LPS synthesis. The clinical isolates were then shown to induce itaconate production in a murine model of pneumonia. Itaconate was shown to promote the growth of these isolates, increasing their biomass. They had a marked preference for itaconate over succinate as a nutrient. Itaconate also suppressed LPS synthesis but promoted EPS and alginate synthesis (the genes for which are co-transcribed from the EPS alginate operon) in the laboratory strain PAO1. Mutants that were unable to synthesize alginate were unable to increase itaconate or suppress IL-1β production, suggesting that alginate might be able to induce Irg1 and increase itaconate production. These results suggested that the clinical isolates were somehow able to metabolize itaconate, since it would normally be bactericidal, in a manner akin to laboratory strains that upregulate itaconate-metabolizing enzymes. Seventeen CF clinical isolates of P. aeruginosa were analyzed in detail, and all had genetic features predicting an increased ability to metabolize itaconate, as well as alginate synthesis. This was not a feature of multiple other respiratory pathogens, suggesting that P. aeruginosa is somewhat unique in its ability to metabolize host itaconate. A mutant strain of P. aeruginosa, which mimics the clinical isolates by having no LPS on its surface, was also tested. Growth in itaconate was shown to boost biofilm production in this mutant. Other pathogenic mutant strains were also shown to produce biofilm, but only if they could metabolize itaconate. In vivo, growth of the LPS-deficient mutant strain in Irg1-deficient mice was shown to be 5% of that in wild-type mice, indicating that this mutant needs itaconate to grow in vivo. The same was true of host-adapted CF strains, which were unable to grow in Irg1-deficient mice, indicating their reliance on itaconate metabolism to grow. Airway-adapted strains of P. aeruginosa are therefore dependent on itaconate. Furthermore, in infected mice, clinical isolates were shown to be relatively resistant to itaconate administered intra-nasally. Host-adapted strains also promoted a higher recruitment of Irg1-expressing monocytes to the airways compared to the control strain, implying that they are actively recruiting cells to supply them with itaconate. Individuals with CF infected with alginate-producing P. aeruginosa also had increased numbers of Irg1-expressing monocytes in their peripheral blood and sputum, and increased itaconate levels in the sputum. Taken together, these findings provide us with important new information on itaconate during infection. As shown in Figure 1, the study suggests that in response to infection with P. aeruginosa, inflammatory macrophages release itaconate, which leads to an adaptation such that strains emerge that can use itaconate as a fuel source for biofilm production, decrease LPS expression, and induce the expression of protective EPSs on the surface of the bacteria, promoting their survival. The decrease in LPS also limits the pro-inflammatory response against the adapted strains. Alginates in the biofilm promote further itaconate production from monocytes recruited to the site of infection, leading to a positive feedback loop, enhanced bacterial survival, and ultimately disease pathogenesis in CF and COPD. This paper is therefore an intriguing example of a host-pathogen interaction, whereby a pathogen promotes a metabolite, which might limit its survival (by being bactericidal or by modulating host defense). However, the pathogen adapts to use the metabolite as a fuel source to promote biofilm formation. Pathogenic Pseudomonas aeruginosa are particularly prone to antibiotic resistance, so if itaconate levels could be reduced in the infectious microenvironment, this might cut off the fuel supply for the bacteria. Likewise, while boosting itaconate pharmacologically might have therapeutic potential as an anti-inflammatory strategy, this study highlights that an unintended consequence might be to promote bacterial growth in certain contexts. Future studies are warranted on the interplay between itaconate and other host-derived immunometabolites and bacteria, including in the microbiome. Such studies may provide us with new therapeutic options for infectious and inflammatory diseases, while potentially avoiding pitfalls of other therapeutic approaches. Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm FormationRiquelme et al.Cell MetabolismMay 18, 2020In BriefThe ability of Pseudomonas aeruginosa to chronically infect the lungs of immunocompetent individuals suggests that these organisms might utilize aspects of the host defense system to their advantage. Riquelme et al. demonstrate how P. aeruginosa exploits itaconate, a major mitochondrial metabolite produced by immune cells to thwart effective immune clearance, to promote biofilm formation. Full-Text PDF Open Archive