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Staphylococcus aureus small colony variants impair host immunity by activating host cell glycolysis and inducing necroptosis

Nature Microbiology volume 5, pages 141–153 (2020)Cite this article

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Abstract

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.

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Fig. 1: The S. aureus SCV prototype ΔhemB stimulates a distinct metabolic response in host cells.
Fig. 2: S. aureus stimulates necroptosis via the induction of host glycolysis.
Fig. 3: S. aureus-induced necroptosis is independent of autophagy or ligand–receptor interactions.
Fig. 4: ΔhemB-induced necroptosis impairs bacterial clearance.
Fig. 5: The ΔhemB SCV fails to induce trained immunity.
Fig. 6: Overexpression of fumC in ΔhemB SCVs inhibits trained immunity.

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Data availability

Whole genome sequence data of the ΔhemB, ΔfumC and ΔhemBΔfumC LAC mutants and the complemented strains ΔhemB (hemB) and ΔfumC (fumC) were submitted to the European Nucleotide Archive under BioProject number PRJEB30093, accession numbers ERS3011129, ERS3011131, ERS3398722, ERS3011130 and ERS3011132, respectively. Data that support the findings of this study are available from the corresponding author upon request.

References

  1. Kahl, B. C., Becker, K. & Loffler, B. Clinical significance and pathogenesis of staphylococcal small colony variants in persistent infections. Clin. Microbiol. Rev. 29, 401–427 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kennedy, A. D. et al. Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model. J. Infect. Dis. 202, 1050–1058 (2010).

    PubMed  PubMed Central  Google Scholar 

  3. Kernodle, D. S., Voladri, R. K., Menzies, B. E., Hager, C. C. & Edwards, K. M. Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model. Infect. Immun. 65, 179–184 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kobayashi, S. D. et al. Comparative analysis of USA300 virulence determinants in a rabbit model of skin and soft tissue infection. J. Infect. Dis. 204, 937–941 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Menzies, B. E. & Kernodle, D. S. Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model. Infect. Immun. 62, 1843–1847 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Proctor, R. A. & Peters, G. Small colony variants in staphylococcal infections: diagnostic and therapeutic implications. Clin. Infect. Dis. 27, 419–422 (1998).

    CAS  PubMed  Google Scholar 

  7. Proctor, R. A. et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4, 295–305 (2006).

    CAS  PubMed  Google Scholar 

  8. Kriegeskorte, A. et al. Staphylococcus aureus small colony variants show common metabolic features in central metabolism irrespective of the underlying auxotrophism. Front. Cell. Infect. Microbiol. 4, 141 (2014).

    PubMed  PubMed Central  Google Scholar 

  9. Proctor, R. A. et al. Staphylococcus aureus small colony variants (SCVs): a road map for the metabolic pathways involved in persistent infections. Front. Cell. Infect. Microbiol. 4, 99 (2014).

    PubMed  PubMed Central  Google Scholar 

  10. Tuchscherr, L. et al. Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence. J. Infect. Dis. 202, 1031–1040 (2010).

    PubMed  Google Scholar 

  11. Arts, R. J., Joosten, L. A. & Netea, M. G. Immunometabolic circuits in trained immunity. Semin. Immunol. 28, 425–430 (2016).

    CAS  PubMed  Google Scholar 

  12. O’Neill, L. A., Kishton, R. J. & Rathmell, J. A guide to immunometabolism for immunologists. Nat. Rev. Immunol. 16, 553–565 (2016).

    PubMed  PubMed Central  Google Scholar 

  13. Wickersham, M. et al. Metabolic stress drives keratinocyte defenses against Staphylococcus aureus infection. Cell Rep. 18, 2742–2751 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kitur, K. et al. Necroptosis promotes Staphylococcus aureus clearance by inhibiting excessive inflammatory signaling. Cell Rep. 16, 2219–2230 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Baek, K. T. et al. Genetic variation in the Staphylococcus aureus 8325 strain lineage revealed by whole-genome sequencing. PLoS ONE 8, e77122 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Herbert, S. et al. Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates. Infect. Immun. 78, 2877–2889 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Horsburgh, M. J. et al. σB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J. Bacteriol. 184, 5457–5467 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Giachino, P., Engelmann, S. & Bischoff, M. ςB activity depends on RsbU in Staphylococcus aureus. J. Bacteriol. 183, 1843–1852 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Anzaldi, L. L. & Skaar, E. P. Overcoming the heme paradox: heme toxicity and tolerance in bacterial pathogens. Infect. Immun. 78, 4977–4989 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Atalla, H., Gyles, C. & Mallard, B. Staphylococcus aureus small colony variants (SCVs) and their role in disease. Anim. Health Res. Rev. 12, 33–45 (2011).

    PubMed  Google Scholar 

  21. von Eiff, C. et al. A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J. Bacteriol. 179, 4706–4712 (1997).

    Google Scholar 

  22. Palma Medina, L. M. et al. Metabolic cross-talk between human bronchial epithelial cells and internalized Staphylococcus aureus as a driver for infection. Mol. Cell. Proteom. 18, 892–908 (2019).

    Google Scholar 

  23. LaRocca, T. J., Sosunov, S. A., Shakerley, N. L., Ten, V. S. & Ratner, A. J. Hyperglycemic conditions prime cells for RIP1-dependent Necroptosis. J. Biol. Chem. 291, 13753–13761 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. McCaig, W. D. et al. Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis. Cell Death Discov. 4, 55 (2018).

    PubMed  PubMed Central  Google Scholar 

  25. Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat. Cell Biol. 16, 55–65 (2014).

    CAS  PubMed  Google Scholar 

  26. Chen, X. et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 24, 105–121 (2014).

    CAS  PubMed  Google Scholar 

  27. Gonzalez-Juarbe, N. et al. Pore-forming toxins induce macrophage necroptosis during acute bacterial pneumonia. PLoS Pathog. 11, e1005337 (2015).

    PubMed  PubMed Central  Google Scholar 

  28. Kitur, K. et al. Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage. PLoS Pathog. 11, e1004820 (2015).

    PubMed  PubMed Central  Google Scholar 

  29. Vitko, N. P., Spahich, N. A. & Richardson, A. R. Glycolytic dependency of high-level nitric oxide resistance and virulence in Staphylococcus aureus. mBio 6, e00045–15 (2015).

    PubMed  Google Scholar 

  30. Yang, Z. et al. RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis. Nat. Cell Biol. 20, 186–197 (2018).

    CAS  PubMed  Google Scholar 

  31. Kucera, O. et al. The effect of tert-butyl hydroperoxide-induced oxidative stress on lean and steatotic rat hepatocytes in vitro. Oxid. Med. Cell. Longev. 2014, 752506 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Galluzzi, L., Kepp, O., Chan, F. K. & Kroemer, G. Necroptosis: mechanisms and relevance to disease. Annu. Rev. Pathol. 12, 103–130 (2017).

    CAS  PubMed  Google Scholar 

  33. Goodall, M. L. et al. The autophagy machinery controls cell death switching between apoptosis and necroptosis. Dev. Cell 37, 337–349 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Bhattacharyya, S. et al. Small colony variants of Staphylococcus aureus isolated from a patient with infective endocarditis: a case report and review of the literature. Iran. J. Microbiol. 4, 98–99 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Jonsson, I. M. et al. Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis. Microb. Pathog. 34, 73–79 (2003).

    CAS  PubMed  Google Scholar 

  36. Chan, L. C. et al. Innate immune memory contributes to host defense against recurrent skin and skin structure infections caused by methicillin-resistant staphylococcus aureus. Infect. Immun. 85, e00876–16 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Chan, L. C. et al. Protective immunity in recurrent Staphylococcus aureus infection reflects localized immune signatures and macrophage-conferred memory. Proc. Natl Acad. Sci. USA 115, E11111–E11119 (2018).

    CAS  PubMed  Google Scholar 

  38. van der Heijden, C. et al. Epigenetics and trained immunity. Antioxid. Redox Signal. 29, 1023–1040 (2018).

    PubMed  PubMed Central  Google Scholar 

  39. Arts, R. J. et al. Glutaminolysis and fumarate accumulation integrate immunometabolic and epigenetic programs in trained immunity. Cell Metab. 24, 807–819 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kornberg, M. D. et al. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity. Science 360, 449–453 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Naik, S. et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature 550, 475–480 (2017); erratum 560, E2 (2018).

  42. Monk, I. R., Tree, J. J., Howden, B. P., Stinear, T. P. & Foster, T. J. Complete bypass of restriction systems for major Staphylococcus aureus lineages. mBio 6, e00308–e00315 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Monk, I. R., Howden, B. P., Seemann, T. & Stinear, T. P. Correspondence: Spontaneous secondary mutations confound analysis of the essential two-component system WalKR in Staphylococcus aureus. Nat. Commun. 8, 14403 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 111, A3.B.1–A3.B.3 (2015).

  45. Melamud, E., Vastag, L. & Rabinowitz, J. D. Metabolomic analysis and visualization engine for LC-MS data. Anal. Chem. 82, 9818–9826 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Barker, M. & Rayens, W. Partial least squares for discrimination. J. Chemom. 17, 166–173 (2003).

    CAS  Google Scholar 

  47. Rohart, F., Gautier, B., Singh, A. & Le Cao, K. A. mixOmics: an R package for ‘omics feature selection and multiple data integration. PLoS Comput. Biol. 13, e1005752 (2017).

    PubMed  PubMed Central  Google Scholar 

  48. Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).

    CAS  PubMed  Google Scholar 

  49. Berger, S. B. et al. Cutting edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J. Immunol. 192, 5476–5480 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Soong, G. et al. Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes. mBio 6, e00289–15 (2015).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank I. Lewis (University of Calgary), R. Groves (University of Calgary) and D. P. De Souza (Metabolomics Australia) for their support with the metabolomics studies and V. J. Torres (New York University School of Medicine) for his gift of antibodies against staphylococci toxins. This work was supported by NIH grant R01AI103854 to A.P., NIH grant S10RR027050 to the Columbia Center for Translational Immunology Flow Cytometry Core and NHMRC grant (Australia) APP1066791 to B.P.H.

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Authors and Affiliations

  1. Department of Pediatrics, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA

    Tania Wong Fok Lung, Karen P. Acker, Sebastián A. Riquelme, Silvia Pires, Loreani P. Noguera, Felix Dach, Stanislaw J. Gabryszewski & Alice Prince

  2. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Ian R. Monk, Andre Mu, Nancy Wang & Benjamin P. Howden

  3. Microbiological Diagnostic Unit Public Health Laboratory, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Andre Mu & Benjamin P. Howden

  4. Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia

    Benjamin P. Howden

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Contributions

T.W.F.L. and A.P. conceived the project, designed the experiments and interpreted the data. T.W.F.L. performed the in vitro and in vivo experiments and data analysis. I.R.M. constructed the bacterial mutants and contributed to the transcriptomics studies. K.P.A. contributed to the in vitro and in vivo work pertaining to Aim2. N.W. performed the in vitro THP-1 infection and A.M. analysed the metabolomics data. S.A.R., S.P., L.P.N., F.D. and S.J.G. assisted with the in vitro and in vivo experiments and data analysis. T.W.F.L., B.P.H. and A.P. wrote and edited the paper.

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Correspondence to Alice Prince.

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Extended data

Extended data 1 ΔhemB LAC SCVs stimulate a distinct metabolic response in THP-1 cells.

(A) Construction of the ΔhemB mutant in the USA300 LAC background. (B) White light image of WT LAC and the ΔhemB LAC mutant on BHI and blood agar plates to better illustrate pinpoint size of the ΔhemB LAC mutant on BHI agar and β (complete)-hemolysis on blood agar, image shown is from 1 representative experiment, n = 3 independent experiments. (C) Representative PCA score plots showing principal component 1 (PC1/variate 1) versus PC2/variate 2 of the total intracellular and extracellular THP-1 metabolomes, n = 2 independent experiments in triplicate. (D) Heatmaps of the total intracellular and extracellular THP-1 metabolomes, n = 2. (E) Heatmap of lower abundance metabolites in the extracellular THP-1 metabolomes, n = 2. (F) Relative quantification of fumarate in the extracellular metabolome of uninfected and infected THP-1 cells. Data represent median with 95% CI, n = 2 independent experiments in triplicate.

Extended Data Fig. 1.

Extended data 2 Necroptosis promotes ΔhemB SCV persistence.

(A) A representative immunoblot showing the induction of PMLKL and PS6 in HEKn cells exposed to PBS, WT LAC, ΔhemB LAC or the complemented strain for 4 h in the presence and absence of 2-DG or BHA, n = 3 independent experiments. (B) Bacterial load in skin biopsies 5 days following infection of WT C57BL/6 or Ripk1d or Mlkl−/− mice with WT 8325-4 and ΔhemB 8325-4. (C) Innate immune cell recruitment from (B) at day 5. (D) Cytokine measurements from mouse skin biopsies from (B) at day 5 For (B-D), data represent mean ± SEM, n = 15 (C57BL/6 WT), n = 17 (C57BL/6 ΔhemB). n = 15 (Ripk1d WT), n = 12 (Ripk1d ΔhemB), n = 9 (Mlkl−/− WT) or n = 10 (Mlkl−/− ΔhemB) independent animals. Statistical analysis was performed by one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Extended Data Fig. 2.

Extended data 3 Characterization of the ΔhemBΔfumC double mutant.

(A) Growth of the ΔhemBΔfumC double mutant as compared to WT LAC, the ΔhemB or ΔfumC single mutants on BHI and blood agar plates. Various dilutions of bacterial cultures were plated. Image shown is from 1 representative experiment, n = 3 independent experiments. (B) Growth of the ΔhemBΔfumC double mutant as compared to WT LAC and the ΔhemB single mutant in liquid LB culture. n = 3 independent experiments. (C) Cytokine measurements from the skin of mice 5 days post infection with WT LAC or the ΔhemBΔfumC mutant. Data represent mean ± SEM, n = 4 independent animal samples.. Statistical analysis was performed by one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Extended Data Fig. 3.

Extended data 4 Protection against secondary S. aureus infection is independent of IL1R1-Aim2 activation.

(A-B) (A) Dermonecrosis and (B) bacterial load in WT C57BL/6 and Il1r1−/− mice treated with PBS or infected with WT LAC on day 0 and challenged at the same site on day 28 with WT LAC; black asterisks denote statistical differences in lesion size with respect to uninfected mice, purple asterisks show statistical difference between WT C57BL/6 and Il1r1−/−-infected mice and green asterisks show statistical differences between WT C57BL/6 and Il1r1−/−-naïve mice upon secondary challenge, n = 2 (C57BL/6 or Il1r1−/− PBS PBS), n = 5 (C57BL/6 PBS WT or C57BL/6 WT WT), n = 4 (Il1r1−/− PBS WT) or n = 6 (Il1r1−/− WT WT) independent animals. (C-D) (C) Dermonecrosis and (D) bacterial load in the skin of WT C57BL/6 and Aim2−/− mice 5 days post infection with WT LAC, ns: not significant, n = 3 or 4 independent animals (day 1) or n = 10 independent animals (day 5). (E) Intracellular mean fluorescence intensity (MFI) of Aim2 measured by flow cytometry in keratinocytes infected with WT LAC or transfected with poly dA:dT for 24 h, n = 1 representative experiment from 3 independent experiments. (F) Expression of Aim2 in HEKn cells infected with WT LAC or transfected with poly dA:dT for 24 h, compared to uninfected cells (PBS) by qRT-PCR, n = 1 representative experiment from 3 independent experiments, For (A-F), data represent mean ± SEM. Statistical analysis was performed by one-way (B) or two-way ANOVA (A, C and D), ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns; p = 0.9999 (C, day 1), 0.9051 (C, day 2), 0.6533 (C, day 3), 0.9820 (C, day 4), 0.9899 (C, day 5) and p = 0.0892 (D, day 1) and 0.4037 (D, day 5).

Supplementary information

Supplementary Tables 1–6

Supplementary Table 1: bacterial strains used in this study. Supplementary Table 2: primers used in this study. Supplementary Table 3: plasmids used in this study. Supplementary Table 4: list of extracellular metabolites from infected human PBMCs. Supplementary Table 5: list of intracellular metabolites from infected THP-1 cells. Supplementary Table 6: list of extracellular metabolites from infected THP-1 cells.

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Wong Fok Lung, T., Monk, I.R., Acker, K.P. et al. Staphylococcus aureus small colony variants impair host immunity by activating host cell glycolysis and inducing necroptosis. Nat Microbiol 5, 141–153 (2020). https://doi.org/10.1038/s41564-019-0597-0

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