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Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia

Nature Microbiology volume 3, pages 600–610 (2018)Cite this article

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Abstract

Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169+ splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169+ splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.

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Fig. 1: The eclipse phase of pneumococcal bacteraemia.
Fig. 2: Numbers of pneumococci within clusters, founded by single cells, increase over time in the infected spleens.
Fig. 3: Tissue localization of infectious foci in the spleen.
Fig. 4: The intracellular phase of S. pneumoniae in the early stages before overt septicaemia.
Fig. 5: The pig spleen perfusion as a model of infection with S. pneumoniae.

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References

  1. Gratz, N., Loh, L. N. & Tuomanen, E. in Streptococcus Pneumoniae (eds Hammerschmidt, S. & Orihuela, C.) 433-451 (Academic Press, London, 2015).

  2. van der Poll, T. Future of sepsis therapies. Crit. Care 20, 106 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Pneumococcal Vaccines: WHO Position Paper WER Vol. 87, 14 (WHO, 2012).

  4. Musher, D. M. & Thorner, A. R. Community-acquired pneumonia. New Engl. J. Med. 371, 1619–1628 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Lim, W. S., Smith, D. L., Wise, M. P. & Welham, S. A. British Thoracic Society community acquired pneumonia guideline and the NICE pneumonia guideline: how they fit together. Thorax https://doi.org/10.1136/thoraxjnl-2015-206881 (2015).

  6. Simell, B. et al. The fundamental link between pneumococcal carriage and disease. Expert Rev. Vaccin. 11, 841–855 (2012).

    Article  CAS  Google Scholar 

  7. Rogers, D. E. Host mechanisms which act to remove bacteria from the blood stream. Bacteriol. Rev. 24, 50–66 (1960).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Gerlini, A. et al. The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck. PLoS Pathog. 10, e1004026 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Brown, E. J., Hosea, S. W. & Frank, M. M. The role of the spleen in experimental pneumococcal bacteremia. J. Clin. Investig. 67, 975–982 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Deniset, J. F., Surewaard, B. G., Lee, W.-Y. & Kubes, P. Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae. J. Exp. Med. 214, 1333–1350 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kono, M. et al. Single cell bottlenecks in the pathogenesis of Streptococcus pneumoniae. PLoS Pathog. 12, e1005887 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Horan, M. & Colebatch, J. H. Relation between splenectomy and subsequent infection: a clinical study. Arch. Dis. Child. 37, 398–414 (1962).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Theilacker, C. et al. Overwhelming postsplenectomy infection: a prospective multicenter cohort study. Clin. Infect. Dis. 62, 871–878 (2016).

    Article  PubMed  Google Scholar 

  14. Shinefield, H. R., Steinberg, C. R. & Kaye, D. Effect of splenectomy on the susceptibility of mice inoculated with diplococcus pneumoniae. J. Exp. Med. 123, 777–794 (1966).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Martinez-Pomares, L. et al. Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers. J. Exp. Med. 184, 1927–1937 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Oetke, C., Vinson, M. C., Jones, C. & Crocker, P. R. Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels. Mol. Cell. Biol. 26, 1549–1557 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kjos, M. et al. Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions. J. Bacteriol. 197, 807–818 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Aichele, P. et al. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J. Immunol. 171, 1148–1155 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Jones, C., Virji, M. & Crocker, P. R. Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake. Mol. Microbiol. 49, 1213–1225 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Heikema, A. P. et al. Enhanced, sialoadhesin-dependent uptake of Guillain-Barré syndrome-associated Campylobacter jejuni strains by human macrophages. Infect. Immun. 81, 2095–2103 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chang, Y.-C. et al. Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B streptococcus.J. Mol. Med. 92, 951–959 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Uchiyama, S. et al. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion. J. Exp. Med. 206, 1845–1852 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vanderheijden, N. et al. Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages. J. Virol. 77, 8207–8215 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Maurin, M. & Raoult, D. in Antimicrobial Agents and Intracellular Pathogens (ed. Raoult, D.) 21–37 (CRC Press Inc, Boca Raton, 1993).

  25. Steiniger, B., Barth, P. & Hellinger, A. The perifollicular and marginal zones of the human splenic white pulp: do fibroblasts guide lymphocyte immigration? Am. J. Pathol. 159, 501–512 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Steiniger, B. S. Human spleen microanatomy: why mice do not suffice. Immunology 145, 334–346 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fairbairn, L., Kapetanovic, R., Sester, D. P. & Hume, D. A. The mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease. J. Leukoc. Biol. 89, 855–871 (2011).

  28. Meurens, F., Summerfield, A., Nauwynck, H., Saif, L. & Gerdts, V. The pig: a model for human infectious diseases. Trends Microbiol. 20, 50–57 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Ezquerra, A. et al. Porcine myelomonocytic markers and cell populations. Dev. Comp. Immunol. 33, 284–298 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Alvarez, B. et al. Phenotypic and functional heterogeneity of CD169+and CD163+macrophages from porcine lymph nodes and spleen. Dev. Comp. Immunol. 44, 44–49 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. de Greeff, A. et al. Pneumococcal colonization and invasive disease studied in a porcine model. BMC Microbiol. 16, 102 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Chung, W. Y. et al. Steps for the autologous ex vivo perfused porcine liver-kidney experiment. J. Vis. Exp 82, e50567 (2013).

    Google Scholar 

  33. Moxon, E. R. & Murphy, P. A. Haemophilus influenzae bacteremia and meningitis resulting from survival of a single organism. Proc. Natl Acad. Sci. USA 75, 1534–1536 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lehar, S. M. et al. Novel antibody–antibiotic conjugate eliminates intracellular S. aureus. Nature 527, 323–328 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Heikema, A. P. et al. Characterization of the specific interaction between sialoadhesin and sialylated Campylobacter jejuni lipooligosaccharides. Infect. Immun. 78, 3237–3246 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Klaas, M. et al. Sialoadhesin promotes rapid proinflammatory and type I IFN responses to a sialylated pathogen, Campylobacter jejuni . J. Immunol. 189, 2414–2422 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. De Schryver, M. et al. Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages. Cell. Immunol. 312, 51–60 (2017).

    Article  PubMed  Google Scholar 

  38. Bewley, M. A. et al. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation. mBio 5, e01710-14 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Dockrell, D. H., Lee, M., Lynch, D. H. & Read, R. C. Immune-mediated phagocytosis and killing of Streptococcus pneumoniae are associated with direct and bystander macrophage apoptosis. J. Infect. Dis. 184, 713–722 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Gordon, S. B., Irving, G. R. B., Lawson, R. A., Lee, M. E. & Read, R. C. Intracellular trafficking and killing of Streptococcus pneumoniae by human alveolar macrophages are influenced by opsonins. Infect. Immun. 68, 2286–2293 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Davis, K. M., Nakamura, S. & Weiser, J. N. Nod2 sensing of lysozyme-digested peptidoglycan promotes macrophage recruitment and clearance of S. pneumoniae colonization in mice. J. Clin. Invest. 121, 3666–3676 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Honke, N. et al. Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus. Nat. Immunol. 13, 51–57 (2012).

    Article  CAS  Google Scholar 

  43. Van Breedam, W., Verbeeck, M., Christiaens, I., Van Gorp, H. & Nauwynck, H. J. Porcine, murine and human sialoadhesin (Sn/Siglec-1/CD169): portals for porcine reproductive and respiratory syndrome virus entry into target cells. J. General. Virol. 94, 1955–1960 (2013).

    Article  Google Scholar 

  44. Backer, R. et al. Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells. Proc. Natl Acad. Sci. USA 107, 216–221 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Veninga, H. et al. Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B-cell responses. Eur. J. Immunol. 45, 747–757 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mastroeni, P., Grant, A., Restif, O. & Maskell, D. A dynamic view of the spread and intracellular distribution of Salmonella enterica. Nat. Rev. Micro. 7, 73–80 (2009).

    Article  CAS  Google Scholar 

  47. Levin, B. R. & Antia, R. Why we don’t get sick: the within-host population dynamics of bacterial infections. Science 292, 1112–1115 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Shaw, S., Smith, A. L., Anderson, P. & Smith, D. H. The paradox of Hemophilus infuenzae type B bacteremia in the presence of serum bactericidal activity. J. Clin. Invest. 58, 1019–1029 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Grant, A. J. et al. Modelling within-host spatiotemporal dynamics of invasive bacterial disease. PLoS Biol. 6, e74 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Surewaard, B. G. J. et al. Identification and treatment of the Staphylococcus aureus reservoir in vivo. J. Exp. Med. 213, 1141–1151 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Avery, O. T., MacLeod, C. M. & McCarty, M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J. Exp. Med. 79, 137–158 (1944).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Iannelli, F., Pearce, B. J. & Pozzi, G. The type 2 capsule locus of Streptococcus pneumoniae. J. Bacteriol. 181, 2652–2654 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Manco, S. et al. Pneumococcal neuraminidases A and B both have essential roles during infection of the respiratory tract and sepsis. Infect. Immun. 74, 4014–4020 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Tettelin, H. et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498–506 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Pearce, B. J., Iannelli, F. & Pozzi, G. Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae. Res. Microbiol. 153, 243–247 (2002).

    Article  CAS  PubMed  Google Scholar 

  56. Morton, D. & Griffiths, P. Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet. Rec. 116, 431–436 (1985).

    Article  CAS  PubMed  Google Scholar 

  57. Kadioglu, A. et al. Sex-based differences in susceptibility to respiratory and systemic pneumococcal disease in mice. J. Infect. Dis. 204, 1971–1979 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Oggioni, M. R. et al. Antibacterial activity of a competence-stimulating peptide in experimental sepsis caused by Streptococcus pneumoniae. Antimicrob. Agents Chemother. 48, 4725–4732 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Oggioni, M. R. et al. Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol. Microbiol. 61, 1196–1210 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Lee, E.-J., Pontes, M. H. & Groisman, E. A. A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium’s own F1Fo ATP synthase. Cell 154, 146–156 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Taylor, P. R. et al. Development of a specific system for targeting protein to metallophilic macrophages. Proc. Natl Acad. Sci. USA 101, 1963–1968 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

G.E. was funded through an academic collaboration agreement between the University of Oxford and University of Leicester and in part by MRC grant MR/M003078/1. The authors thank J.W. Veening for providing GFP and RFP pneumococci, F. Focarelli for construction of the non-encapsulated GFP-expressing strain, M. De Ste Croix for mutant construction, S. Glenn for help with the infection experiments, the Electron Microscopy Facility, the University of Leicester for technical support and R. Kumar and J. Isherwood for help with the perfusion of the porcine organs at explant, the staff of Joseph Morris Butchers Ltd, Michael F Wood Butchers and the staff of Leicester Preclinical Research Facility for support.

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Authors and Affiliations

  1. Department of Genetics and Genome Biology, University of Leicester, Leicester, UK

    Giuseppe Ercoli, Joseph J. Wanford, Christopher D. Bayliss & Marco R. Oggioni

  2. Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK

    Vitor E. Fernandes & Peter W. Andrew

  3. Hepato-Pancreato-Biliary Unit, Leicester General Hospital, University of Hospitals of Leicester, NHS Trust, Leicester, UK

    Wen Y. Chung & Ashley Dennison

  4. Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, UK

    Sarah Thomson & Paul R. Crocker

  5. Centre for Core Biotechnology Services, University of Leicester, Leicester, UK

    Kornelis Straatman

  6. School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham, UK

    Luisa Martinez-Pomares

  7. Department of Pediatrics, University of Oxford, Oxford, UK

    E. Richard Moxon

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  1. Giuseppe Ercoli
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Contributions

G.E. performed almost all experiments and wrote the manuscript. V.E.F. led and performed the animal infections. W.Y.C. led and performed the porcine spleen perfusion experiments. J.J.W. contributed to microscopy and animal infection work, and contributed to the writing of the manuscript. C.D.B. contributed to the design of the experimental work. K.S. led the microscopy work. S.T. performed the infections of the CD169 KO mice. P.R.C. discussed the work and supervised the infections in the KO mice, and contributed to the writing of the manuscript. A.D. designed and led the porcine perfusion work, and contributed to the writing of the manuscript. L.M.-P. designed the immunological work and overall setup of experimentation, and contributed to the writing of the manuscript. P.W.A. participated in the overall design and setup of the experimentation, and contributed to the writing of the manuscript. E.R.M. initiated and participated in the overall design and setup of the experimentation, and contributed to the writing of the manuscript. M.R.O. led the design and setup of the project, and contributed to the writing of the manuscript.

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Correspondence to Marco R. Oggioni.

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Ercoli, G., Fernandes, V.E., Chung, W.Y. et al. Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia. Nat Microbiol 3, 600–610 (2018). https://doi.org/10.1038/s41564-018-0147-1

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