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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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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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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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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Marco R. Oggioni.

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