Abstract

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody–antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody–antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Protein Data Bank

Data deposits

The structure of the anti-β-WTA Fab bound to the synthetic WTA fragment (β-GlcNAc anomer) has been deposited in the Protein Data Bank under accession number 5D6C.

References

  1. 1.

    et al. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin. Infect. Dis . 32 (suppl. 2), S114–S132 (2001)

  2. 2.

    Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998)

  3. 3.

    et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009)

  4. 4.

    , & Resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillin-resistant Staphylococcus aureus. Curr. Opin. Pharmacol. 10, 516–521 (2010)

  5. 5.

    & Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nature Rev. Microbiol. 9, 215–222 (2011)

  6. 6.

    & The survival of staphylococci within human leukocytes. J. Exp. Med. 95, 209–230 (1952)

  7. 7.

    et al. Survival of Staphylococcus aureus inside neutrophils contributes to infection. J. Immunol. 164, 3713–3722 (2000)

  8. 8.

    & Intracellular survival of staphylococci. J. Exp. Med. 110, 123–138 (1959)

  9. 9.

    , , , & The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes. Clin. Exp. Immunol. 157, 216–224 (2009)

  10. 10.

    & Intracellular Staphylococcus aureus: live-in and let die. Front. Cell. Infect. Microbiol . 2, 43 (2012)

  11. 11.

    & Return of the Trojan horse: intracellular phenotype switching and immune evasion by Staphylococcus aureus. EMBO Mol. Med. 3, 115–117 (2011)

  12. 12.

    Studies on bacteriemia. I. Mechanisms relating to the persistence of bacteriemia in rabbits following the intravenous injection of staphylococci. J. Exp. Med. 103, 713–742 (1956)

  13. 13.

    et al. Comparative study of clinical characteristics of neutropenic and non-neutropenic adult cancer patients with bloodstream infections. Eur. J. Clin. Microbiol. Infect. Dis. 25, 1–7 (2006)

  14. 14.

    et al. Staphylococcus aureus bacteremia in patients with hematologic malignancies: a retrospective case-control study. Haematologica 88, 923–930 (2003)

  15. 15.

    , & Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report. J. Bone Joint Surg. Am. 87, 1343–1347 (2005)

  16. 16.

    et al. Evidence of an intracellular reservoir in the nasal mucosa of patients with recurrent Staphylococcus aureus rhinosinusitis. J. Infect. Dis. 192, 1023–1028 (2005)

  17. 17.

    , & The expression of α-haemolysin is required for Staphylococcus aureus phagosomal escape after internalization in CFT-1 cells. Cell. Microbiol. 10, 1801–1814 (2008)

  18. 18.

    et al. Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis. J. Exp. Med. 201, 1627–1635 (2005)

  19. 19.

    et al. Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis. J. Immunol. 192, 4709–4717 (2014)

  20. 20.

    et al. Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus. J. Innate Immun. 2, 560–575 (2010)

  21. 21.

    , , , & Pharmacodynamic evaluation of the intracellular activities of antibiotics against Staphylococcus aureus in a model of THP-1 macrophages. Antimicrob. Agents Chemother. 50, 841–851 (2006)

  22. 22.

    , , , & Intracellular activity of antibiotics against Staphylococcus aureus in a mouse peritonitis model. Antimicrob. Agents Chemother. 53, 1874–1883 (2009)

  23. 23.

    et al. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. 13, 855–869 (2002)

  24. 24.

    , & Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus. Int. J. Med. Microbiol. 304, 215–221 (2014)

  25. 25.

    et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901–912 (2001)

  26. 26.

    et al. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503, 365–370 (2013)

  27. 27.

    , & A doubly labeled penetratin analogue as a ratiometric sensor for intracellular proteolytic stability. Bioconjug. Chem. 21, 64–73 (2010)

  28. 28.

    & Extracellular and intracellular killing in neutrophil granulocytes of Staphylococcus aureus with rifampicin in combination with dicloxacillin or fusidic acid. J. Antimicrob. Chemother. 43, 407–410 (1999)

  29. 29.

    , , & Impact of vancomycin exposure on outcomes in patients with methicillin-resistant Staphylococcus aureus bacteremia: support for consensus guidelines suggested targets. Clin. Infect. Dis. 52, 975–981 (2011)

  30. 30.

    et al. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N. Engl. J. Med. 355, 653–665 (2006)

  31. 31.

    , , , & Predictors of persistent methicillin-resistant Staphylococcus aureus bacteraemia in patients treated with vancomycin. J. Antimicrob. Chemother. 65, 1015–1018 (2010)

  32. 32.

    , , , & Staphylococcus aureus bacteremia: compliance with standard treatment, long-term outcome and predictors of relapse. Scand. J. Infect. Dis. 35, 782–789 (2003)

  33. 33.

    Noninherited resistance to antibiotics. Science 305, 1578–1579 (2004)

  34. 34.

    , , , & Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc. Natl Acad. Sci. USA 109, 12147–12152 (2012)

  35. 35.

    Persister cells. Annu. Rev. Microbiol. 64, 357–372 (2010)

  36. 36.

    et al. Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment. PLoS Biol. 12, e1001793 (2014)

  37. 37.

    Anti-MRSA agents: under investigation, in the exploratory phase and clinically available. Expert Rev. Anti Infect. Ther. 3, 505–553 (2005)

  38. 38.

    et al. Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins. PLoS Pathog. 9, e1003653 (2013)

  39. 39.

    , , , & Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3, e00277–11 (2012)

  40. 40.

    et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006)

  41. 41.

    , , & Human antibody repertoires. Methods Mol. Biol. 525, 261–277 (2009)

  42. 42.

    et al. Rifamycin Analogs and Uses Thereof (Activbiotics, 2005)

  43. 43.

    et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechnol. 26, 925–932 (2008)

  44. 44.

    , , & Synthesis of 4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-ribitol, antigenic determinant of Staphylococcus aureus. Carbohydr. Res. 110, 153–158 (1982)

  45. 45.

    & Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrob. Agents Chemother. 16, 743–749 (1979)

Download references

Acknowledgements

This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

Author information

Affiliations

  1. Infectious Diseases Department, Genentech Inc., South San Francisco, California 94080, USA

    • Sophie M. Lehar
    • , Kimberly K. Kajihara
    • , Wouter L. Hazenbos
    • , J. Hiroshi Morisaki
    • , Man Wah Tan
    • , Eric J. Brown
    •  & Sanjeev Mariathasan
  2. Medicinal Chemistry Department, Genentech Inc., South San Francisco, California 94080, USA

    • Thomas Pillow
    • , Leanna Staben
    • , Joseph P. Lyssikatos
    •  & John A. Flygare
  3. Translational Immunology Department, Genentech Inc., South San Francisco, California 94080, USA

    • Min Xu
    • , Janice Kim
    • , Summer Park
    •  & Donghong Yan
  4. Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080, USA

    • Richard Vandlen
    • , Laura DePalatis
    • , Helga Raab
    • , Martine Darwish
    • , Byoung-Chul Lee
    •  & Elizabeth Luis
  5. Biochemical and Cellular Pharmacology Department, Genentech Inc., South San Francisco, California 94080, USA

    • Hilda Hernandez
    • , Kelly M. Loyet
    •  & Yana Khalfin
  6. Structural Biology Department, Genentech Inc., South San Francisco, California 94080, USA

    • Patrick Lupardus
    •  & Rina Fong
  7. Pathology Department, Genentech Inc., South San Francisco, California 94080, USA

    • Cecile Chalouni
  8. Drug metabolism and Pharmacokinetics Department, Genentech Inc., South San Francisco, California 94080, USA

    • Emile Plise
    •  & Jonathan Cheong
  9. Symphogen A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark

    • Magnus Strandh
    • , Klaus Koefoed
    •  & Peter S. Andersen

Authors

  1. Search for Sophie M. Lehar in:

  2. Search for Thomas Pillow in:

  3. Search for Min Xu in:

  4. Search for Leanna Staben in:

  5. Search for Kimberly K. Kajihara in:

  6. Search for Richard Vandlen in:

  7. Search for Laura DePalatis in:

  8. Search for Helga Raab in:

  9. Search for Wouter L. Hazenbos in:

  10. Search for J. Hiroshi Morisaki in:

  11. Search for Janice Kim in:

  12. Search for Summer Park in:

  13. Search for Martine Darwish in:

  14. Search for Byoung-Chul Lee in:

  15. Search for Hilda Hernandez in:

  16. Search for Kelly M. Loyet in:

  17. Search for Patrick Lupardus in:

  18. Search for Rina Fong in:

  19. Search for Donghong Yan in:

  20. Search for Cecile Chalouni in:

  21. Search for Elizabeth Luis in:

  22. Search for Yana Khalfin in:

  23. Search for Emile Plise in:

  24. Search for Jonathan Cheong in:

  25. Search for Joseph P. Lyssikatos in:

  26. Search for Magnus Strandh in:

  27. Search for Klaus Koefoed in:

  28. Search for Peter S. Andersen in:

  29. Search for John A. Flygare in:

  30. Search for Man Wah Tan in:

  31. Search for Eric J. Brown in:

  32. Search for Sanjeev Mariathasan in:

Contributions

S.M.L. designed and executed the in vitro and in vivo analysis of the AAC mechanism of action. T.P., L.S. and J.A.F. designed and synthesized antibiotics and linker drugs. M.X., J.K., S.P. and D.Y. designed and analysed in vivo models for intravenous infection. H.R., L.D., M.D. and R.V. designed and conjugated linker antibiotic to antibodies. K.K.K., W.L.H., J.H.M. and S.M. characterized the anti-MRSA antibodies. Y.K., H.H., K.M.L., E.P. and J.C. did mass spectrometry analysis of the rifalogues during in vitro efficacy studies. P.L. and R.F. performed X-ray crystallography of anti-β-WTA monoclonal antibody. J.P.L. designed the synthesis of β-phospho-ribitol. B.-C.L. and C.C. characterized FRET constructs and helped with video microscopy. E.L. determined the number of antibody-binding sites on MRSA. M.S., K.K. and P.S.A. isolated anti-MRSA antibodies from patients. M.W.T. contributed to bacterial genetics and data analysis. E.J.B. and S.M. initiated the project and S.M. led the project. S.M.L., E.J.B. and S.M. composed the paper with input from all authors.

Competing interests

As employees of either Genentech or Symphogen, all authors declare competing financial interests.

Corresponding authors

Correspondence to Eric J. Brown or Sanjeev Mariathasan.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature16057

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.