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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Diekema, D. J. 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)
Lowy, F. D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998)
Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009)
Nannini, E., Murray, B. E. & Arias, C. A. Resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillin-resistant Staphylococcus aureus. Curr. Opin. Pharmacol. 10, 516–521 (2010)
Thwaites, G. E. & Gant, V. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nature Rev. Microbiol. 9, 215–222 (2011)
Rogers, D. E. & Tompsett, R. The survival of staphylococci within human leukocytes. J. Exp. Med. 95, 209–230 (1952)
Gresham, H. D. et al. Survival of Staphylococcus aureus inside neutrophils contributes to infection. J. Immunol. 164, 3713–3722 (2000)
Kapral, F. A. & Shayegani, M. G. Intracellular survival of staphylococci. J. Exp. Med. 110, 123–138 (1959)
Anwar, S., Prince, L. R., Foster, S. J., Whyte, M. K. & Sabroe, I. The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes. Clin. Exp. Immunol. 157, 216–224 (2009)
Fraunholz, M. & Sinha, B. Intracellular Staphylococcus aureus: live-in and let die. Front. Cell. Infect. Microbiol . 2, 43 (2012)
Garzoni, C. & Kelley, W. L. Return of the Trojan horse: intracellular phenotype switching and immune evasion by Staphylococcus aureus. EMBO Mol. Med. 3, 115–117 (2011)
Rogers, D. E. 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)
Velasco, E. 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)
Venditti, M. et al. Staphylococcus aureus bacteremia in patients with hematologic malignancies: a retrospective case-control study. Haematologica 88, 923–930 (2003)
Bosse, M. J., Gruber, H. E. & Ramp, W. K. 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)
Clement, S. 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)
Jarry, T. M., Memmi, G. & Cheung, A. L. The expression of α-haemolysin is required for Staphylococcus aureus phagosomal escape after internalization in CFT-1 cells. Cell. Microbiol. 10, 1801–1814 (2008)
Que, Y. A. et al. Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis. J. Exp. Med. 201, 1627–1635 (2005)
Greenlee-Wacker, M. C. et al. Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis. J. Immunol. 192, 4709–4717 (2014)
Kobayashi, S. D. et al. Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus. J. Innate Immun. 2, 560–575 (2010)
Barcia-Macay, M., Seral, C., Mingeot-Leclercq, M. P., Tulkens, P. M. & Van Bambeke, F. 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)
Sandberg, A., Hessler, J. H., Skov, R. L., Blom, J. & Frimodt-Møller, N. Intracellular activity of antibiotics against Staphylococcus aureus in a mouse peritonitis model. Antimicrob. Agents Chemother. 53, 1874–1883 (2009)
Dubowchik, G. M. 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)
Winstel, V., Xia, G. & Peschel, A. Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus. Int. J. Med. Microbiol. 304, 215–221 (2014)
Campbell, E. A. et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901–912 (2001)
Conlon, B. P. et al. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503, 365–370 (2013)
Fischer, R., Hufnagel, H. & Brock, R. A doubly labeled penetratin analogue as a ratiometric sensor for intracellular proteolytic stability. Bioconjug. Chem. 21, 64–73 (2010)
Nielsen, S. L. & Black, F. T. 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)
Kullar, R., Davis, S. L., Levine, D. P. & Rybak, M. J. 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)
Fowler, V. G. Jr et al. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N. Engl. J. Med. 355, 653–665 (2006)
Yoon, Y. K., Kim, J. Y., Park, D. W., Sohn, J. W. & Kim, M. J. Predictors of persistent methicillin-resistant Staphylococcus aureus bacteraemia in patients treated with vancomycin. J. Antimicrob. Chemother. 65, 1015–1018 (2010)
Johnson, L. B., Almoujahed, M. O., Ilg, K., Maolood, L. & Khatib, R. Staphylococcus aureus bacteremia: compliance with standard treatment, long-term outcome and predictors of relapse. Scand. J. Infect. Dis. 35, 782–789 (2003)
Levin, B. R. Noninherited resistance to antibiotics. Science 305, 1578–1579 (2004)
Grant, S. S., Kaufmann, B. B., Chand, N. S., Haseley, N. & Hung, D. T. Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc. Natl Acad. Sci. USA 109, 12147–12152 (2012)
Lewis, K. Persister cells. Annu. Rev. Microbiol. 64, 357–372 (2010)
Kaiser, P. et al. Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment. PLoS Biol. 12, e1001793 (2014)
Bryskier, A. Anti-MRSA agents: under investigation, in the exploratory phase and clinically available. Expert Rev. Anti Infect. Ther. 3, 505–553 (2005)
Hazenbos, W. L. et al. Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins. PLoS Pathog. 9, e1003653 (2013)
Monk, I. R., Shah, I. M., Xu, M., Tan, M. W. & Foster, T. J. Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3, e00277–11 (2012)
Meijer, P. J. et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006)
Meijer, P. J., Nielsen, L. S., Lantto, J. & Jensen, A. Human antibody repertoires. Methods Mol. Biol. 525, 261–277 (2009)
Van Duzer, J. et al. Rifamycin Analogs and Uses Thereof (Activbiotics, 2005)
Junutula, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechnol. 26, 925–932 (2008)
Boullanger, P., Descotes, G., Flandrois, J. P. & Marmet, D. Synthesis of 4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-ribitol, antigenic determinant of Staphylococcus aureus. Carbohydr. Res. 110, 153–158 (1982)
Vaudaux, P. & Waldvogel, F. A. Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrob. Agents Chemother. 16, 743–749 (1979)
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.
As employees of either Genentech or Symphogen, all authors declare competing financial interests.
Extended data figures and tables
In vivo infection of mice shown in Fig. 1a. Mice (n = 5) were infected with equivalent doses of free bacteria or intracellular bacteria and treated with vancomycin at 110 mg kg−1 10 min after infection and then once per day. Bacterial burden was monitored in the brain 4 days after infection. Bars show geometric mean.
Extended Data Figure 2 MRSA is able to grow in the presence of vancomycin when cultured on a monolayer of infectable cells.
a, b, Similar to the set up in Fig. 1d, planktonic MRSA were either seeded in media alone, or in the presence of vancomycin. Intracellular bacteria were generated by infecting a monolayer of either A549 bronchial epithelial cells (a) or HBMECs (b) in the presence of vancomycin (vanco). In these experiments plates were centrifuged to promote contact of the bacteria with the monolayer to enhance intracellular infection. At each time point, the culture supernatant was collected to recover extracellular bacteria and adherent cells were lysed to release intracellular bacteria. Extracellular bacteria (planktonic bacteria) grew well in media alone, but were killed by vancomycin. In wells containing a monolayer of mammalian cells (intracellular MRSA + vanco) a fraction of the bacteria were protected from vancomycin during the first 5 h after infection and were able to expand within the intracellular compartment over 24 h. Error bars show s.d. from triplicate wells. Representative of three independent experiments.
a, Determining the intracellular MIC for rifalogue and rifampicin. MRSA was allowed to infect peritoneal macrophages and macrophages were cultured overnight in gentamycin to kill extracellular bacteria. Various doses of rifalogue (red) or rifampicin (grey) were added to the culture medium 1 day after infection and the number of viable intracellular bacteria was determined 24 h later by spotting macrophage lysates onto agar plates. Data shown are representative of more than three independent experiments. b, Diffusion of rifalogue versus rifampicin into murine macrophages. Murine peritoneal macrophages were incubated with rifalogue or rifampicin in the culture media. Wells were harvested at 10 and 60 min and the total amount of antibiotic associated with the cells was determined by quantitative mass spectrometry. Results are shown as percentage of input. Error bars show s.d. from triplicate wells. Representative of two experiments. Rifalogue is more lipophilic than rifampicin as a result of its two additional fused aromatic rings, determined by measuring logDs at pH 7, with rifalogue at 3.4, being 100-fold higher than rifampicin (logD 1.3). Additionally, rifalogue has a more basic amine (pKa 9.7) compared to that found in rifampicin (pKa 8.2). This balance of lipophilicity and basicity in rifalogue allows it to localize preferentially in lysosomes. It is challenging to develop antibiotics with these properties for systemic administration due to poor pharmacokinetic (PK) properties and toxicity profiles associated with indiscriminate accumulation of these molecules in all host cells. However, appending such antibiotics to an anti-MRSA specific antibody both extends its half-life in the circulation and converts it into an inactive pro-drug whose properties are manifest only after it has been released in phagolysosomes of cells infected with MRSA.
In vivo infection of mice as shown in Fig. 1a. Mice were infected with equivalent doses of free bacteria or intracellular bacteria and treated with either saline (PBS) or vancomycin (vanco) at 110 mg kg−1, 10 min after infection and then once per day. Selected mice were given vancomycin as described earlier and also treated with a single dose of AAC at 50 mg kg−1 10 min after infection. Four days after infection, bacterial burden was monitored in the brain. Each point represents data from a single mouse (n = 5). Bars show geometric mean. *P < 0.05, Mann–Whitney U-test.
Extended Data Figure 5 Human serum contains high levels of anti-S. aureus antibodies that can compete with the AAC for binding.
a, To estimate the concentration of antibodies that could potentially compete for binding with the anti-β-WTA antibody used in the AAC, human IgG from various sources (normal human serum, serum from MRSA infected patients, purified human IgG (Sigma) or IGIV derived from pooled normal donors) was tested for binding to various bacterial cell wall preparations (CWPs) by enzyme-linked immunosorbent assay (ELISA). CWPs were made from either USA300 (wild type (WT)) or ΔtarMΔtarS (DKO) USA300; the latter strain is deficient in the WTA-GlcNAc antigen recognized by the anti-WTA antibodies. For these studies protein-A-deficient USA300 background strains were used to minimize non-specific antibody binding. A standard curve was generated by titrating known amounts of an anti-MRSA antibody directed against peptidoglycan on both cell wall extracts. b, Estimated concentration of anti-S. aureus antibodies in human serum. The amount of anti-MRSA antibodies in each sample was estimated by comparing the signal obtained for each sample with the standard curve. In the absence of WTA GlcNAc antigens, ~60–70% less serum IgG binding was observed (DKO ELISA; red bars). This indicates the high prevalence of natural antibodies against WTA in adult human serum. Results are reported as μg ml−1 of antibody per 10 mg ml−1 of total IgG. Error bars show mean ± s.d. from triplicate wells. Data are representative of two independent experiments.
a, Titration of AAC in intravenous infection model shown in Fig. 4c (n = 8 mice per group). b, Efficacy of AACs specific for β-WTA or α-WTA in SCID-IGIV model. SCID mice (n = 8 mice per group) were reconstituted with IGIV Immune Globulin using a dosing regimen optimized to achieve constant serum levels of >10 mg ml−1 of human IgG and infected with MRSA. Mice were treated with 60 mg kg−1 of the indicated AACs in a single intravenous injection 1 day after infection and bacterial burden was monitored in kidneys 4 days after infection. Each point represents data from a single animal. Bars show geometric mean. Mann–Whitney test: ***P < 0.005, not significant (NS) P > 0.05.
a, Rifampicin–AAC and rifalogue–AAC release equivalent amounts of free antibiotic after treatment with exogenous cathepsin-B. Released antibiotics from AACs made with rifalogue (red) and rifampicin (grey) are equally active as they can kill USA300 grown in broth culture. Error bars show s.d. from triplicate wells. b, Intracellular killing assay in primary mouse macrophages as shown in Fig. 3c indicates that the rifalogue–AAC, but not rifampicin–AAC is able to kill intracellular S. aureus. Error bars show s.d. from triplicate wells. c, Greater intracellular retention of unconjugated rifalogue compared with rifampicin after release from AAC inside macrophage cells. MRSA was opsonized with AACs and incubated with macrophages (RAW 264.7 cells) to permit phagocytosis. The macrophages were washed to remove extracellular bacteria and samples of cell lysates or supernatants were collected in triplicate at indicated time points and the total amount of released antibiotic was determined by quantitative mass spectrometry. Error bars represent means ± s.d. from triplicate wells. a–c, Representative of two or more independent experiments. d, Rifampicin–AAC is not efficacious in the SCID-IGIV intravenous infection model as shown in Fig. 4d. Each point represents data from a single mouse (n = 8 mice per group). Bars show geometric mean. Mann–Whitney U-test: not significant (NS), P > 0.05.
About this article
Cite this article
Lehar, S., Pillow, T., Xu, M. et al. Novel antibody–antibiotic conjugate eliminates intracellular S. aureus. Nature 527, 323–328 (2015). https://doi.org/10.1038/nature16057
Journal of Synthetic Organic Chemistry, Japan (2020)
Determining the immunological characteristics of a novel human monoclonal antibody developed against staphylococcal enterotoxin B
Human Vaccines & Immunotherapeutics (2020)
Clinical Microbiology Reviews (2020)
Advanced Drug Delivery Reviews (2020)
Intracellular bacteria destruction via traceable enzymes-responsive release and deferoxamine-mediated ingestion of antibiotics
Journal of Controlled Release (2020)