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.
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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.
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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.
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Extended data figures and tables
Extended Data Figure 1 Intracellular MRSA can infect the brain even in the presence of vancomycin.
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.
Extended Data Figure 3 Rifalogue can also kill intracellular bacteria.
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.
Extended Data Figure 4 AAC kills MRSA that survive treatment with vancomycin.
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.
Extended Data Figure 6 Optimization of the in vivo model of bacteraemia.
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.
Extended Data Figure 8 Comparison of anti-β-WTA AACs made with rifalogue and rifampicin.
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.
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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
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DOI: https://doi.org/10.1038/nature16057
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