Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens

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Abstract

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2,3,4,5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6,7,8,9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.

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Fig. 1: Presence of a high-affinity biotin transporter in pathogens accounts for variation in biotin requirements.
Fig. 2: Biotin biosynthesis is essential for A. baumannii, K. pneumoniae and P. aeruginosa during an infection mimicking human biotin levels.
Fig. 3: Crystal structure of E. coli BioA in complex with MAC13772.
Fig. 4: In vivo efficacy of biotin biosynthesis inhibition.

Data availability

The crystal structure of E. coli BioA in complex with MAC13772 solved to 2.4 Å has been deposited to the Protein Data Bank with accession code 6ED7. The source data underlying Figs. 1d and 4a–c, are provided as Supplementary Data Tables 29. The data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank S. McCusker from the Centre for Microbial Chemical Biology and G. Wright for bacterial strains from the Institute for Infectious Disease Research clinical collection, A. Fiebig-Comyn for amendments to the animal use protocol, National Institute of Allergy and Infectious Diseases preclinical services for pharmacokinetic testing, A. Eakin for advice and guidance (contract HHSN272201100022I awarded to SRI, International) and R. Melano at Public Health Ontario for bacterial strains GB687 and C0064. This research was supported by a Foundation grant from the Canadian Institutes for Health Research (FDN-143215), a donation from the Boris Family Foundation and by funding from the Ontario Research Fund Research Excellence program (RE07-048). E.D.B. was supported by a salary award from the Canada Research Chairs program. L.A.C. was supported by an Ontario Graduate Scholarship Award and a Canadian Institutes for Health Research scholarship.

Author information

L.A.C. conceived the research, designed and carried out experiments and data analysis, and wrote the manuscript. C.R.M. assisted with data acquisition and interpretation and manuscript editing. L.A.C designed and conducted the in vitro assays to determine biotin requirement and in vitro enzyme assays. C.N.T. performed the orthologue search and phylogenetic analysis. L.A.C. and B.S.W. designed and performed the plasma growth assays. C.M.B., S.Z. and J.C. designed and performed crystallization experiments and C.M.B performed the analysis with input from M.S.J. L.A.C. and V.N.R. performed susceptibility testing. L.A.C and C.R.M. designed and performed in vivo infection model experiments. B.S.W., M.S.J. and B.K.C. assisted with data interpretation. E.D.B. conceived the research and assisted with data interpretation and manuscript editing.

Correspondence to Eric D. Brown.

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Extended data

Extended Data Fig. 1 Biotin requirements of BioA deficient strains represent the entirety of the biosynthetic pathway.

Analysis of the biotin concentrations required to restore growth of (a) A. baumannii transposon mutants in bioACDF and (b) E. coli ΔbioABCDF in M9 minimal media supplemented with amino acids after 18 hours. Points show the mean of 8 replicates ± s.d. and the solid lines show a four-parameter dose response curve. (c) Presence or absence of a high-affinity biotin transporter in a phylogenetic tree of pathogenic bacteria with an ortholog to E. coli BioA. The presence of an ortholog to biotin transporters, E. coli BioP and S. aureus BioY was determined using OrtholugeDB. Species with a predicted ortholog to E. coli BioP (green) or S. aureus BioY (blue) transporters are demarked by a circle.

Extended Data Fig. 2 Biotin levels are comparable among three common mouse strains.

(a) Quantification of biotin levels in purchased human and mouse plasma from Innovative Research. Each point represents a technical replicate (n = 4) and the line is the mean. (b) Quantification of biotin in the plasma of BALB/c (n = 6), CD-1 (n = 7), and C57Bl/6 (n = 8) mice. Each point represents the biotin levels of a single mouse and the line is the mean. (c) Fold change in viable cell counts of BioA deficient (grey) and wild-type (black) A. baumannii (AB), K. pneumoniae (KP), P. aeruginosa (PA), and S. Typhimurium (ST) growth after 24 hours in 50% human plasma supplemented with biotin (10 µg/mL). BioA deficient or wild-type strains were separately inoculated into plasma diluted with M9 salts supplemented with 25 mM of sodium bicarbonate. Viable cell counts were calculated at time 0 and 24 hours. Fold change was calculated by dividing the viable cell count at 24 hours by the viable cell count at time 0. The bars indicate the mean of 6 replicates ± s.d.

Extended Data Fig. 3 Biotin biosynthesis is dispensable for A. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and S. Typhimurium during standard infection models.

Bacterial load in the blood, spleen, kidneys, liver, and lungs following a systemic infection with (a) A. baumannii (AB; n = 5), (b) K. pneumoniae (KP; n=10), (c) P. aeruginosa (PA; n=9), (d) S. aureus (SA; n=6), and (e) S. Typhimurium (ST; n=5). Mice were co-infected with a mixed inoculum of wild- type (grey) and BioA deficient (black) bacteria inoculated by intraperitoneal injection. (f) Bacterial load in the lungs of mice co-infected with a mixed inoculum of wild-type (grey) and ΔbioA (black) bacteria. Infection was established by intranasal administration of 2×107 CFU by micropipette and mice (n=10) euthanized 36 hours later. (g) Bacterial load in the thighs of mice co-infected with a mixed inoculum of wild-type (grey) and BioA deficient (black) A. baumannii (AB; n=6), K. pneumoniae (KP; n=8), P. aeruginosa (PA; n=4), and S. aureus (SA; n=10). 5×105 (KP and SA) or 5×106 (AB and PA) CFU were injected into the thighs of mice. In all instances box plot whiskers show the minimum to maximum values, the box denotes the interquartile range, and the line in the box shows the median.

Extended Data Fig. 4 Determination of an optimal dose of streptavidin to mimic human biotin levels.

Quantification of biotin in the plasma of CD-1 mice following intraperitoneal administration of (a) streptavidin (1 mg/kg) or (b) streptavidin (4 mg/kg) treatment. Each point represents the biotin levels of a single mouse (n = 2–7) and the line indicates the mean. The grey area indicates the typical range of human biotin concentration in plasma accounting for daily fluctuations. (c) Streptavidin pretreatment has no effect on the ability of wild-type bacteria to cause infection. A. baumannii bacterial load in the blood, spleen, kidney, liver, and lungs in a standard infection model (n=6) or mice pretreated with streptavidin (2 mg/kg; n=4). Mice were infected by intraperitoneal injection and euthanized 10 hours post- infection. Each point represents the bacterial load from an individual mouse and the line indicates the mean.

Extended Data Fig. 5 Biotin biosynthesis is a critical fitness determinant for A. baumannii, K. pneumoniae, and P. aeruginosa during a systemic infection with human biotin levels.

Competitive index (CI) for the co-infection of BioA deficient and wild-type strains of (a) A. baumannii (n=5, 8), (b) K. pneumoniae (n=10, 8), (c) P. aeruginosa (n=9, 8), and (d) S. aureus (n=6, 9) in the blood, spleen, kidneys, liver, and lungs in a standard systemic infection model (light blue) or systemic infection in mice pretreated with streptavidin (2 mg/kg; blue) 1-hour prior to inoculation. CI is calculated by dividing the bacterial load of the query strain (BioA deficient) by the bacterial load of the wild-type strain. Each point represents the CI from an individual mouse and the line indicates the mean. The dotted line represents a CI of 1 indicating the two strains are proliferating equally in vivo. Groups were analyzed with an unpaired two-tailed t-test and corrected for multiple comparisons with a Holm-Sidak test, * indicates a p<0.01.

Extended Data Fig. 6 Biotin biosynthesis is a critical fitness determinant for A. baumannii, K. pneumoniae, and P. aeruginosa during a systemic infection with human biotin levels.

Bacterial load in the blood, spleen, kidneys, liver, and lungs following a systemic infection with human biotin levels with (a) A. baumannii (n=8), (b) K. pneumoniae (n=8), (c) P. aeruginosa (n=8), and (d) S. aureus (n=9). Mice were pretreated with streptavidin (2 mg/kg) 1-hour prior to co- infection with a mixed inoculum of wild-type (grey) and BioA deficient (black) bacteria. Box plot whiskers show the minimum to maximum values, the box denotes the interquartile range, and the line in the box shows the median of each group. Groups were analyzed with an unpaired two-tailed t-test and corrected for multiple comparisons with a Holm-Sidak test, * indicates a p<0.01.

Extended Data Fig. 7 Biotin biosynthesis is dispensable during colonization of a systemic murine infection mimicking human biotin levels.

Bacterial load in the blood, spleen, kidneys, liver, and lungs 1- hour post-infection following a systemic infection with human biotin levels with (a) A. baumannii (AB; n=3), (b) K. pneumoniae (KP; n=2), and (c) P. aeruginosa (PA; n=3). Mice were pretreated with streptavidin (2 mg/kg) 1-hour prior to co- infection with a mixed inoculum of wild-type (grey) and BioA deficient (black) bacteria. Each point represents the bacterial load from an individual mouse and the line indicates the mean.

Extended Data Fig. 8 Resistance to inhibition of biotin biosynthesis imparts a fitness cost.

Kinetics of (7,8)-di-amino-pelargonic acid production. Initial velocities of (7,8)-di-amino-pelargonic acid production with BioA, BioAW52A, BioAK274A, BioAF393A, and BioAY398A were determined at various (a) (S)-keto-amino-pelargonic acid (KAPA; n=4) and (b) S-adenosyl-L-methionine (SAM; n=6) concentrations. Points show the mean of replicates ± s.d. and the solid lines show a Michaelis-Menten curve. (c) Analysis of the biotin concentrations required to restore growth of BioA deficient A. baumannii passaged into the lowest biotin concentration supporting growth for 14 days. Points show the mean of 8 replicates ± s.d. and the solid lines show a four- parameter dose response curve.

Extended Data Fig. 9 MAC13772 is efficacious against A. baumannii in a systemic infection with human biotin levels.

Bacterial load of A. baumannii in the blood, spleen, kidneys, liver, and lungs following a systemic infection with (a) standard biotin levels and (b) human biotin levels, treated with MAC13772 (15 mg/kg). Mice were pretreated with streptavidin (2 mg/kg) 1-hour prior infection and treated with MAC13772 (15 mg/kg) 1-hour following infection. Groups were analyzed with an unpaired two-tailed t-test and corrected for multiple comparisons with a Holm-Sidak test, * indicates a p<0.01. (c) Time course of plasma concentrations of MAC13772 in male (black) and female (grey) Sprague Dawley rats, after intravenous administration. Each data point represents the mean ± s.d. concentration from n=3 rats, the dotted line indicates the limit of quantification (LOQ).

Supplementary information

Supplementary Information

Supplementary Tables 1, 3–6 and 8–11, legends for Supplementary Tables 2 and 7, Supplementary Figures 1–3 and Supplementary References.

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Supplementary Tables 2 and 7

Supplemental Table 2: OrtholugeDB predictions for the presence of an orthologue to E. coli BioP and S. aureus BioY. Supplemental Table 7: prediction of the presence of Phe393, Tyr144, Trp52, Tyr398 and Lys274 in 100 bacteria.

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Carfrae, L.A., MacNair, C.R., Brown, C.M. et al. Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens. Nat Microbiol (2019) doi:10.1038/s41564-019-0595-2

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