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Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality

Abstract

Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)—p85α (PIK3R1) and p85β (PIK3R2)3,4—are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.

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Fig. 1: Alignments and constructs.
Fig. 2: p85 proteins are cleaved by LF.
Fig. 3: LT affects PI3K signalling.
Fig. 4: Mutant p85 mice expressing an LT-resistant variant are more resistant to toxin challenge, and LT-induced lethality in mice requires targeting of both MEK and PI3K pathways.

Data availability

Full-length blot scans, data from animal studies and associated statistical analyses can be found within the source data files. Any other data generated during and/or analysed for the current study are available from the corresponding author on reasonable request. Reported structural analogues used by iTASSER 5.1 in creation of the model in Fig. 1 are available in the Protein Data Bank under accession codes 1XA6, 3CXI, 3BYI, 5C5S, 2QV2, 3QIS, 3FK2, 2OSA, 1OW3, 2EE4 and 3CXL. Source data are provided with this paper.

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Acknowledgements

We thank members of the Mouse Genetics and Gene Modification Section at the National Institute of Allergy and Infectious Diseases and animal care staff and veterinarians in the Comparative Medicine Branch in National Institutes of Health Building 33. This research was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research, National Institutes of Health.

Author information

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Authors

Contributions

M.M. conceived the project. M.M. and M.A.M. designed and performed experiments and analysed the data. M.K.P. and D.O. performed experiments. S.L. designed experiments, performed preliminary mutant LF studies and analysed the data. S.H.L. designed constructs, contributed reagents and analysed the data. R.F. purified proteins and performed mass spectrometry analyses. R.S., T.H.B. and J.S.K. designed and created the knockin mouse model. M.M., M.A.M. and S.H.L. wrote and edited the manuscript with input from all authors.

Corresponding author

Correspondence to Mahtab Moayeri.

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Extended data

Extended Data Fig. 1 Mass spectrometry on LF-generated cleavage products.

SH3-BH protein was incubated with LF (molar ratio of 100:1, SH3-BH: LF) overnight at 37 °C. Mass spectrometry on cleavage products shown as F1 and F2 on gel yielded molecular masses labeled above each peak. Top graph shows molecular mass of uncleaved SH3-BH, middle graph shows molecular mass of F1 cleavage product, and bottom graph shows molecular mass of F2 cleavage product.

Source data

Extended Data Fig. 2 LT effects on MKK4 and MKK7.

a, C57BL/6J were injected IP with protective antigen (PA) (35 µg) or LT (35 µg) and spleens harvested at 24 h, lysed and analyzed for MEK1, MKK4 and MKK7 cleavage (sequentially, in order shown, re-probing the same blot, using antibodies reactive to N-termini of the proteins). The MEK1 and MKK7 antibodies were detected with an anti-rabbit secondary antibody tagged with same wavelength IR800 label, while MKK4 was detected with an anti-rabbit antibody tagged with an IR680 dye. In the lower panel both secondary antibodies react with all three primary antibodies, resulting in yellow color where both MKK7 and MEK1 are present (lanes 1,2,5), orange where MKK7 alone is present (lanes 3,4, 6) and a lower migrating (red) MKK4 band present equally in all samples (lanes 1-6). b, BMDMs from C57BL/6J mice were treated with LT (1 µg/mL, 1h and 18 h) and analyzed for MKK4 and MKK3 cleavage. The MKK3 band is not seen in spleen lysates, processed as in (a). In panel a MEK1 and MKK7 bands overlap with each other as well as with MKK4, thus densitometry quantification of MKK4 bands was performed only for panel b (see Supplementary Table 1).

Source data

Extended Data Fig. 3 Signaling and survival consequences of LF cleavage.

a, C57BL/6J bone marrow-derived macrophages (BMDMs) were treated with LT or LTW271A (1 µg/ml) for indicated times, followed by LPS (1 µg/ml, 90 min). p38 inhibitor PH-797804 treatment was at 50 nM for 16 h, then at 100 nM for 1 h prior to LPS stimulation. Lysates were analyzed using antibodies to the N-terminus of MEK1 and re-probed with anti-pAkt (Thr308). b, c, Human foreskin fibroblasts (b) and HT1080 cells (c) were treated with LT (1 µg/ml, 12 h) prior to addition of insulin (200 nM, 45 min) and lysate analyses as in (a). d, Pik3r1GVAA/GVAA (KI) or wild-type (WT) mice were injected with LT (35 µg) and organs harvested at 24 h were analyzed for p85α and MEK1 cleavage. In the color panel, KI or WT BMDMs were treated with LT (1 µg/mL, 12 h). e, BMDMs were pre-treated with LT (1 µg/ml, 2 or 18 h), followed by stimulation with H2O2 (1 mM, 30 min) or insulin (200 nM, 45 min). Lysates were analyzed for pAkt, MEK1 cleavage, and actin. Top blot is initial probe with p-Akt (Thr308) antibody, middle gel is probe of same gel with MEK1 N-terminal antibody and lower gel is a third re-probe with anti β-actin, where β-actin runs with MEK1. f, Organs of mice challenged with mutant toxin LTW271A or LT (35 µg, IP, 18 h) were analyzed for p85α and MEK1 cleavage (g) KI mice and WT littermates were challenged with high dose LT (75 µg, IV) and monitored for survival. P-value comparing curves is 0.8114 by Log-rank test. h, C57BL/6J mice were challenged with LT (100 µg, IP) or LTW271A (100 µg or 250 µg, IP) and monitored for survival. P-value comparing LT (100 µg) to LTW271A (100 µg) is 0.0926, LT (100 µg) to LTW271A (250 µg) is 0.0128, and comparing the two LTW271A curves is 0.7301 (all by Log-rank test). Blue arrows in panels a-e point to cross-reactive bands which serve as internal equal loading controls. For densitometry quantifications see Supplementary Table 1.

Source data

Extended Data Fig. 4 Test of p38 inhibitor function in vivo.

C57BL/6J mice (n=6/group) were treated with either p38 inhibitor Losmapimod (LOS) (25 mg/kg/200 µl by oral gavage) or vehicle at 18 h and 2 h prior to injection of LPS (5 mg/kg/500 µl, IP). TNF-α levels in serum were measured at 2 h post-LPS treatment to assess inhibition of p38 pathway. Serum from two untreated animals was also measured to establish baseline. **** indicates that P-value comparing the vehicle vs. drug treated group is <0.0001 by unpaired t-test (two-tailed).

Source data

Supplementary information

Supplementary Information

Supplementary Table 1 and densitometry.

Reporting Summary

Source data

Source Data Fig. 2

Full-length blot scans.

Source Data Fig. 2

Densitometry readings, enzyme assays and statistical analyses.

Source Data Fig. 3

Full-length blot scans.

Source Data Fig. 4

Full-length blot scans.

Source Data Fig. 4

Animal study data and statistical analyses.

Source Data Extended Data Fig. 1

Full-length blot scans.

Source Data Extended Data Fig. 2

Full-length blot scans.

Source Data Extended Data Fig. 3

Full-length blot scans.

Source Data Extended Data Fig. 3

Animal study data and statistical analyses.

Source Data Extended Data Fig. 4

ELISA data and statistical analyses.

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Mendenhall, M.A., Liu, S., Portley, M.K. et al. Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality. Nat Microbiol 5, 1464–1471 (2020). https://doi.org/10.1038/s41564-020-0782-1

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